Sample records for gag facilitates hiv-1

  1. Human endogenous retrovirus K Gag coassembles with HIV-1 Gag and reduces the release efficiency and infectivity of HIV-1.

    PubMed

    Monde, Kazuaki; Contreras-Galindo, Rafael; Kaplan, Mark H; Markovitz, David M; Ono, Akira

    2012-10-01

    Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

  2. Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

    PubMed

    Barajas, Brook C; Tanaka, Motoko; Robinson, Bridget A; Phuong, Daryl J; Chutiraka, Kasana; Reed, Jonathan C; Lingappa, Jaisri R

    2018-04-01

    During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing

  3. Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging

    PubMed Central

    Barajas, Brook C.; Tanaka, Motoko; Robinson, Bridget A.; Phuong, Daryl J.; Reed, Jonathan C.

    2018-01-01

    During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing

  4. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.

    2012-05-09

    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV),more » a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.« less

  5. Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination▿

    PubMed Central

    Balamurugan, Arumugam; Lewis, Martha J.; Kitchen, Christina M. R.; Robertson, Michael N.; Shiver, John W.; Daar, Eric S.; Pitt, Jacqueline; Ali, Ayub; Ng, Hwee L.; Currier, Judith S.; Yang, Otto O.

    2008-01-01

    Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016. PMID:18199650

  6. Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

    PubMed

    Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

    2017-08-15

    Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly

  7. HIV-1 subtype A gag variability and epitope evolution.

    PubMed

    Abidi, Syed Hani; Kalish, Marcia L; Abbas, Farhat; Rowland-Jones, Sarah; Ali, Syed

    2014-01-01

    The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

  8. An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

    PubMed Central

    La Porte, Annalena; Cano, Jennifer; Wu, Xuhong; Mitra, Doyel

    2016-01-01

    ABSTRACT INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE Significant gaps exist in our

  9. The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

    PubMed Central

    Waddington, Lynne; Hijnen, Marcel; Velkov, Tony; McKinstry, William J.

    2017-01-01

    The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. PMID:28222188

  10. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

    PubMed Central

    French, Martyn A.; Abudulai, Laila N.; Fernandez, Sonia

    2013-01-01

    The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-α-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection. PMID:26344116

  11. Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection.

    PubMed

    French, Martyn A; Abudulai, Laila N; Fernandez, Sonia

    2013-08-09

    The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of "protective" immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8⁺ T-cell responses restricted by "protective" HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

  12. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag▿

    PubMed Central

    Datta, Siddhartha A. K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan

    2011-01-01

    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed. PMID:21917964

  13. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

  14. Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

    PubMed Central

    2012-01-01

    Background Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. Results A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named AnkGAG1D4 (16.5 kDa) was isolated. AnkGAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of Kd ~ 1 μM, and the AnkGAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing AnkGAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. AnkGAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The AnkGAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of AnkGAG1D4-CA with the Gag assembly and budding pathway. Conclusions The resistance of AnkGAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin AnkGAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules. PMID:22348230

  15. Lipid biosensor interactions with wild type and matrix deletion HIV-1 Gag proteins.

    PubMed

    Barklis, Eric; Staubus, August O; Mack, Andrew; Harper, Logan; Barklis, Robin Lid; Alfadhli, Ayna

    2018-05-01

    The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

    PubMed Central

    Becker, Jordan T.

    2017-01-01

    ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency

  17. Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

    PubMed

    Becker, Jordan T; Sherer, Nathan M

    2017-03-15

    Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis -acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1

  18. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  19. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

    PubMed

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

    2016-01-01

    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

  20. Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

    PubMed

    Llewellyn, G Nicholas; Hogue, Ian B; Grover, Jonathan R; Ono, Akira

    2010-10-28

    T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.

  1. HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice.

    PubMed

    Keller, Paul W; Adamson, Catherine S; Heymann, J Bernard; Freed, Eric O; Steven, Alasdair C

    2011-02-01

    Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.

  2. Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

    PubMed Central

    Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

    2015-01-01

    facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. PMID:26018170

  3. HIV-1 Gag as an Antiviral Target: Development of Assembly and Maturation Inhibitors.

    PubMed

    Spearman, Paul

    2016-01-01

    HIV-1 Gag is the master orchestrator of particle assembly. The central role of Gag at multiple stages of the HIV lifecycle has led to efforts to develop drugs that directly target Gag and prevent the formation and release of infectious particles. Until recently, however, only the catalytic site protease inhibitors have been available to inhibit late stages of HIV replication. This review summarizes the current state of development of antivirals that target Gag or disrupt late events in the retrovirus lifecycle such as maturation of the viral capsid. Maturation inhibitors represent an exciting new series of antiviral compounds, including those that specifically target CA-SP1 cleavage and the allosteric integrase inhibitors that inhibit maturation by a completely different mechanism. Numerous small molecules and peptides targeting CA have been studied in attempts to disrupt steps in assembly. Efforts to target CA have recently gained considerable momentum from the development of small molecules that bind CA and alter capsid stability at the post-entry stage of the lifecycle. Efforts to develop antivirals that inhibit incorporation of genomic RNA or to inhibit late budding events remain in preliminary stages of development. Overall, the development of novel antivirals targeting Gag and the late stages in HIV replication appears much closer to success than ever, with the new maturation inhibitors leading the way.

  4. Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals.

    PubMed

    Mendiratta, Sanjay; Vajpayee, Madhu; Malhotra, Uma; Kaushik, Shweta; Dar, Lalit; Mojumdar, Kamalika; Chauhan, Neeraj Kumar; Sreenivas, Vishnubhatla

    2009-02-01

    Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

  5. Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges.

    PubMed

    Khattar, Sunil K; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C; Montefiori, David C; Samal, Siba K

    2015-07-21

    Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. A safe and effective vaccine that can induce both systemic and mucosal immune responses is needed to control HIV-1. In this study, we showed that coexpression of Env and Gag proteins of HIV-1 performed using a single

  6. Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

    PubMed

    Yang, Yantao; Qu, Na; Tan, Jie; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

    2018-06-11

    During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag ZiL ) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and Gag ZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while Gag ZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not Gag ZiL , was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses. Copyright © 2018 the Author(s). Published by PNAS.

  7. Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

    PubMed

    Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José; Muriaux, Delphine

    2015-08-01

    During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly

  8. Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

    PubMed

    Nikolaitchik, Olga A; Hu, Wei-Shau

    2014-04-01

    A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

  9. Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

    PubMed Central

    Nikolaitchik, Olga A.

    2014-01-01

    ABSTRACT A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5′ untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important

  10. Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.

    PubMed

    Tomo, Naoki; Goto, Toshiyuki; Morikawa, Yuko

    2013-03-26

    Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells

  11. Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression.

    PubMed

    Kiguoya, Marion W; Mann, Jaclyn K; Chopera, Denis; Gounder, Kamini; Lee, Guinevere Q; Hunt, Peter W; Martin, Jeffrey N; Ball, T Blake; Kimani, Joshua; Brumme, Zabrina L; Brockman, Mark A; Ndung'u, Thumbi

    2017-07-01

    There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates ( r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases ( P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants ( P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes

  12. Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame.

    PubMed

    Deforges, Jules; de Breyne, Sylvain; Ameur, Melissa; Ulryck, Nathalie; Chamond, Nathalie; Saaidi, Afaf; Ponty, Yann; Ohlmann, Theophile; Sargueil, Bruno

    2017-07-07

    In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

    PubMed

    Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan

    2017-07-20

    Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

  14. Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method.

    PubMed

    Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A; Wolinsky, Steven M

    2008-06-01

    Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse

  15. How HIV-1 Gag assembles in cells: putting together pieces of the puzzle

    PubMed Central

    Lingappa, Jaisri R; Reed, Jonathan C; Tanaka, Motoko; Chutiraka, Kasana; Robinson, Bridget A

    2014-01-01

    During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly-translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions. Particular emphasis is placed on reconciling data suggesting that Gag assembles by two different paths, depending on the assembly environment. Specifically, in assembly systems that lack cellular proteins, high concentrations of Gag can spontaneously assemble using purified nucleic acid as a scaffold. However, in the more complex intracellular environment, barriers that limit self-assembly are present in the form of cellular proteins, organelles, host defenses, and the absence of free nucleic acid. To overcome these barriers and promote efficient immature capsid formation in an unfavorable environment, Gag appears to utilize an energy-dependent, host-catalyzed, pathway of assembly intermediates in cells. Overall, we show how data obtained using a variety of techniques has led to our current understanding of HIV assembly. PMID:25066606

  16. Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

    PubMed

    Datta, Siddhartha A K; Clark, Patrick K; Fan, Lixin; Ma, Buyong; Harvin, Demetria P; Sowder, Raymond C; Nussinov, Ruth; Wang, Yun-Xing; Rein, Alan

    2016-02-15

    HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the

  17. Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

    PubMed Central

    Clark, Patrick K.; Fan, Lixin; Ma, Buyong; Harvin, Demetria P.; Sowder, Raymond C.; Nussinov, Ruth; Wang, Yun-Xing

    2015-01-01

    ABSTRACT HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a “spacer” between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly

  18. Interaction of HIV-1 Gag protein components with single DNA molecules

    NASA Astrophysics Data System (ADS)

    Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

    2003-03-01

    The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

  19. Tsg101 regulates PI(4,5)P2/Ca2+ signaling for HIV-1 Gag assembly

    PubMed Central

    Ehrlich, Lorna S.; Medina, Gisselle N.; Photiadis, Sara; Whittredge, Paul B.; Watanabe, Susan; Taraska, Justin W.; Carter, Carol A.

    2014-01-01

    Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an

  20. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

    PubMed Central

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-01-01

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane. PMID:27120610

  1. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

    PubMed

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-04-25

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  2. Critical Role of the HTLV-1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

    PubMed

    Martin, Jessica L; Mendonça, Luiza; Marusinec, Rachel; Zuczek, Jennifer; Angert, Isaac; Blower, Ruth J; Mueller, Joachim D; Perilla, Juan R; Zhang, Wei; Mansky, Louis M

    2018-04-25

    The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD) as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses. Importance Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The capsid (CA) domain of Gag is generally thought to possess the key

  3. Induction of strong anti-HIV cellular immunity by a combination of Clostridium perfringens expressing HIV gag and virus like particles.

    PubMed

    Pegu, Poonam; Helmus, Ruth; Gupta, Phalguni; Tarwater, Patrick; Caruso, Lori; Shen, Chengli; Ross, Ted; Chen, Yue

    2011-12-01

    The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8(+) and CD4(+) T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

  4. Lack of a significant impact of Gag-Protease-mediated HIV-1 replication capacity on clinical parameters in treatment-naive Japanese individuals.

    PubMed

    Sakai, Keiko; Chikata, Takayuki; Brumme, Zabrina L; Brumme, Chanson J; Gatanaga, Hiroyuki; Gatanag, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2015-11-19

    HLA class I-associated escape mutations in HIV-1 Gag can reduce viral replication, suggesting that associated fitness costs could impact HIV-1 disease progression. Previous studies in North American and African cohorts have reported reduced Gag-Protease mediated viral replication capacity (Gag-Pro RC) in individuals expressing protective HLA class I alleles including HLA-B*57:01, B*27:05, and B*81:01. These studies also reported significant positive associations between Gag-Pro RCs and plasma viral load (pVL). However, these HLA alleles are virtually absent in Japan, and the importance of Gag as an immune target is not clearly defined in this population. We generated chimeric NL4-3 viruses carrying patient-derived Gag-Protease from 306 treatment-naive Japanese individuals chronically infected with HIV-1 subtype B. We analyzed associations between Gag-Pro RC and clinical markers of HIV-1 infection and host HLA expression. We observed no significant correlation between Gag-Pro RC and pVL in Japan in the overall cohort. However, upon exclusion of individuals expressing Japanese protective alleles HLA-B*52:01 and B*67:01, Gag-Pro RC correlated positively with pVL and negatively with CD4 T-cell count. Our results thus contrast with studies from other global cohorts reporting significantly lower Gag-Pro RC among persons expressing protective HLA alleles, and positive relationships between Gag-Pro RC and pVL in the overall study populations. We also identified five amino acids in Gag-Protease significantly associated with Gag-Pro RC, whose effects on RC were confirmed by site-directed mutagenesis. However, of the four mutations that decreased Gag-Pro RC, none were associated with reductions in pVL in Japan though two were associated with lower pVL in North America. These data indicate that Gag fitness does not affect clinical outcomes in subjects with protective HLA class I alleles as well as the whole Japanese population. Moreover, the impact of Gag fitness costs on HIV

  5. Modeling the dynamics and kinetics of HIV-1 Gag during viral assembly.

    PubMed

    Tomasini, Michael D; Johnson, Daniel S; Mincer, Joshua S; Simon, Sanford M

    2018-01-01

    We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results.

  6. Modeling the dynamics and kinetics of HIV-1 Gag during viral assembly

    PubMed Central

    Tomasini, Michael D.; Johnson, Daniel S.; Mincer, Joshua S.

    2018-01-01

    We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results. PMID:29677208

  7. Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges

    PubMed Central

    Khattar, Sunil K.; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C.; Montefiori, David C.

    2015-01-01

    ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. PMID:26199332

  8. HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA.

    PubMed

    Ren, Xiao-Xin; Wang, Hai-Bo; Li, Chuan; Jiang, Jin-Feng; Xiong, Si-Dong; Jin, Xia; Wu, Li; Wang, Jian-Hua

    2016-02-26

    HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Role of Gag and lipids during HIV-1 assembly in CD4+ T cells and macrophages

    PubMed Central

    Mariani, Charlotte; Desdouits, Marion; Favard, Cyril; Benaroch, Philippe; Muriaux, Delphine M.

    2014-01-01

    HIV-1 is an RNA enveloped virus that preferentially infects CD4+ T lymphocytes and also macrophages. In CD4+ T cells, HIV-1 mainly buds from the host cell plasma membrane. The viral Gag polyprotein targets the plasma membrane and is the orchestrator of the HIV assembly as its expression is sufficient to promote the formation of virus-like particles carrying a lipidic envelope derived from the host cell membrane. Certain lipids are enriched in the viral membrane and are thought to play a key role in the assembly process and the envelop composition. A large body of work performed on infected CD4+ T cells has provided important knowledge about the assembly process and the membrane virus lipid composition. While HIV assembly and budding in macrophages is thought to follow the same general Gag-driven mechanism as in T-lymphocytes, the HIV cycle in macrophage exhibits specific features. In these cells, new virions bud from the limiting membrane of seemingly intracellular compartments, where they accumulate while remaining infectious. These structures are now often referred to as Virus Containing Compartments (VCCs). Recent studies suggest that VCCs represent intracellularly sequestered regions of the plasma membrane, but their precise nature remains elusive. The proteomic and lipidomic characterization of virions produced by T cells or macrophages has highlighted the similarity between their composition and that of the plasma membrane of producer cells, as well as their enrichment in acidic lipids, some components of raft lipids and in tetraspanin-enriched microdomains. It is likely that Gag promotes the coalescence of these components into an assembly platform from which viral budding takes place. How Gag exactly interacts with membrane lipids and what are the mechanisms involved in the interaction between the different membrane nanodomains within the assembly platform remains unclear. Here we review recent literature regarding the role of Gag and lipids on HIV-1

  10. Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates.

    PubMed

    Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

    2012-01-01

    Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

  11. Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

    PubMed Central

    Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

    2012-01-01

    Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods. PMID:22355381

  12. Plasmacytoid dendritic cell and functional HIV Gag p55-specific T cells before treatment interruption can inform set-point plasma HIV viral load after treatment interruption in chronically suppressed HIV-1(+) patients.

    PubMed

    Papasavvas, Emmanouil; Foulkes, Andrea; Yin, Xiangfan; Joseph, Jocelin; Ross, Brian; Azzoni, Livio; Kostman, Jay R; Mounzer, Karam; Shull, Jane; Montaner, Luis J

    2015-07-01

    The identification of immune correlates of HIV control is important for the design of immunotherapies that could support cure or antiretroviral therapy (ART) intensification-related strategies. ART interruptions may facilitate this task through exposure of an ART partially reconstituted immune system to endogenous virus. We investigated the relationship between set-point plasma HIV viral load (VL) during an ART interruption and innate/adaptive parameters before or after interruption. Dendritic cell (DC), natural killer (NK) cell and HIV Gag p55-specific T-cell functional responses were measured in paired cryopreserved peripheral blood mononuclear cells obtained at the beginning (on ART) and at set-point of an open-ended interruption from 31 ART-suppressed chronically HIV-1(+) patients. Spearman correlation and linear regression modeling were used. Frequencies of plasmacytoid DC (pDC), and HIV Gag p55-specific CD3(+)  CD4(-)  perforin(+)  IFN-γ(+) cells at the beginning of interruption associated negatively with set-point plasma VL. Inclusion of both variables with interaction into a model resulted in the best fit (adjusted R(2)  = 0·6874). Frequencies of pDC or HIV Gag p55-specific CD3(+)  CD4(-)  CSFE(lo)  CD107a(+) cells at set-point associated negatively with set-point plasma VL. The dual contribution of pDC and anti-HIV T-cell responses to viral control, supported by our models, suggests that these variables may serve as immune correlates of viral control and could be integrated in cure or ART-intensification strategies. © 2015 John Wiley & Sons Ltd.

  13. HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

    PubMed

    Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D; Ndung'u, Thumbi; Ndhlovu, Zaza M

    2017-04-01

    Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

  14. HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection

    PubMed Central

    Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D.; Ndung'u, Thumbi

    2017-01-01

    ABSTRACT Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = −0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

  15. The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation.

    PubMed

    Checkley, Mary Ann; Luttge, Benjamin G; Soheilian, Ferri; Nagashima, Kunio; Freed, Eric O

    2010-04-25

    The human immunodeficiency virus type 1 (HIV-1) maturation inhibitor bevirimat disrupts virus replication by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) Gag processing intermediate to mature CA. The observation that bevirimat delays but does not completely block CA-SP1 processing suggests that the presence of uncleaved CA-SP1 may disrupt the maturation process in trans. In this study, we validate this hypothesis by using a genetic approach to demonstrate that a non-cleavable CA-SP1 mutant exerts a dominant-negative effect on maturation of wild-type HIV-1. In contrast, a mutant in which cleavage can occur internally within SP1 is significantly less potent as a dominant-negative inhibitor. We also show that bevirimat blocks processing at both the major CA-SP1 cleavage site and the internal site. These data underscore the importance of full CA-SP1 processing for HIV-1 maturation and highlight the therapeutic potential of inhibitors that target this Gag cleavage event. Published by Elsevier Inc.

  16. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

    PubMed

    Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

    2013-08-01

    Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

  17. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    PubMed Central

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  18. Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

    PubMed

    Harro, Clayton D; Robertson, Michael N; Lally, Michelle A; O'Neill, Lori D; Edupuganti, Srilatha; Goepfert, Paul A; Mulligan, Mark J; Priddy, Frances H; Dubey, Sheri A; Kierstead, Lisa S; Sun, Xiao; Casimiro, Danilo R; DiNubile, Mark J; Shiver, John W; Leavitt, Randi Y; Mehrotra, Devan V

    2009-01-01

    Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus > or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus > or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen

  19. Safety and Immunogenicity of Adenovirus-Vectored Near-Consensus HIV Type 1 Clade B gag Vaccines in Healthy Adults

    PubMed Central

    Robertson, Michael N.; Lally, Michelle A.; O'Neill, Lori D.; Edupuganti, Srilatha; Goepfert, Paul A.; Mulligan, Mark J.; Priddy, Frances H.; Dubey, Sheri A.; Kierstead, Lisa S.; Sun, Xiao; Casimiro, Danilo R.; DiNubile, Mark J.; Shiver, John W.; Leavitt, Randi Y.; Mehrotra, Devan V.

    2009-01-01

    Abstract Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-γ ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus ≥200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus ≥200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the

  20. Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner.

    PubMed

    Hahn, Friedrich; Schmalen, Adrian; Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Fraedrich, Kirsten; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

    2017-01-01

    There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

  1. PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.

    PubMed

    Sharma, Shilpee; Arunachalam, Prabhu S; Menon, Malini; Ragupathy, Viswanath; Satya, Ravi Vijaya; Jebaraj, Joshua; Ganeshappa Aralaguppe, Shambhu; Rao, Chaitra; Pal, Sreshtha; Saravanan, Shanmugam; Murugavel, Kailapuri G; Balakrishnan, Pachamuthu; Solomon, Suniti; Hewlett, Indira; Ranga, Udaykumar

    2018-05-17

    HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation - an enhanced effect at low concentration and an inhibitory effect only at higher concentrations - unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

  2. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

    PubMed

    Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

    2011-12-07

    Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

  3. Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DELTAV1V2 is strongly immunogenic

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guerbois, Mathilde; Moris, Arnaud; Combredet, Chantal

    Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DELTAV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity tomore » both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.« less

  4. Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

    PubMed Central

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

  5. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    PubMed

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  6. Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring

    PubMed Central

    Mücksch, Frauke; Laketa, Vibor; Müller, Barbara; Schultz, Carsten; Kräusslich, Hans-Georg

    2017-01-01

    HIV-1 assembles at the plasma membrane (PM) of infected cells. PM association of the main structural protein Gag depends on its myristoylated MA domain and PM PI(4,5)P2. Using a novel chemical biology tool that allows rapidly tunable manipulation of PI(4,5)P2 levels in living cells, we show that depletion of PI(4,5)P2 completely prevents Gag PM targeting and assembly site formation. Unexpectedly, PI(4,5)P2 depletion also caused loss of pre-assembled Gag lattices from the PM. Subsequent restoration of PM PI(4,5)P2 reinduced assembly site formation even in the absence of new protein synthesis, indicating that the dissociated Gag molecules remained assembly competent. These results reveal an important role of PI(4,5)P2 for HIV-1 morphogenesis beyond Gag recruitment to the PM and suggest a dynamic equilibrium of Gag-lipid interactions. Furthermore, they establish an experimental system that permits synchronized induction of HIV-1 assembly leading to induced production of infectious virions by targeted modulation of Gag PM targeting. DOI: http://dx.doi.org/10.7554/eLife.25287.001 PMID:28574338

  7. Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

    PubMed

    Kharytonchyk, Siarhei; Brown, Joshua D; Stilger, Krista; Yasin, Saif; Iyer, Aishwarya S; Collins, John; Summers, Michael F; Telesnitsky, Alice

    2018-07-06

    The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features. We previously defined minimal 5' leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5' leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5' leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. A Temporospatial Map That Defines Specific Steps at Which Critical Surfaces in the Gag MA and CA Domains Act during Immature HIV-1 Capsid Assembly in Cells

    PubMed Central

    Robinson, Bridget A.; Reed, Jonathan C.; Geary, Clair D.; Swain, J. Victor

    2014-01-01

    ABSTRACT During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used

  9. C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain.

    PubMed

    Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

    2017-05-15

    HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6

  10. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates

    NASA Astrophysics Data System (ADS)

    Wille-Reece, Ulrike; Flynn, Barbara J.; Loré, Karin; Koup, Richard A.; Kedl, Ross M.; Mattapallil, Joseph J.; Weiss, Walter R.; Roederer, Mario; Seder, Robert A.

    2005-10-01

    Induction and maintenance of antibody and T cell responses will be critical for developing a successful vaccine against HIV. A rational approach for generating such responses is to design vaccines or adjuvants that have the capacity to activate specific antigen-presenting cells. In this regard, dendritic cells (DCs) are the most potent antigen-presenting cells for generating primary T cell responses. Here, we report that Toll-like receptor (TLR) agonists and ligands that activate DCs in vitro influence the magnitude and quality of the cellular immune response in nonhuman primates (NHPs) when administered with HIV Gag protein. NHPs immunized with HIV Gag protein and a TLR7/8 agonist or a TLR9 ligand [CpG oligodeoxynucleotides (CpG ODN)] had significantly increased Gag-specific T helper 1 and antibody responses, compared with animals immunized with HIV Gag protein alone. Importantly, conjugating the HIV Gag protein to the TLR7/8 agonist (Gag-TLR7/8 conjugate) dramatically enhanced the magnitude and altered the quality of the T helper 1 response, compared with animals immunized with HIV Gag protein and the TLR7/8 agonist or CpG ODN. Furthermore, immunization with the Gag-TLR7/8 conjugate vaccine elicited Gag-specific CD8+ T responses. Collectively, our results show that conjugating HIV Gag protein to a TLR7/8 agonist is an effective way to elicit broad-based adaptive immunity in NHPs. This type of vaccine formulation should have utility in preventive or therapeutic vaccines in which humoral and cellular immunity is required. vaccine | dendritic cell | cross-presentation | cellular immunity

  11. NC-Mediated Nucleolar Localization of Retroviral Gag Proteins

    PubMed Central

    Lochmann, Timothy L.; Bann, Darrin V.; Ryan, Eileen P.; Beyer, Andrea R.; Mao, Annie; Cochrane, Alan

    2012-01-01

    The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5′ untranslated RU5 region of the viral RNA genome, suggesting the psi (ψ packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that the there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection. PMID:23036987

  12. Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus

    PubMed Central

    Khattar, Sunil K; Palaniyandi, Senthilkumar; Samal, Sweety; LaBranche, Celia C; Montefiori, David C; Zhu, Xiaoping; Samal, Siba K

    2015-01-01

    The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8+ T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4+ T cells. The level of Gag-specific CD8+ and CD4+ T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins. PMID:25695657

  13. Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia

    2010-10-10

    Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses,more » as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.« less

  14. A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meador, Lydia R.

    Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viralmore » vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.« less

  15. Genetic diversity of HIV-1 non-B strains in Sicily: evidence of intersubtype recombinants by sequence analysis of gag, pol, and env genes.

    PubMed

    Tramuto, Fabio; Bonura, Filippa; Perna, Anna Maria; Mancuso, Salvatrice; Firenze, Alberto; Romano, Nino; Vitale, Francesco

    2007-09-01

    The molecular epidemiology of HIV-1 strains in Sicily (Italy) was phylogenetically investigated by the analysis of HIV-1 gag, pol, and env gene sequences from 11 HIV-1 non-B strains from 408 HIV-1-seropositive patients observed from September 2001 to August 2006. Sequences suggestive of recombination were further investigated by bootscanning analysis of various fragments. Overall, we identified several second-generation recombinant (SGRs) strains, which contained genetic material of CRF02_AG in at least one gene. Notably, three individuals were found to be infected with subsubtype A3, and one of them showed genetic recombination with subsubtype A4. The current study emphasizes the genetic analysis of gag, pol, and env genes as a powerful tool to trace the spread of complex HIV-1 recombinant forms, and highlight the genetic diversity of HIV-1 non-B strains in Italy.

  16. Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription

    PubMed Central

    Beerens, Nancy; Kjems, Jørgen

    2010-01-01

    Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer involves a jump from the 5′ to the 3′ terminal repeat (R) region positioned at each end of the viral genome. The process depends on base pairing between the cDNA synthesized from the 5′ R region and the 3′ R RNA. The tertiary conformation of the viral RNA genome may facilitate strand transfer by juxtaposing the 5′ R and 3′ R sequences that are 9 kb apart in the linear sequence. In this study, RNA sequences involved in an interaction between the 5′ and 3′ ends of the HIV-1 genome were mapped by mutational analysis. This interaction appears to be mediated mainly by a sequence in the extreme 3′ end of the viral genome and in the gag open reading frame. Mutation of 3′ R sequences was found to inhibit the 5′–3′ interaction, which could be restored by a complementary mutation in the 5′ gag region. Furthermore, we find that circularization of the HIV-1 genome does not affect the initiation of reverse transcription, but stimulates the first strand transfer during reverse transcription in vitro, underscoring the functional importance of the interaction. PMID:20430859

  17. Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

    PubMed

    Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

    2016-07-01

    BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1

  18. Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

    PubMed

    Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

    2017-09-15

    Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a

  19. Drug evaluation: bevirimat--HIV Gag protein and viral maturation inhibitor.

    PubMed

    Temesgen, Zelalem; Feinberg, Judith E

    2006-08-01

    Panacos Pharmaceuticals Inc is developing the HIV Gag protein and viral maturation inhibitor bevirimat for the potential oral treatment of HIV infection. Phase II clinical trials are underway and phase III trials expected to commence in 2007.

  20. Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

    PubMed Central

    Chopera, Denis R.; Cotton, Laura A.; Zawaira, Alexander; Mann, Jaclyn K.; Ngandu, Nobubelo K.; Ntale, Roman; Carlson, Jonathan M.; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D.; Gray, Clive M.; Martin, Darren P.; Ndung'u, Thumbi; Brockman, Mark A.; Karim, Salim Abdool

    2012-01-01

    Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. PMID:23015721

  1. Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

    PubMed

    Grewe, Bastian; Hoffmann, Bianca; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grunwald, Thomas; Uberla, Klaus

    2012-03-01

    In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

  2. Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese.

    PubMed

    Naruto, Takuya; Murakoshi, Hayato; Chikata, Takayuki; Koyanagi, Madoka; Kawashima, Yuka; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2011-08-01

    We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  3. Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.

    PubMed

    Esteves, Aida; Parreira, Ricardo; Piedade, João; Venenno, Teresa; Franco, Margarida; Germano de Sousa, José; Patrício, Luis; Brum, Paula; Costa, António; Canas-Ferreira, Wanda F

    2003-06-01

    We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.

  4. Modeling the full length HIV-1 Gag polyprotein reveals the role of its p6 subunit in viral maturation and the effect of non-cleavage site mutations in protease drug resistance.

    PubMed

    Su, Chinh Tran-To; Kwoh, Chee-Keong; Verma, Chandra Shekhar; Gan, Samuel Ken-En

    2017-12-27

    HIV polyprotein Gag is increasingly found to contribute to protease inhibitor resistance. Despite its role in viral maturation and in developing drug resistance, there remain gaps in the knowledge of the role of certain Gag subunits (e.g. p6), and that of non-cleavage mutations in drug resistance. As p6 is flexible, it poses a problem for structural experiments, and is hence often omitted in experimental Gag structural studies. Nonetheless, as p6 is an indispensable component for viral assembly and maturation, we have modeled the full length Gag structure based on several experimentally determined constraints and studied its structural dynamics. Our findings suggest that p6 can mechanistically modulate Gag conformations. In addition, the full length Gag model reveals that allosteric communication between the non-cleavage site mutations and the first Gag cleavage site could possibly result in protease drug resistance, particularly in the absence of mutations in Gag cleavage sites. Our study provides a mechanistic understanding to the structural dynamics of HIV-1 Gag, and also proposes p6 as a possible drug target in anti-HIV therapy.

  5. Measuring the binding stoichiometry of HIV-1 Gag to very-low-density oligonucleotide surfaces using surface plasmon resonance spectroscopy.

    PubMed

    Stephen, Andrew G; Datta, Siddhartha A K; Worthy, Karen M; Bindu, Lakshman; Fivash, Matthew J; Turner, Kevin B; Fabris, Daniele; Rein, Alan; Fisher, Robert J

    2007-09-01

    The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.

  6. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    PubMed

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  7. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

    PubMed Central

    Lakhashe, Samir K.; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B.; DiPasquale, Janet M.; Hemashettar, Girish; Yoon, John K.; Rasmussen, Robert A.; Yang, Feng; Lee, Sandra J.; Montefiori, David C.; Novembre, Francis J.; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R.; Robert-Guroff, Marjorie; Johnson, Welkin E.; Lieberman, Judy; Ruprecht, Ruth M.

    2011-01-01

    We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus >90%; these RM also had strong SIV Gag-specific proliferation of CD8+ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4+ T cells; the latter have been implicated as preferential virus targets in-vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

  8. Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

    PubMed

    Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

    2011-08-05

    We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

    PubMed

    Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Kramberger, Petra; Tover, Andres; Berger, Eva; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

    2017-02-01

    The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10 12 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10 11 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10 9 particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity

    PubMed Central

    Naidoo, Vanessa L.; Mann, Jaclyn K.; Noble, Christie; Adland, Emily; Carlson, Jonathan M.; Thomas, Jake; Brumme, Chanson J.; Thobakgale-Tshabalala, Christina F.; Brumme, Zabrina L.; Goulder, Philip J. R.

    2017-01-01

    ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT

  11. Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat.

    PubMed

    Adamson, Catherine S; Sakalian, Michael; Salzwedel, Karl; Freed, Eric O

    2010-04-20

    The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance. Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

  12. Cross-reactivity of anti-HIV-1 T cell immune responses among the major HIV-1 clades in HIV-1-positive individuals from 4 continents.

    PubMed

    Coplan, Paul M; Gupta, Swati B; Dubey, Sheri A; Pitisuttithum, Punnee; Nikas, Alex; Mbewe, Bernard; Vardas, Efthyia; Schechter, Mauro; Kallas, Esper G; Freed, Dan C; Fu, Tong-Ming; Mast, Christopher T; Puthavathana, Pilaipan; Kublin, James; Brown Collins, Kelly; Chisi, John; Pendame, Richard; Thaler, Scott J; Gray, Glenda; Mcintyre, James; Straus, Walter L; Condra, Jon H; Mehrotra, Devan V; Guess, Harry A; Emini, Emilio A; Shiver, John W

    2005-05-01

    The genetic diversity of human immunodeficiency virus type 1 (HIV-1) raises the question of whether vaccines that include a component to elicit antiviral T cell immunity based on a single viral genetic clade could provide cellular immune protection against divergent HIV-1 clades. Therefore, we quantified the cross-clade reactivity, among unvaccinated individuals, of anti-HIV-1 T cell responses to the infecting HIV-1 clade relative to other major circulating clades. Cellular immune responses to HIV-1 clades A, B, and C were compared by standardized interferon- gamma enzyme-linked immunospot assays among 250 unvaccinated individuals, infected with diverse HIV-1 clades, from Brazil, Malawi, South Africa, Thailand, and the United States. Cross-clade reactivity was evaluated by use of the ratio of responses to heterologous versus homologous (infecting) clades of HIV-1. Cellular immune responses were predominantly focused on viral Gag and Nef proteins. Cross-clade reactivity of cellular immune responses to HIV-1 clade A, B, and C proteins was substantial for Nef proteins (ratio, 0.97 [95% confidence interval, 0.89-1.05]) and lower for Gag proteins (ratio, 0.67 [95% confidence interval, 0.62-0.73]). The difference in cross-clade reactivity to Nef and Gag proteins was significant (P<.0001). Cross-clade reactivity of cellular immune responses can be substantial but varies by viral protein.

  13. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

    PubMed Central

    Jones, Christopher P.; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-01-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNALys3. Host cell tRNALys is selectively packaged into HIV-1 through a specific interaction between the major tRNALys-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNALys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNALys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNALys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNALys to increase the efficiency of tRNALys3 annealing to viral RNA. PMID:23264568

  14. Polyfunctional analysis of Gag and Nef specific CD8+ T-cell responses in HIV-1 infected Indian individuals.

    PubMed

    Mendiratta, Sanjay; Vajpayee, Madhu; Mojumdar, Kamalika; Chauhan, Neeraj K; Sreenivas, Vishnubhatla

    2011-02-01

    Polyfunctional CD8+ T-cells have been described as most competent in controlling viral replication. We studied the impact of antigen persistence on the polyfunctional immune responses of CD8+ T-lymphocytes to HIV Gag and Nef peptides and polyclonal stimuli in 40 ART naïve HIV infected individuals and analyzed the alterations in T-cell functionality in early and late stages of infection. Significantly elevated level of global response and polyfunctional profile of CD8+ T-cells were observed to polyclonal stimulation, than HIV specific antigens in chronically infected individuals. However no key differences were observed in CD8+ T-cell functional profile in any of the 15 unique subsets for Gag and Nef specific antigens. The subjects in early stage of infection (defined as a gap of 6 months or less between seroconversion and enrolment and with no apparent clinical symptoms) had a higher degree of response functionality (4+ or 3+ different functions simultaneously) than in the late stage infection (defined as time duration since seroconversion greater than 6 months). The data suggest that persistence of antigen during chronic infection leads to functional impairment of HIV specific responses. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

    PubMed

    Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

    2017-12-01

    The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

    PubMed

    Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

    2005-11-01

    We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

  17. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

    PubMed

    Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-02-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

  18. Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells.

    PubMed

    Gomez, Candace Y; Hope, Thomas J

    2006-09-01

    Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

  19. Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

    PubMed

    Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

    2017-11-28

    Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently

  20. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, whichmore » is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt

  1. On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?▿

    PubMed Central

    Datta, Siddhartha A. K.; Temeselew, Lakew G.; Crist, Rachael M.; Soheilian, Ferri; Kamata, Anne; Mirro, Jane; Harvin, Demetria; Nagashima, Kunio; Cachau, Raul E.; Rein, Alan

    2011-01-01

    Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch. PMID:21325421

  2. HIV type 1 subtypes among bar and hotel workers in Moshi, Tanzania.

    PubMed

    Kiwelu, Ireen E; Renjifo, Boris; Chaplin, Beth; Sam, Noel; Nkya, Watoky M M M; Shao, John; Kapiga, Saidi; Essex, Max

    2003-01-01

    The HIV-1 prevalence among bar and hotel workers in Tanzania suggests they are a high-risk group for HIV-1 infection. We determined the HIV-1 subtype of 3'-p24/5'-p7 gag and C2-C5 env sequences from 40 individuals representing this population in Moshi. Genetic patterns composed of A(gag)-A(env), C(gag)-C(env), and D(gag)-D(env) were found in 19 (48.0%), 8 (20.0%), and 3 (8.0%) samples, respectively. The remaining 10 samples (25%) had different subtypes in gag and env, indicative of intersubtype recombinants. Among these recombinants, two contained sequences from HIV-1 subsubtype A2, a new genetic variant in Tanzania. Five bar and hotel workers may have been infected with viruses from a common source, based on phylogenetic analysis. The information obtained by surveillance of HIV-1 subtypes in a high-risk population should be useful in the design and evaluation of behavioral, therapeutic, and vaccine trial interventions aimed at reducing HIV-1 transmission.

  3. MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

    PubMed Central

    Janes, Holly; Frahm, Nicole; DeCamp, Allan; Rolland, Morgane; Gabriel, Erin; Wolfson, Julian; Hertz, Tomer; Kallas, Esper; Goepfert, Paul; Friedrich, David P.; Corey, Lawrence; Mullins, James I.; McElrath, M. Juliana; Gilbert, Peter

    2012-01-01

    Background The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1) but the difference was not significant (p = 0·27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30). Interpretation Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression. PMID

  4. Pharmacological intervention of HIV-1 maturation.

    PubMed

    Wang, Dan; Lu, Wuxun; Li, Feng

    2015-11-01

    Despite significant advances in antiretroviral therapy, increasing drug resistance and toxicities observed among many of the current approved human immunodeficiency virus (HIV) drugs indicate a need for discovery and development of potent and safe antivirals with a novel mechanism of action. Maturation inhibitors (MIs) represent one such new class of HIV therapies. MIs inhibit a late step in the HIV-1 Gag processing cascade, causing defective core condensation and the release of non-infectious virus particles from infected cells, thus blocking the spread of the infection to new cells. Clinical proof-of-concept for the MIs was established with betulinic acid derived bevirimat, the prototype HIV-1 MI. Despite the discontinuation of its further clinical development in 2010 due to a lack of uniform patient response caused by naturally occurring drug resistance Gag polymorphisms, several second-generation MIs with improved activity against viruses exhibiting Gag polymorphism mediated resistance have been recently discovered and are under clinical evaluation in HIV/AID patients. In this review, current understanding of HIV-1 MIs is described and recent progress made toward elucidating the mechanism of action, target identification and development of second-generation MIs is reviewed.

  5. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

    PubMed

    Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

    2015-11-01

    To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

  6. Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

    PubMed Central

    Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

    2005-01-01

    We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

  7. Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

    PubMed Central

    Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

    2013-01-01

    Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263

  8. High prevalence of natural polymorphisms in Gag (CA-SP1) associated with reduced response to Bevirimat, an HIV-1 maturation inhibitor.

    PubMed

    Seclén, Eduardo; González, María Del Mar; Corral, Angélica; de Mendoza, Carmen; Soriano, Vincent; Poveda, Eva

    2010-01-28

    Mutations H358Y, L363F/M, A364V and A366T/V confer in-vitro resistance to bevirimat. Moreover, polymorphisms at the Glutamine-Valine-Threonine (QVT) motif (369-371) have been associated with reduced bevirimat activity in vivo. The rate of these changes was assessed in 389 HIV+ patients naïve for bevirimat. QVT polymorphisms were frequent (47%), especially in non-B subtypes (93%). Conversely, only four patients (1%) harbored major bevirimat resistance mutations. Finally, specific gag changes were associated with protease inhibitor resistance mutations in subtype B viruses.

  9. HIV-1 transmission linkage in an HIV-1 prevention clinical trial

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Leitner, Thomas; Campbell, Mary S; Mullins, James I

    2009-01-01

    HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determinationmore » in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

  10. Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media.

    PubMed

    Lynch, Alisson; Meyers, Ann E; Williamson, Anna-Lise; Rybicki, Edward P

    2012-09-18

    HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4°C, -20°C and -70°C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70°C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70°C for 12 months is most effective in retaining VLP stability.

  11. Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

    PubMed Central

    Shahid, Aniqa; Olvera, Alex; Anmole, Gursev; Kuang, Xiaomei T.; Cotton, Laura A.; Plana, Montserrat; Brander, Christian; Brockman, Mark A.

    2015-01-01

    ABSTRACT HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24Gag (GQMVHQAIGag140–147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other

  12. HIV-1 subtype C is not associated with higher risk of heterosexual HIV-1 transmission: a multinational study among HIV-1 serodiscordant couples.

    PubMed

    Kahle, Erin; Campbell, Mary; Lingappa, Jairam; Donnell, Deborah; Celum, Connie; Ondondo, Raphael; Mujugira, Andrew; Fife, Kenneth; Mugo, Nelly; Kapiga, Saidi; Mullins, James I; Baeten, Jared M

    2014-01-14

    HIV-1 subtype C has emerged as the most prevalent strain of HIV-1 worldwide, leading to speculation that subtype C may be more transmissible than other subtypes. We compared the risk of HIV-1 transmission for subtype C versus non-C subtypes (A, D, G and recombinant forms) among heterosexual African HIV-1 serodiscordant couples. We conducted a nested case-control analysis using data from two prospective cohort studies of heterosexual HIV-1 serodiscordant couples from six countries in eastern and southern Africa. Cases (N = 121) included incident HIV-1 transmissions that were established as linked within the serodiscordant partnership by viral sequencing; controls (N = 501) were nontransmitting HIV-1-infected partners. Subtype was determined for partial env and gag genes. Multiple logistic regression controlled for age and gender of the HIV-1-nfected partner and self-reported unprotected sex. Plasma and genital HIV-1 RNA concentrations were compared between subtype C and non-C subtypes using generalized estimating equations. HIV-1 subtype C was not associated with increased risk of HIV-1 transmission compared with non-C subtypes: env adjusted odds ratio (adjOR) 1.14 [95% confidence interval (CI) 0.74-1.75, P = 0.6] and gag adjOR 0.98 (95% CI 0.63-1.52, P = 0.9). Plasma and genital HIV-1 RNA levels did not differ significantly for subtype C versus non-C. In a geographically diverse population of heterosexual African HIV-1 serodiscordant couples, subtype C was not associated with greater risk of HIV-1 transmission compared with non-C subtypes, arguing against the hypothesis that subtype C is more transmissible compared with other common subtypes.

  13. Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania☆,☆☆

    PubMed Central

    Bakari, Muhammad; Aboud, Said; Nilsson, Charlotta; Francis, Joel; Buma, Deus; Moshiro, Candida; Aris, Eric A.; Lyamuya, Eligius F.; Janabi, Mohamed; Godoy-Ramirez, Karina; Joachim, Agricola; Polonis, Victoria R.; Bråve, Andreas; Earl, Patricia; Robb, Merlin; Marovich, Mary; Wahren, Britta; Pallangyo, Kisali; Biberfeld, Gunnel; Mhalu, Fred; Sandström, Eric

    2016-01-01

    Background We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. Methods Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1 mg intradermally (id), n = 20, or 3.8 mg intramuscularly (im), n = 20, or placebo, n = 20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. Results The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01 AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher

  14. The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

    PubMed

    Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

    1994-12-01

    The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

  15. Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

    PubMed Central

    Wang, Margaret Q; Kim, Wankee; Gao, Guangxia; Torrey, Ted A; Morse, Herbert C; De Camilli, Pietro; Goff, Stephen P

    2004-01-01

    Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production. PMID:14659004

  16. HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China.

    PubMed

    Lu, Xinli; Kang, Xianjiang; Liu, Yongjian; Cui, Ze; Guo, Wei; Zhao, Cuiying; Li, Yan; Chen, Suliang; Li, Jingyun; Zhang, Yuqi; Zhao, Hongru

    2017-01-01

    New human immunodeficiency virus type 1 (HIV-1) diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF)01_AE (53.4%), CRF07_BC (23.4%), subtype B (15.9%), and unique recombinant forms URFs (4.9%). Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx), unknown before in Hebei, were first found among men who have sex with men (MSM). All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%), CRF01_AE/B (23.3%), B/C (16.7%), CRF01_AE/C (13.3%), CRF01_AE/B/A2 (3.3%) and CRF01_AE/BC/A2 (3.3%), plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China.

  17. HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China

    PubMed Central

    Lu, Xinli; Kang, Xianjiang; Liu, Yongjian; Cui, Ze; Guo, Wei; Zhao, Cuiying; Li, Yan; Chen, Suliang; Li, Jingyun; Zhang, Yuqi; Zhao, Hongru

    2017-01-01

    New human immunodeficiency virus type 1 (HIV-1) diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF)01_AE (53.4%), CRF07_BC (23.4%), subtype B (15.9%), and unique recombinant forms URFs (4.9%). Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx), unknown before in Hebei, were first found among men who have sex with men (MSM). All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%), CRF01_AE/B (23.3%), B/C (16.7%), CRF01_AE/C (13.3%), CRF01_AE/B/A2 (3.3%) and CRF01_AE/BC/A2 (3.3%), plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China. PMID:28178737

  18. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-boxmore » of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.« less

  19. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  20. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    NASA Astrophysics Data System (ADS)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  1. Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation

    PubMed Central

    Soto-Rifo, Ricardo; Limousin, Taran; Rubilar, Paulina S.; Ricci, Emiliano P.; Décimo, Didier; Moncorgé, Olivier; Trabaud, Mary-Anne; André, Patrice; Cimarelli, Andrea; Ohlmann, Théophile

    2012-01-01

    The 5′-untranslated region (5′-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5′-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5′-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens. PMID:22121214

  2. Characterization of New Cationic N,N-Dimethyl[70]fulleropyrrolidinium Iodide Derivatives as Potent HIV-1 Maturation Inhibitors.

    PubMed

    Castro, Edison; Martinez, Zachary S; Seong, Chang-Soo; Cabrera-Espinoza, Andrea; Ruiz, Mauro; Hernandez Garcia, Andrea; Valdez, Federico; Llano, Manuel; Echegoyen, Luis

    2016-12-22

    HIV-1 maturation can be impaired by altering protease (PR) activity, the structure of the Gag-Pol substrate, or the molecular interactions of viral structural proteins. Here we report the synthesis and characterization of new cationic N,N-dimethyl[70]fulleropyrrolidinium iodide derivatives that inhibit more than 99% of HIV-1 infectivity at low micromolar concentrations. Analysis of the HIV-1 life cycle indicated that these compounds inhibit viral maturation by impairing Gag and Gag-Pol processing. Importantly, fullerene derivatives 2a-c did not inhibit in vitro PR activity and strongly interacted with HIV immature capsid protein in pull-down experiments. Furthermore, these compounds potently blocked infectivity of viruses harboring mutant PR that are resistant to multiple PR inhibitors or mutant Gag proteins that confer resistance to the maturation inhibitor Bevirimat. Collectively, our studies indicate fullerene derivatives 2a-c as potent and novel HIV-1 maturation inhibitors.

  3. Dynamics of HIV-1 Assembly and Release

    PubMed Central

    Ivanchenko, Sergey; Godinez, William J.; Lampe, Marko; Kräusslich, Hans-Georg; Eils, Roland; Rohr, Karl; Bräuchle, Christoph; Müller, Barbara; Lamb, Don C.

    2009-01-01

    Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics. PMID:19893629

  4. Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0

    PubMed Central

    Swanson, Priscilla; Soriano, Vincent; Devare, Sushil G.; Hackett, John

    2001-01-01

    The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O. PMID:11230396

  5. Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

    PubMed

    Reed, Jonathan C; Westergreen, Nick; Barajas, Brook C; Ressler, Dylan T B; Phuong, Daryl J; Swain, John V; Lingappa, Vishwanath R; Lingappa, Jaisri R

    2018-05-01

    During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules. IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether

  6. HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

    PubMed

    Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L; Stout, Richard R; Robb, Merlin L; Shattock, Robin J; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

    2015-01-01

    We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

  7. HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

    PubMed Central

    Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L.; Stout, Richard R.; Robb, Merlin L.; Shattock, Robin J.; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

    2015-01-01

    Background We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. Methods HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. Results The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Conclusion Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. Trial Registration International Standard

  8. Early evolution of HLA-associated escape mutations in variable Gag proteins predicts CD4+ decline in HIV-1 subtype C infected women

    PubMed Central

    Chopera, Denis R.; Ntale, Roman; Ndabambi, Nonkululeko; Garrett, Nigel; Gray, Clive M.; Matten, David; Karim, Quarraisha Abdool; Karim, Salim Abdool; Williamson, Carolyn

    2016-01-01

    Objective HIV-1 escape from cytotoxic T-lymphocytes (CTL) results in the accumulation of HLA-associated mutations in the viral genome. To understand the contribution of early escape to disease progression, this study investigated the evolution and pathogenic implications of CTL escape in a cohort followed from infection for five years. Methods Viral loads and CD4+ counts were monitored in 78 subtype C infected individuals from onset of infection until CD4+ decline to <350 cells/μl or five years post-infection. The gag gene was sequenced and HLA-associated changes between enrolment and 12 months post-infection were mapped. Results HLA-associated escape mutations were identified in 48 (62%) of the participants and were associated with CD4+ decline to <350 copies/ml (p=0.05). Escape mutations in variable Gag proteins (p17 and p7p6) had a greater impact on disease progression than escape in more conserved regions (p24) (p=0.03). The association between HLA-associated escape mutations and CD4+ decline was independent of protective HLA allele (B*57, B*58:01, B*81) expression. Conclusion The high frequency of escape contributed to rapid disease progression in this cohort. While HLA-adaption in both conserved and variable Gag domains in the first year of infection was detrimental to long term clinical outcome, escape in variable domains had greater impact. PMID:27755110

  9. A single polymorphism in HIV-1 subtype C SP1 is sufficient to confer natural resistance to the maturation inhibitor bevirimat.

    PubMed

    Lu, Wuxun; Salzwedel, Karl; Wang, Dan; Chakravarty, Suvobrata; Freed, Eric O; Wild, Carl T; Li, Feng

    2011-07-01

    3-O-(3',3'-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.

  10. Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

    PubMed

    Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

    2007-11-01

    The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

  11. Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C.

    PubMed

    Ghimire, Dibya; Timilsina, Uddhav; Srivastava, Tryambak Pratap; Gaur, Ritu

    2017-03-02

    HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics.

  12. Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag.

    PubMed

    Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

    2006-12-01

    The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

  13. The HIV-1 Rev/RRE system is required for HIV-1 5' UTR cis elements to augment encapsidation of heterologous RNA into HIV-1 viral particles

    PubMed Central

    2011-01-01

    Background The process of HIV-1 genomic RNA (gRNA) encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ). Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site) from the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV) vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles. Results We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ). Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form. Conclusions Premised on encapsidation of a heterologous RNA into HIV-1 viral particles

  14. Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection. AGIS Group and L'Agence Nationale de Recherches sur Le Sida.

    PubMed

    Salmon-Céron, D; Excler, J L; Finkielsztejn, L; Autran, B; Gluckman, J C; Sicard, D; Matthews, T J; Meignier, B; Valentin, C; El Habib, R; Blondeau, C; Raux, M; Moog, C; Tartaglia, J; Chong, P; Klein, M; Milcamps, B; Heshmati, F; Plotkin, S

    1999-05-01

    A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol. This activity was mediated by both CD4+ and CD8+ lymphocytes. On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.

  15. Structural and functional insights into the HIV-1 maturation inhibitor binding pocket.

    PubMed

    Waki, Kayoko; Durell, Stewart R; Soheilian, Ferri; Nagashima, Kunio; Butler, Scott L; Freed, Eric O

    2012-01-01

    Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for in vitro. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased

  16. Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

    PubMed Central

    Ghiglione, Yanina; Falivene, Juliana; Socias, María Eugenia; Laufer, Natalia; Coloccini, Romina Soledad; Rodriguez, Ana María; Ruiz, María Julia; Pando, María Ángeles; Giavedoni, Luis David; Cahn, Pedro; Sued, Omar; Salomon, Horacio; Gherardi, María Magdalena

    2013-01-01

    The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection. PMID:23616666

  17. Contact-induced mitochondrial polarization supports HIV-1 virological synapse formation.

    PubMed

    Groppelli, Elisabetta; Starling, Shimona; Jolly, Clare

    2015-01-01

    Rapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes. HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell

  18. Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

    PubMed

    Ataie Kachoie, Elham; Behjatnia, Seyed Ali Akbar; Kharazmi, Sara

    2018-01-01

    It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants.

  19. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    PubMed

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. Identification of potent maturation inhibitors against HIV-1 clade C.

    PubMed

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J; Wild, Carl T; Freed, Eric O; Gaur, Ritu

    2016-06-06

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic.

  1. Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

    PubMed

    Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D; Lichterfeld, Mathias; Yu, Xu G

    2015-06-01

    The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

  2. Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

    PubMed

    Munseri, Patricia J; Kroidl, Arne; Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

    2015-01-01

    Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. World Health Organization International Clinical Trials Registry Platform PACTR

  3. HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

    PubMed

    Kanagavelu, Saravana; Termini, James M; Gupta, Sachin; Raffa, Francesca N; Fuller, Katherine A; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S; Stone, Geoffrey W

    2014-01-01

    Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia-Gag

  4. HIV-1 subtypes D and F are prevalent in Guinea Conakry.

    PubMed

    Freimanis, G L; Loua, A; Allain, J P

    2012-04-01

    Limited data is available upon the distribution of different HIV-1/2 genotypes in the blood donor population from Guinea Conakry. To investigate the prevalence of HIV-1/2 subtypes in asymptomatic blood donors in Guinea Conakry, in order to update knowledge of HIV-1/2 epidemiology within this country. Samples from 104 blood donors seropositive for HIV-1/2 were tested for HIV-1 by real-time RT-PCR. Those negative for HIV-1 were tested with HIV-2 nested RT-PCR. Positive samples were further amplified in the HIV-1 gag and pol regions and sequenced. Subtypes were determined by phylogenetic analysis on amplicon sequences. 61 samples were positive by HIV-1 real-time RT-PCR. Of the 43 negative, 2 (4.6%) were positive for HIV-2. 52/61 (85.3%) samples were positive by nested RT-PCR. Of the 52, 43 (70.5%) and 31(59.6%) sequences were obtained in the gag and pol regions, respectively; 23 for both regions. HIV-1 subtype distribution was 1 B (2.1%), 8 F (17%), 8 D (17%) and 28 CRF02_AG (59.6%) with 2 unclassified recombinants (4.3%). Unique clusters for subtype D and F distinguished Guinea from HIV-1 subtype distribution in neighboring countries. Subtype F and subtype D strains, uncommon in West Africa, are a substantial part of HIV-1 epidemiology in Guinea. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.

    PubMed

    Schur, Florian K M; Obr, Martin; Hagen, Wim J H; Wan, William; Jakobi, Arjen J; Kirkpatrick, Joanna M; Sachse, Carsten; Kräusslich, Hans-Georg; Briggs, John A G

    2016-07-29

    Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1. Copyright © 2016, American Association for the Advancement of Science.

  6. Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

    PubMed

    Viegas, Edna Omar; Tembe, Nelson; Nilsson, Charlotta; Meggi, Bindiya; Maueia, Cremildo; Augusto, Orvalho; Stout, Richard; Scarlatti, Gabriella; Ferrari, Guido; Earl, Patricia L; Wahren, Britta; Andersson, Sören; Robb, Merlin L; Osman, Nafissa; Biberfeld, Gunnel; Jani, Ilesh; Sandström, Eric

    2017-11-27

    We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 μg (n = 10; 2 × 0.1 ml) or 1,200 μg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 10 8 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 μg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

  7. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus.

    PubMed

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

    2016-05-01

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa.

    PubMed

    Hölzemer, Angelique; Thobakgale, Christina F; Jimenez Cruz, Camilo A; Garcia-Beltran, Wilfredo F; Carlson, Jonathan M; van Teijlingen, Nienke H; Mann, Jaclyn K; Jaggernath, Manjeetha; Kang, Seung-gu; Körner, Christian; Chung, Amy W; Schafer, Jamie L; Evans, David T; Alter, Galit; Walker, Bruce D; Goulder, Philip J; Carrington, Mary; Hartmann, Pia; Pertel, Thomas; Zhou, Ruhong; Ndung'u, Thumbi; Altfeld, Marcus

    2015-11-01

    Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I-presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the

  9. Nucleic acid chaperone activity of retroviral Gag proteins.

    PubMed

    Rein, Alan

    2010-01-01

    Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive

  10. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretchmore » of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.« less

  11. HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

    PubMed Central

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L.; Seaman, Michael S.; Montefiori, David C.; Yates, Nicole L.; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; Roederer, Mario; Self, Steven G.; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony D.; Weiss, Deborah E.; Lee, Carter; Kibler, Karen V.; Jacobs, Bertram L.; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

    2017-01-01

    ABSTRACT The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions. IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1

  12. Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons.

    PubMed

    Schmechel, S C; Russell, N; Hladik, F; Lang, J; Wilson, A; Ha, R; Desbien, A; McElrath, M J

    2001-11-01

    Rare individuals who are repeatedly exposed to HIV-1 through unprotected sexual contact fail to acquire HIV-1 infection. These persons represent a unique study population to evaluate mechanisms by which HIV-1 replication is either prevented or controlled. We followed longitudinally a group of healthy HIV-1 seronegative persons each reporting repeated high-risk sexual activities with their HIV-1-infected partner at enrollment. The volunteers were primarily (90%) male homosexuals, maintaining high risk activities with their known infected partner (45%) or multiple other partners (61%). We evaluated the quantity and specificity of HIV-1-specific T cells in 31 exposed seronegatives (ES) using a IFN-gamma ELISPOT assay to enumerate T cells recognizing epitopes within HIV-1 Env, Gag, Pol and Nef. PBMC from only three of the 31 volunteers demonstrated ex vivo HIV-1-specific IFN-gamma secretion, in contrast to nearly 30% exhibiting cytolytic responses in previous studies. These findings suggest that if T cell responses in ES are induced by HIV-1 exposure, the frequency is at low levels in most of them, and below the level of detection using the ELISPOT assay. Alternative approaches to improve the sensitivity of detection may include use of dendritic cells as antigen-presenting cells in the ex vivo assay and more careful definition of the risk behavior and extent of HIV-1 exposure in conjunction with the evaluation of T cell responses.

  13. Productive HIV-1 Infection of Human Cervical Tissue Ex Vivo is Associated with the Secretory Phase of the Menstrual Cycle

    PubMed Central

    Saba, Elisa; Origoni, Massimo; Taccagni, Gianluca; Ferrari, Davide; Doglioni, Claudio; Nava, Alice; Lisco, Andrea; Grivel, Jean-Charles; Margolis, Leonid; Poli, Guido

    2013-01-01

    Cervical tissue explants (CTE) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTE were productively infected in terms of HIV-1 p24Gag release in culture supernatants whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag+ CD4+ T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTE. Nonproductive CTE differed from productive CTE for higher secretion of CCL3 and CCL5. A post-hoc analysis revealed that all productive CTE were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTE derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle. PMID:23385427

  14. Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

    PubMed

    Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

    2014-07-03

    The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

  15. Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of Alpha Interferon

    PubMed Central

    Akiyama, Hisashi; Ramirez, Nora-Guadalupe Pina; Gibson, Gregory; Kline, Christopher; Watkins, Simon; Ambrose, Zandrea

    2017-01-01

    ABSTRACT A hallmark of human immunodeficiency virus type 1 (HIV-1) infection in vivo is chronic immune activation concomitant with type I interferon (IFN) production. Although type I IFN induces an antiviral state in many cell types, HIV-1 can replicate in vivo via mechanisms that have remained unclear. We have recently identified a type I IFN-inducible protein, CD169, as the HIV-1 attachment factor on dendritic cells (DCs) that can mediate robust infection of CD4+ T cells in trans. Since CD169 expression on macrophages is also induced by type I IFN, we hypothesized that type I IFN-inducible CD169 could facilitate productive HIV-1 infection in myeloid cells in cis and CD4+ T cells in trans and thus offset antiviral effects of type I IFN. In support of this hypothesis, infection of HIV-1 or murine leukemia virus Env (MLV-Env)-pseudotyped HIV-1 particles was enhanced in IFN-α-treated THP-1 monocytoid cells, and this enhancement was primarily dependent on CD169-mediated enhancement at the virus entry step, a phenomenon phenocopied in HIV-1 infections of IFN-α-treated primary monocyte-derived macrophages (MDMs). Furthermore, expression of CD169, a marker of type I IFN-induced immune activation in vivo, was enhanced in lymph nodes from pigtailed macaques infected with simian immunodeficiency virus (SIV) carrying HIV-1 reverse transcriptase (RT-SHIV), compared to uninfected macaques, and interestingly, there was extensive colocalization of p27gag and CD169, suggesting productive infection of CD169+ myeloid cells in vivo. While cell-free HIV-1 infection of IFN-α-treated CD4+ T cells was robustly decreased, initiation of infection in trans via coculture with CD169+ IFN-α-treated DCs restored infection, suggesting that HIV-1 exploits CD169 in cis and in trans to attenuate a type I IFN-induced antiviral state. IMPORTANCE HIV-1 infection in humans causes immune activation characterized by elevated levels of proinflammatory cytokines, including type I interferons (IFN

  16. A trans-Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

    PubMed Central

    Saha, Agniva; Mitchell, Jessica A.; Nishida, Yuri; Hildreth, Jonathan E.; Ariberre, Joshua A.; Gilbert, Wendy V.

    2015-01-01

    ABSTRACT Saccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation. IMPORTANCE Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both “host and parasite.” To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate

  17. Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

    PubMed

    Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

    1994-01-01

    Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

  18. Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

    PubMed

    Martins, Angelica N; Waheed, Abdul A; Ablan, Sherimay D; Huang, Wei; Newton, Alicia; Petropoulos, Christos J; Brindeiro, Rodrigo D M; Freed, Eric O

    2016-01-15

    HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are

  19. Promiscuous, Multi-Target Lupane-Type Triterpenoids Inhibits Wild Type and Drug Resistant HIV-1 Replication Through the Interference With Several Targets.

    PubMed

    Bedoya, Luis M; Beltrán, Manuela; García-Pérez, Javier; Obregón-Calderón, Patricia; Callies, Oliver; Jímenez, Ignacio A; Bazzocchi, Isabel L; Alcamí, José

    2018-01-01

    Current research on antiretroviral therapy is mainly focused in the development of new formulations or combinations of drugs belonging to already known targets. However, HIV-1 infection is not cured by current therapy and thus, new approaches are needed. Bevirimat was developed by chemical modification of betulinic acid, a lupane-type pentacyclic triterpenoid (LPT), as a first-in-class HIV-1 maturation inhibitor. However, in clinical trials, bevirimat showed less activity than expected because of the presence of a natural mutation in Gag protein that conferred resistance to a high proportion of HIV-1 strains. In this work, three HIV-1 inhibitors selected from a set of previously screened LPTs were investigated for their targets in the HIV-1 replication cycle, including their maturation inhibitor effect. LPTs were found to inhibit HIV-1 infection acting as promiscuous compounds with several targets in the HIV-1 replication cycle. LPT12 inhibited HIV-1 infection mainly through reverse transcription, integration, viral transcription, viral proteins (Gag) production and maturation inhibition. LPT38 did it through integration, viral transcription or Gag production inhibition and finally, LPT42 inhibited reverse transcription, viral transcription or Gag production. The three LPTs inhibited HIV-1 infection of human primary lymphocytes and infections with protease inhibitors and bevirimat resistant HIV-1 variants with similar values of IC 50 . Therefore, we show that the LPTs tested inhibited HIV-1 infection through acting on different targets depending on their chemical structure and the activities of the different LPTs vary with slight structural alterations. For example, of the three LPTs under study, we found that only LPT12 inhibited infectivity of newly-formed viral particles, suggesting a direct action on the maturation process. Thus, the multi-target behavior gives a potential advantage to these compounds since HIV-1 resistance can be overcome by modulating more

  20. HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

    PubMed

    Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

    2011-08-24

    Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different

  1. [Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

    PubMed

    Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

    2004-09-01

    HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

  2. Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors

    PubMed Central

    Quakkelaar, Esther D.; Redeker, Anke; Haddad, Elias K.; Harari, Alexandre; McCaughey, Stella Mayo; Duhen, Thomas; Filali-Mouhim, Abdelali; Goulet, Jean-Philippe; Loof, Nikki M.; Ossendorp, Ferry; Perdiguero, Beatriz; Heinen, Paul; Gomez, Carmen E.; Kibler, Karen V.; Koelle, David M.; Sékaly, Rafick P.; Sallusto, Federica; Lanzavecchia, Antonio; Pantaleo, Giuseppe; Esteban, Mariano; Tartaglia, Jim; Jacobs, Bertram L.; Melief, Cornelis J. M.

    2011-01-01

    Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. PMID:21347234

  3. A HIV-1 heterosexual transmission chain in Guangzhou, China: a molecular epidemiological study.

    PubMed

    Han, Zhigang; Leung, Tommy W C; Zhao, Jinkou; Wang, Ming; Fan, Lirui; Li, Kai; Pang, Xinli; Liang, Zhenbo; Lim, Wilina W L; Xu, Huifang

    2009-09-25

    We conducted molecular analyses to confirm four clustering HIV-1 infections (Patient A, B, C & D) in Guangzhou, China. These cases were identified by epidemiological investigation and suspected to acquire the infection through a common heterosexual transmission chain. Env C2V3V4 region, gag p17/p24 junction and partial pol gene of HIV-1 genome from serum specimens of these infected cases were amplified by reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequenced. Phylogenetic analyses indicated that their viral nucleotide sequences were significantly clustered together (bootstrap value is 99%, 98% and 100% in env, gag and pol tree respectively). Evolutionary distance analysis indicated that their genetic diversities of env, gag and pol genes were significantly lower than non-clustered controls, as measured by unpaired t-test (env gene comparison: p < 0.005; gag gene comparison: p < 0.005; pol gene comparison: p < 0.005). Epidemiological results and molecular analyses consistently illustrated these four cases represented a transmission chain which dispersed in the locality through heterosexual contact involving commercial sex worker.

  4. HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses

    PubMed Central

    Soghoian, Damien Z.; Lindqvist, Madelene; Ghebremichael, Musie; Donaghey, Faith; Carrington, Mary; Seaman, Michael S.; Kaufmann, Daniel E.; Walker, Bruce D.

    2015-01-01

    ABSTRACT Antigen-specific CD4+ T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4+ T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4+ T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4+ T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4+ T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4+ T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4+ T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4+ T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4+ T cells and, to a lesser extent, gp41-specific CD4+ T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE One of the earliest discoveries related to CD4+ T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4+ T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4+ T cells on the generation of antibodies that can neutralize

  5. Novel Acylguanidine-Based Inhibitor of HIV-1

    PubMed Central

    Mwimanzi, Philip; Tietjen, Ian; Miller, Scott C.; Shahid, Aniqa; Cobarrubias, Kyle; Kinloch, Natalie N.; Baraki, Bemuluyigza; Richard, Jonathan; Finzi, Andrés; Fedida, David; Brumme, Zabrina L.

    2016-01-01

    ABSTRACT The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24Gag production was unaffected, but virion release (measured as extracellular p24Gag) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of

  6. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

    PubMed

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Conserved Elements Vaccine for HIV | NCI Technology Transfer Center | TTC

    Cancer.gov

    Researchers at the National Cancer Institute (NCI) developed a DNA vaccine using conserved elements of HIV-1 Gag, administered in a prime-boost vaccination protocol. Two of the HIV Gag CE DNA vectors have been tested in a rhesus macaque model. Priming with the Gag CE vaccine and boosting with full length Gag DNA showed increased immune responses when compared to vaccination with Gag alone. Researchers seek licensing and/or co-development research collaborations for development this DNA vaccine.

  8. APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

    PubMed

    Dussart, Sylvie; Douaisi, Marc; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne

    2005-01-21

    APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

  9. Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

    PubMed

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

    2012-05-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

  10. Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

    PubMed Central

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C.

    2012-01-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site. PMID:22754649

  11. Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6 ▿

    PubMed Central

    Cano, Jennifer; Kalpana, Ganjam V.

    2011-01-01

    INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions. A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55gag) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding. PMID:21159874

  12. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

    PubMed

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-10-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

  13. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

    NASA Astrophysics Data System (ADS)

    Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

  14. Cyclophilin B enhances HIV-1 Infection

    PubMed Central

    DeBoer, Jason; Madson, Christian J.; Belshan, Michael

    2016-01-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

  15. Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging

    PubMed Central

    Angert, Isaac; Cao, Sheng; Berk, Serkan; Zhang, Wei; Mueller, Joachim D.

    2017-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur

  16. Functional Redundancy in HIV-1 Viral Particle Assembly

    PubMed Central

    O'Carroll, Ina P.; Crist, Rachael M.; Mirro, Jane; Harvin, Demetria; Soheilian, Ferri; Kamata, Anne; Nagashima, Kunio

    2012-01-01

    Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly. PMID:22993163

  17. Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

    PubMed Central

    Errazuriz, Elisabeth; Coric, Pascale; Souquet, Florence; Turcaud, Serge; Boulanger, Pierre; Bouaziz, Serge; Hong, Saw See

    2011-01-01

    The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the

  18. Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

    Cancer.gov

    During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the

  19. HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

    PubMed

    Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

    2015-01-01

    Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

  20. On the Selective Packaging of Genomic RNA by HIV-1.

    PubMed

    Comas-Garcia, Mauricio; Davis, Sean R; Rein, Alan

    2016-09-12

    Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

  1. An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

    PubMed

    Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

    2018-03-14

    NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594

  2. Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

    PubMed Central

    Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

    2010-01-01

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

  3. Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

    PubMed

    Edmonds, Tara G; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S; Conway, Joan A; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T; Montefiori, David C; Kappes, John C; Ochsenbauer, Christina

    2010-12-05

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. MAS NMR of HIV-1 protein assemblies

    NASA Astrophysics Data System (ADS)

    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  5. Oral Immunization with a Recombinant Lactococcus lactis-Expressing HIV-1 Antigen on Group A Streptococcus Pilus Induces Strong Mucosal Immunity in the Gut.

    PubMed

    Chamcha, Venkateswarlu; Jones, Andrew; Quigley, Bernard R; Scott, June R; Amara, Rama Rao

    2015-11-15

    The induction of a potent humoral and cellular immune response in mucosal tissue is important for the development of an effective HIV vaccine. Most of the current HIV vaccines under development use the i.m. route for immunization, which is relatively poor in generating potent and long-lived mucosal immune responses. In this article, we explore the ability of an oral vaccination with a probiotic organism, Lactococcus lactis, to elicit HIV-specific immune responses in the mucosal and systemic compartments of BALB/c mice. We expressed the HIV-1 Gag-p24 on the tip of the T3 pilus of Streptococcus pyogenes as a fusion to the Cpa protein (LL-Gag). After four monthly LL-Gag oral immunizations, we observed strong Gag-specific IgG and IgA responses in serum, feces, and vaginal secretions. However, the Gag-specific CD8 T cell responses in the blood were at or below our detection limit. After an i.m. modified vaccinia Ankara/Gag boost, we observed robust Gag-specific CD8 T cell responses both in systemic and in mucosal tissues, including intraepithelial and lamina propria lymphocytes of the small intestine, Peyer's patches, and mesenteric lymph nodes. Consistent with strong immunogenicity, the LL-Gag induced activation of CD11c(+) CD11b(+) dendritic cells in the Peyer's patches after oral immunization. Our results demonstrate that oral immunization with L. lactis expressing an Ag on the tip of the group A Streptococcus pilus serves as an excellent vaccine platform to induce strong mucosal humoral and cellular immunity against HIV. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFN{gamma} production by CD4+ T cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Caivano, Antonella; Doria-Rose, Nicole A.; Dept. of Molecular and Cell Biology, University of Washington, Seattle, WA 98124-6108

    2010-11-25

    We have constructed stable virus-like particles displaying the HIV-1 Gag(p17) protein as an N-terminal fusion with an engineered protein domain from the Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2. Mice immunized with the Gag(p17)-E2 60-mer scaffold particles mounted a strong and sustained antibody response. Antibodies directed to Gag(p17) were boosted significantly with additional immunizations, while anti-E2 responses reached a plateau. The isotype of the induced antibodies was biased towards IgG1, and the E2-primed CD4+ T cells did not secrete IFN{gamma}. Using transgenic mouse model systems, we demonstrated that CD8+ T cells primed with E2 particles were able to exert lytic activitymore » and produce IFN{gamma}. These results show that the E2 scaffold represents a powerful vaccine delivery system for whole antigenic proteins or polyepitope engineered proteins, evoking antibody production and antigen specific CTL activity even in the absence of IFN{gamma}-producing CD4+ T cells.« less

  7. Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex.

    PubMed

    Praditwongwan, Warachai; Chuankhayan, Phimonphan; Saoin, Somphot; Wisitponchai, Tanchanok; Lee, Vannajan Sanghiran; Nangola, Sawitree; Hong, Saw See; Minard, Philippe; Boulanger, Pierre; Chen, Chun-Jung; Tayapiwatana, Chatchai

    2014-08-01

    Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated Ank(GAG)1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTD(CA)) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the Ank(GAG)1D4-NTD(CA) interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling. We determined the Ank(GAG)1D4 structure at 2.2 Å resolution, and used the crystal structure in molecular docking with a homology model of HIV-1 capsid. Our results indicated that NTD(CA) alpha-helices H1 and H7 could mediate the formation of the capsid-Ank(GAG)1D4 binary complex, but the interaction involving H7 was predicted to be more stable than with H1. Arginine-18 (R18) in H1, and R132 and R143 in H7 were found to be the key players of the Ank(GAG)1D4-NTD(CA) interaction. This was confirmed by R-to-A mutagenesis of NTD(CA), and by sequence analysis of trimodular ankyrins negative for capsid binding. In Ank(GAG)1D4, major interactors common to H1 and H7 were found to be S45, Y56, R89, K122 and K123. Collectively, our ankyrin-capsid binding analysis implied a significant degree of flexibility within the NTD(CA) domain of the HIV-1 capsid protein, and provided some clues for the design of new antivirals targeting the capsid protein and viral assembly.

  8. Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex

    NASA Astrophysics Data System (ADS)

    Praditwongwan, Warachai; Chuankhayan, Phimonphan; Saoin, Somphot; Wisitponchai, Tanchanok; Lee, Vannajan Sanghiran; Nangola, Sawitree; Hong, Saw See; Minard, Philippe; Boulanger, Pierre; Chen, Chun-Jung; Tayapiwatana, Chatchai

    2014-08-01

    Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated AnkGAG1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTDCA) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the AnkGAG1D4-NTDCA interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling. We determined the AnkGAG1D4 structure at 2.2 Å resolution, and used the crystal structure in molecular docking with a homology model of HIV-1 capsid. Our results indicated that NTDCA alpha-helices H1 and H7 could mediate the formation of the capsid-AnkGAG1D4 binary complex, but the interaction involving H7 was predicted to be more stable than with H1. Arginine-18 (R18) in H1, and R132 and R143 in H7 were found to be the key players of the AnkGAG1D4-NTDCA interaction. This was confirmed by R-to-A mutagenesis of NTDCA, and by sequence analysis of trimodular ankyrins negative for capsid binding. In AnkGAG1D4, major interactors common to H1 and H7 were found to be S45, Y56, R89, K122 and K123. Collectively, our ankyrin-capsid binding analysis implied a significant degree of flexibility within the NTDCA domain of the HIV-1 capsid protein, and provided some clues for the design of new antivirals targeting the capsid protein and viral assembly.

  9. Complex Subtype Diversity of HIV-1 Among Drug Users in Major Kenyan Cities

    PubMed Central

    Gounder, Kamini; Oyaro, Micah; Padayachi, Nagavelli; Zulu, Thando Mbali; de Oliveira, Tulio; Wylie, John

    2017-01-01

    Abstract Drug users are increasingly recognized as a key population driving human immunodeficiency virus (HIV) spread in sub-Saharan Africa. To determine HIV-1 subtypes circulating in this population group and explore possible geographic differences, we analyzed HIV-1 sequences among drug users from Nairobi, Mombasa, and Kisumu in Kenya. We sequenced gag and env from 55 drug users. Subtype analysis from 220 gag clonal sequences from 54 of 55 participants (median = 4/participant) showed that 44.4% were A, 16.7% were C, 3.7% were D, and 35.2% were intersubtype recombinants. Of 156 env clonal sequences from 48 of 55 subjects (median = 3/participant), 45.8% were subtype A, 14.6% were C, 6.3% were D, and 33.3% were recombinants. Comparative analysis of both genes showed that 30 (63.8%) participants had concordant subtypes, while 17 (36.2%) were discordant. We identified one genetically linked transmission pair and two cases of dual infection. These data are indicative of extensive HIV-1 intersubtype recombination in Kenya and suggest decline in subtype D prevalence. PMID:28068781

  10. Complex Subtype Diversity of HIV-1 Among Drug Users in Major Kenyan Cities.

    PubMed

    Gounder, Kamini; Oyaro, Micah; Padayachi, Nagavelli; Zulu, Thando Mbali; de Oliveira, Tulio; Wylie, John; Ndung'u, Thumbi

    2017-05-01

    Drug users are increasingly recognized as a key population driving human immunodeficiency virus (HIV) spread in sub-Saharan Africa. To determine HIV-1 subtypes circulating in this population group and explore possible geographic differences, we analyzed HIV-1 sequences among drug users from Nairobi, Mombasa, and Kisumu in Kenya. We sequenced gag and env from 55 drug users. Subtype analysis from 220 gag clonal sequences from 54 of 55 participants (median = 4/participant) showed that 44.4% were A, 16.7% were C, 3.7% were D, and 35.2% were intersubtype recombinants. Of 156 env clonal sequences from 48 of 55 subjects (median = 3/participant), 45.8% were subtype A, 14.6% were C, 6.3% were D, and 33.3% were recombinants. Comparative analysis of both genes showed that 30 (63.8%) participants had concordant subtypes, while 17 (36.2%) were discordant. We identified one genetically linked transmission pair and two cases of dual infection. These data are indicative of extensive HIV-1 intersubtype recombination in Kenya and suggest decline in subtype D prevalence.

  11. Human cyclophilin has a significantly higher affinity for HIV-1 recombinant p55 than p24.

    PubMed

    Bristow, R; Byrne, J; Squirell, J; Trencher, H; Carter, T; Rodgers, B; Saman, E; Duncan, J

    1999-04-01

    The ability of cyclophilin to bind a panel of recombinant HIV-gag proteins was assessed using sensitive, quantitative, sandwich enzyme-linked immunosorbant assays (ELISAs). Significantly higher binding to cyclophilin was observed when recombinants contained at least 12 carboxy-terminal amino acids of p17 in addition to p24 sequences. These results indicate that the carboxy-terminus of p17 is important for optimal binding of cyclophilin to p24 and support the theory that cyclophilin acts on the uncleaved HIV-1 gag (p17-p24) precursor.

  12. Cyclophilin B enhances HIV-1 infection.

    PubMed

    DeBoer, Jason; Madson, Christian J; Belshan, Michael

    2016-02-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. HIV-1 group P infection: towards a dead-end infection?

    PubMed

    Alessandri-Gradt, Elodie; De Oliveira, Fabienne; Leoz, Marie; Lemee, Véronique; Robertson, David L; Feyertag, Felix; Ngoupo, Paul-Alain; Mauclere, Philippe; Simon, François; Plantier, Jean-Christophe

    2018-06-19

    HIV/1 group P (HIV-1/P) is the last HIV/1 group discovered and, to date, constitutes only two strains. To obtain new insight into this divergent group, we screened for new infections by developing specific tools, and analysed phenotypic and genotypic properties of the prototypic strain RBF168. In addition, the follow-up of the unique infected patient monitored so far has raised the knowledge of the natural history of this infection and its therapeutic management. We developed an HIV-1/P specific seromolecular strategy and screened over 29 498 specimen samples. Infectivity and evolution of the gag-30 position, considered as marker of adaptation to human, were explored by successive passages of RBF168 strain onto human peripheral blood mononuclear cells. Natural history and immunovirological responses to combined antiretroviral therapy (cART) were analysed based on CD4 cells and plasmatic viral load evolution. No new infection was detected. Infectivity of RBF168 was found lower, relative to other main HIV groups and the conservative methionine found in the gag-30 position revealed a lack of adaptation to human. The follow-up of the patient during the 5-year ART-free period, showed a relative stability of CD4 cell count with a mean of 326 cells/μl. Initiation of cART led to rapid RNA undetectability with a significant increase of CD4 cells, reaching 687 cells/μl after 8 years. Our results showed that HIV-1/P strains remain extremely rare and could be less adapted and pathogenic than other HIV strains. These data lead to the hypothesis that HIV-1/P infection could evolve towards, or even already corresponds to, a dead-end infection.

  14. Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

    PubMed Central

    Lin, Ying-Chuan; Brik, Ashraf; de Parseval, Aymeric; Tam, Karen; Torbett, Bruce E.; Wong, Chi-Huey; Elder, John H.

    2006-01-01

    We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations. PMID:16873240

  15. An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

    PubMed

    Fujisaki, Seiichiro; Ibe, Shiro; Hattori, Junko; Shigemi, Urara; Fujisaki, Saeko; Shimizu, Kayoko; Nakamura, Kazuyo; Yokomaku, Yoshiyuki; Mamiya, Naoto; Utsumi, Makoto; Hamaguchi, Motohiro; Kaneda, Tsuguhiro

    2009-01-01

    Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

  16. Barriers and Facilitators to HIV Testing Among Women

    PubMed Central

    McDougall, Graham J.; Dalmida, Safiya George; Foster, Pamela Payne; Burrage, Joe

    2017-01-01

    Aim The purpose of this secondary analysis was to analyze for barriers and facilitators to HIV testing in women attending community health clinics. Introduction The Centers for Disease Control and Prevention (CDC), reported that all women account for 20% or 1 in 5 of new HIV cases (CDC, 2012). Of those new cases in heterosexual women, 5,300 were Black, 1,300 were White, and 1,200 were Hispanic/Latina. The CDC estimated that in 2012 there were 9,268 individuals living with a diagnosis of HIV or AIDS, of which 19% were women. Results The existing de-identified data consisted of thirty individual interviews conducted using a semi-structured interview guide was collected as the initial phase of the parent study, “HIV Testing and Women’s Attitudes on HIV Vaccine Trials”. This secondary analysis addressed the identification of key obstacles to HIV testing and only those related portions of the transcripts were analyzed. The major themes identified were familiarity with testing, stigma, fear, perceived risks, and access to care. Conclusion The themes implicated the need to further assess women for barriers and facilitators to testing, tailor community based interventions that have the ability to decrease fear and stigma, increase trust in testing methods and offer counseling to positive results. PMID:29607406

  17. Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation.

    PubMed

    Buttler, Carmen A; Pezeshkian, Nairi; Fernandez, Melissa V; Aaron, Jesse; Norman, Sofya; Freed, Eric O; van Engelenburg, Schuyler B

    2018-05-10

    Human immunodeficiency virus type 1 (HIV-1) assembly occurs on the inner leaflet of the host cell plasma membrane, incorporating the essential viral envelope glycoprotein (Env) within a budding lattice of HIV-1 Gag structural proteins. The mechanism by which Env incorporates into viral particles remains poorly understood. To determine the mechanism of recruitment of Env to assembly sites, we interrogate the subviral angular distribution of Env on cell-associated virus using multicolor, three-dimensional (3D) superresolution microscopy. We demonstrate that, in a manner dependent on cell type and on the long cytoplasmic tail of Env, the distribution of Env is biased toward the necks of cell-associated particles. We postulate that this neck-biased distribution is regulated by vesicular retention and steric complementarity of Env during independent Gag lattice formation.

  18. Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane

    PubMed Central

    Pak, Alexander J.; Grime, John M. A.; Sengupta, Prabuddha; Chen, Antony K.; Durumeric, Aleksander E. P.; Srivastava, Anand; Yeager, Mark; Briggs, John A. G.; Lippincott-Schwartz, Jennifer; Voth, Gregory A.

    2017-01-01

    The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA–SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding. PMID:29114055

  19. Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

    Cancer.gov

    During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the HIV-1 core, and NC packages the viral RNA within the core. Current models of the development of HIV-1 suggest that when CA is cleaved from Gag it dissociates from the membrane and moves into the virus interior before nucleating, in a concentration-dependent manner, into the core, which is the last step in virus maturation. The core is thought to grow from its narrow end stopping only when it reaches the opposite side of the virus membrane. Since blocking the formation of infectious viral particles is an important therapeutic strategy, it is critical to understand the detailed mechanism of core maturation.

  20. Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

    PubMed

    Chege, Gerald K; Burgers, Wendy A; Stutz, Helen; Meyers, Ann E; Chapman, Rosamund; Kiravu, Agano; Bunjun, Rubina; Shephard, Enid G; Jacobs, William R; Rybicki, Edward P; Williamson, Anna-Lise

    2013-05-01

    We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

  1. Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.

    PubMed

    Kraus, Benjamin; Fischer, Katrin; Sliva, Katja; Schnierle, Barbara S

    2014-03-26

    Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

  2. Nucleic acids encoding mosaic HIV-1 gag proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy

    The disclosure generally relates to an immunogenic composition (e.g., a vaccine) and, in particular, to a polyvalent immunogenic composition, such as a polyvalent HIV vaccine, and to methods of using same.

  3. Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

    PubMed Central

    Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

    2014-01-01

    With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new

  4. TIM-family proteins inhibit HIV-1 release

    PubMed Central

    Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

    2014-01-01

    Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

  5. Cyclophilin B enhances HIV-1 infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DeBoer, Jason; Madson, Christian J.; Belshan, Michael, E-mail: michaelbelshan@creighton.edu

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence,more » putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.« less

  6. Dendritic Cells Exposed to MVA-Based HIV-1 Vaccine Induce Highly Functional HIV-1-Specific CD8+ T Cell Responses in HIV-1-Infected Individuals

    PubMed Central

    Climent, Núria; Guerra, Susana; García, Felipe; Rovira, Cristina; Miralles, Laia; Gómez, Carmen Elena; Piqué, Núria; Gil, Cristina; Gatell, José María; Esteban, Mariano; Gallart, Teresa

    2011-01-01

    Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B. PMID:21625608

  7. Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

    PubMed Central

    Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

    2011-01-01

    Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

  8. Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine.

    PubMed

    Zagury, J F; Sill, A; Blattner, W; Lachgar, A; Le Buanec, H; Richardson, M; Rappaport, J; Hendel, H; Bizzini, B; Gringeri, A; Carcagno, M; Criscuolo, M; Burny, A; Gallo, R C; Zagury, D

    1998-01-01

    To investigate which immune parameters, such as antibodies against HIV-1 specificities, or viral parameters, such as p24 antigenemia, are predictive of disease progression. We performed studies on serum collected from individuals exhibiting two extremes of disease evolution--67 fast progressors (FP) and 182 nonprogressors (NP)--at their enrollment. After a 1- to 2-year clinical follow-up of 104 nonprogressors after their enrollment, we could determine the best serologic predictors for disease progression. We investigated levels of antibodies to tetanus toxoid and to HIV antigens including Env, Gag, Nef, and Tat proteins, as well as p24 antigenemia, viremia, CD4 cell count, and interferon-alpha (IFN-alpha) titers in FPs and NPs, and we correlated these data with clinical and biologic signs of progression. p24 Antigenemia, a marker of viral replication, and anti-Tat antibodies were highly and inversely correlated in both groups (P < .001). Furthermore, anti-p24 antibodies and low serum IFN-alpha levels were correlated to the NP versus the FP cohort. Finally, among NPs, only antibodies to Tat and not to the other HIV specificities (Env, Nef, Gag) were significantly predictive of clinical stability during their follow-up. Antibodies toward HIV-1 Tat, which are inversely correlated to p24 antigenemia, appear as a critical marker for a lack of disease progression. This study strongly suggests that rising anti-Tat antibodies through active immunization may be beneficial in AIDS vaccine development to control viral replication.

  9. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    PubMed Central

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  10. Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

    PubMed Central

    Gerber, Pehuén Pereyra; Cabrini, Mercedes; Jancic, Carolina; Paoletti, Luciana; Banchio, Claudia; von Bilderling, Catalina; Sigaut, Lorena; Pietrasanta, Lía I.; Duette, Gabriel; Freed, Eric O.; de Saint Basile, Genevieve; Moita, Catarina Ferreira; Moita, Luis Ferreira; Amigorena, Sebastian; Benaroch, Philippe; Geffner, Jorge

    2015-01-01

    During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication. PMID:25940347

  11. T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

    PubMed

    Mummert, Christiane; Hofmann, Christian; Hückelhoven, Angela G; Bergmann, Silke; Mueller-Schmucker, Sandra M; Harrer, Ellen G; Dörrie, Jan; Schaft, Niels; Harrer, Thomas

    2016-09-10

    Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.

  12. Modulation of innate and adaptive cellular immunity relevant to HIV-1 vaccine design by seminal plasma.

    PubMed

    Selva, Kevin J; Kent, Stephen J; Parsons, Matthew S

    2017-01-28

    Mucosal exposure to HIV-1 infection generally occurs in the presence of semen. Immunomodulation by seminal plasma is well described in the reproductive biology literature. Little is known, however, about the impact of seminal plasma on innate and adaptive anti-HIV-1 cellular immunity. The study investigated the effects of seminal plasma on immune responses considered important for prophylactic HIV-1 vaccine development, namely innate and adaptive cellular immunity mediated by natural killer (NK) cells and T cells, respectively. The ability of seminal plasma to modulate direct, antibody-dependent and cytokine-stimulated NK cell activation was assessed utilizing intracellular cytokine staining. Direct and antibody-dependent cellular cytotoxicity was assessed using lactate dehydrogenase release assays. The effects of seminal plasma on T-cell activation upon stimulation with staphylococcus enterotoxin B or HIV-1 Gag peptides were assessed by intracellular cytokine staining. The impact of seminal plasma on redirected cytolysis mediated by T cells was measured using lactate dehydrogenase release assays. Both direct and antibody-dependent NK cell activation were dramatically impaired by the presence of either HIV-1-uninfected or HIV-1-infected seminal plasma in a dose-dependent manner. Additionally, seminal plasma suppressed both direct and antibody-dependent NK cell-mediated cytolysis, including anti-HIV-1 antibody-dependent cytolysis of gp120-pulsed CEM.NKr-CCR5 cells. Finally, seminal plasma attenuated both HIV-1 Gag-specific and staphylococcus enterotoxin B-induced CTL activation. Semen contains potent immunosuppressors of both NK cell and CD8 T-cell-mediated anti-HIV-1 immune responses. This could impede attempts to provide vaccine-induced immunity to HIV-1.

  13. HIV-1 adaptation to antigen processing results in population-level immune evasion and affects subtype diversification.

    PubMed

    Tenzer, Stefan; Crawford, Hayley; Pymm, Phillip; Gifford, Robert; Sreenu, Vattipally B; Weimershaus, Mirjana; de Oliveira, Tulio; Burgevin, Anne; Gerstoft, Jan; Akkad, Nadja; Lunn, Daniel; Fugger, Lars; Bell, John; Schild, Hansjörg; van Endert, Peter; Iversen, Astrid K N

    2014-04-24

    The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8(+) T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ~30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ~60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Role of molecular mimicry to HIV-1 peptides in HIV-1–related immunologic thrombocytopenia

    PubMed Central

    Li, Zongdong; Nardi, Michael A.; Karpatkin, Simon

    2005-01-01

    Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the β3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti–GPIIIa49-66–induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated. PMID:15774614

  15. Conformations of the HIV-1 protease: A crystal structure data set analysis.

    PubMed

    Palese, Luigi Leonardo

    2017-11-01

    The HIV protease is an important drug target for HIV/AIDS therapy, and its structure and function have been extensively investigated. This enzyme performs an essential role in viral maturation by processing specific cleavage sites in the Gag and Gag-Pol precursor polyproteins so as to release their mature forms. This 99 amino acid aspartic protease works as a homodimer, with the active site localized in a central cavity capped by two flexible flap regions. The dimer presents closed or open conformations, which are involved in the substrate binding and release. Here the results of the analysis of a HIV-1 protease data set containing 552 dimer structures are reported. Different dimensionality reduction methods have been used in order to get information from this multidimensional database. Most of the structures in the data set belong to two conformational clusters. An interesting observation that comes from the analysis of these data is that some protease sequences are localized preferentially in specific areas of the conformational landscape of this protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

    PubMed Central

    Strauss, Joshua D.; Hammonds, Jason E.; Yi, Hong; Ding, Lingmei

    2015-01-01

    ABSTRACT Tetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes. IMPORTANCE Tetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ∼15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered

  17. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

    PubMed Central

    Omosa-Manyonyi, Gloria; Mpendo, Juliet; Ruzagira, Eugene; Kilembe, William; Chomba, Elwyn; Roman, François; Bourguignon, Patricia; Koutsoukos, Marguerite; Collard, Alix; Voss, Gerald; Laufer, Dagna; Stevens, Gwynn; Hayes, Peter; Clark, Lorna; Cormier, Emmanuel; Dally, Len; Barin, Burc; Ackland, Jim; Syvertsen, Kristen; Zachariah, Devika; Anas, Kamaal; Sayeed, Eddy; Lombardo, Angela; Gilmour, Jill; Cox, Josephine; Fast, Patricia; Priddy, Frances

    2015-01-01

    Background Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses. Methods In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured. Results The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration. Conclusion Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses. Trial Registration ClinicalTrials.gov NCT01264445 PMID:25961283

  18. Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

    PubMed Central

    Milev, Miroslav P.; Ravichandran, Mukunthan; Khan, Morgan F.; Schriemer, David C.; Mouland, Andrew J.

    2012-01-01

    The human immunodeficiency virus type 1 (HIV-1) unspliced, 9 kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as >200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced vRNA in HIV-1-producing cells. PMID:23125841

  19. Facilitators and barriers to discussing HIV prevention with adolescents: perspectives of HIV-infected parents.

    PubMed

    Edwards, Laura L; Reis, Janet S; Weber, Kathleen M

    2013-08-01

    We examined HIV-infected parents' conversations about HIV prevention with their uninfected children, including what facilitated or hindered communication. Parents with HIV/AIDS (n = 90) who had children aged 10 to 18 years were recruited for a mixed method study from 2009 to 2010. Interviews assessed facilitators and barriers to discussing HIV prevention. A questionnaire identified the frequency and content of conversations, parental confidence level, and perceived importance of discussing preventive topics. Eighty-one percent of parents reported "sometimes" or "often" communicating about HIV prevention. A subset of parents found these conversations difficult; 44% indicated their desire for support. Facilitators to communication included utilizing support, focusing on the benefits of talking, and having a previous relationship with one's child. Barriers to discussions included fear of negative consequences, living in denial, and lacking a parental role model who discussed safer sex. Parents varied as to how they believed their HIV status affected communication. Those who did not disclose their HIV status to their children reported less frequent communication; self-efficacy partially mediated this relationship. Findings highlighted the need for communication skills training that support HIV-infected parents in their efforts to discuss HIV-related information with adolescents.

  20. Use of a high resolution melting (HRM) assay to compare gag, pol, and env diversity in adults with different stages of HIV infection.

    PubMed

    Cousins, Matthew M; Laeyendecker, Oliver; Beauchamp, Geetha; Brookmeyer, Ronald; Towler, William I; Hudelson, Sarah E; Khaki, Leila; Koblin, Beryl; Chesney, Margaret; Moore, Richard D; Kelen, Gabor D; Coates, Thomas; Celum, Connie; Buchbinder, Susan P; Seage, George R; Quinn, Thomas C; Donnell, Deborah; Eshleman, Susan H

    2011-01-01

    Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14-540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.

  1. FAITH – Fast Assembly Inhibitor Test for HIV

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hadravová, Romana; Rumlová, Michaela, E-mail: michaela.rumlova@vscht.cz; Department of Biotechnology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague

    Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification ofmore » the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors. - Highlights: • Allows screening of assembly inhibitors of both mature and immature HIV-1 particles. • Based on Gag-derived proteins with CA in mature or immature conformation. • Simple and sensitive method suitable for high-throughput screening of inhibitors. • Unlike in other HIV assembly methods, works under physiological conditions. • No washing steps are necessary.« less

  2. A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN) vectors and multi-colour analyses

    PubMed Central

    2013-01-01

    Background Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs. PMID:23286882

  3. Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatability complex class I-mediated control

    PubMed Central

    Beck, Sarah E.; Queen, Suzanne E.; Viscidi, Raphael; Johnson, Darius; Kent, Stephen J.; Adams, Robert J.; Tarwater, Patrick M.; Mankowski, Joseph L.

    2016-01-01

    In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower CSF, but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies. PMID:26727909

  4. Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatibility complex class I-mediated control.

    PubMed

    Beck, Sarah E; Queen, Suzanne E; Viscidi, Raphael; Johnson, Darius; Kent, Stephen J; Adams, Robert J; Tarwater, Patrick M; Mankowski, Joseph L

    2016-08-01

    In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower cerebrospinal fluid (CSF), but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies.

  5. Identification and genetic characterization of unique HIV-1 A1/C recombinant strain in South Africa.

    PubMed

    Musyoki, Andrew M; Rakgole, Johnny N; Selabe, Gloria; Mphahlele, Jeffrey

    2015-03-01

    HIV isolates from South Africa are predominantly subtype C. Sporadic isolation of non-C strains has been reported mainly in cosmopolitan cities. HIV isolate j51 was recovered from a rural South African heterosexual female aged 51 years. Near full length amplification of the genome was attempted using PCR with primers targeting overlapping segments of the HIV genome. Analysis of 5593 bp (gag to vpu) at a bootstrap value greater than 70% found that all but the vpu gene was HIV-1 subtype A1. The vpu gene was assigned HIV-1 subtype C. The recombination breaking point was estimated at position 6035+/- 15 bp with reference to the beginning of the HXB2 reference strain. Isolate j51 revealed a unique genome constellation to previously reported recombinant strains with parental A/C backbones from South Africa though a common recombination with subtype C within the vpu gene. Identification of recombinant strains supports continued surveillance of HIV genetic diversity.

  6. Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique.

    PubMed

    Martrus, G; Niehrs, A; Cornelis, R; Rechtien, A; García-Beltran, W; Lütgehetmann, M; Hoffmann, C; Altfeld, M

    2016-10-15

    HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection of gagpol mRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4(+) T cells, four cell populations were detected according to their expression levels of viral mRNA and protein. gagpol mRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle. Early after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency

  7. Facilitators and Barriers to Discussing HIV Prevention With Adolescents: Perspectives of HIV-Infected Parents

    PubMed Central

    Reis, Janet S.; Weber, Kathleen M.

    2013-01-01

    Objectives. We examined HIV-infected parents’ conversations about HIV prevention with their uninfected children, including what facilitated or hindered communication. Methods. Parents with HIV/AIDS (n = 90) who had children aged 10 to 18 years were recruited for a mixed method study from 2009 to 2010. Interviews assessed facilitators and barriers to discussing HIV prevention. A questionnaire identified the frequency and content of conversations, parental confidence level, and perceived importance of discussing preventive topics. Results. Eighty-one percent of parents reported “sometimes” or “often” communicating about HIV prevention. A subset of parents found these conversations difficult; 44% indicated their desire for support. Facilitators to communication included utilizing support, focusing on the benefits of talking, and having a previous relationship with one’s child. Barriers to discussions included fear of negative consequences, living in denial, and lacking a parental role model who discussed safer sex. Parents varied as to how they believed their HIV status affected communication. Those who did not disclose their HIV status to their children reported less frequent communication; self-efficacy partially mediated this relationship. Conclusions. Findings highlighted the need for communication skills training that support HIV-infected parents in their efforts to discuss HIV-related information with adolescents. PMID:23763390

  8. Facilitating HIV Disclosure Across Diverse Settings: A Review

    PubMed Central

    Baijal, Parijat; Pegurri, Elisabetta

    2011-01-01

    HIV status disclosure is central to debates about HIV because of its potential for HIV prevention and its links to privacy and confidentiality as human-rights issues. Our review of the HIV-disclosure literature found that few people keep their status completely secret; disclosure tends to be iterative and to be higher in high-income countries; gender shapes disclosure motivations and reactions; involuntary disclosure and low levels of partner disclosure highlight the difficulties faced by health workers; the meaning and process of disclosure differ across settings; stigmatization increases fears of disclosure; and the ethical dilemmas resulting from competing values concerning confidentiality influence the extent to which disclosure can be facilitated. Our results suggest that structural changes, including making more services available, could facilitate HIV disclosure as much as individual approaches and counseling do. PMID:21493947

  9. Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

    PubMed Central

    Yu, Zheng; Beer, Christiane; Koester, Mario; Wirth, Manfred

    2006-01-01

    Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly. PMID:16956408

  10. A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly.

    PubMed

    Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J

    2014-04-01

    The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules

  11. Viral Linkage in HIV-1 Seroconverters and Their Partners in an HIV-1 Prevention Clinical Trial

    PubMed Central

    Campbell, Mary S.; Mullins, James I.; Hughes, James P.; Celum, Connie; Wong, Kim G.; Raugi, Dana N.; Sorensen, Stefanie; Stoddard, Julia N.; Zhao, Hong; Deng, Wenjie; Kahle, Erin; Panteleeff, Dana; Baeten, Jared M.; McCutchan, Francine E.; Albert, Jan; Leitner, Thomas; Wald, Anna; Corey, Lawrence; Lingappa, Jairam R.

    2011-01-01

    Background Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners. Methodology/Principal Findings We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners' sequences and a Bayesian posterior probability of ≥50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters. Conclusions/Significance In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of

  12. Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection

    PubMed Central

    Cousins, Matthew M.; Laeyendecker, Oliver; Beauchamp, Geetha; Brookmeyer, Ronald; Towler, William I.; Hudelson, Sarah E.; Khaki, Leila; Koblin, Beryl; Chesney, Margaret; Moore, Richard D.; Kelen, Gabor D.; Coates, Thomas; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2011-01-01

    Background Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. Methods HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Results Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. Conclusions The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined. PMID:22073290

  13. HIV-1 genetic diversity and its distribution characteristics among newly diagnosed HIV-1 individuals in Hebei province, China.

    PubMed

    Lu, Xinli; Zhao, Cuiying; Wang, Wei; Nie, Chenxi; Zhang, Yuqi; Zhao, Hongru; Chen, Suliang; Cui, Ze

    2016-01-01

    Since the first HIV-1 case in 1989, Hebei province has presented a clearly rising trend of HIV-1 prevalence, and HIV-1 genetic diversity has become the vital barrier to HIV prevention and control in this area. To obtain detailed information of HIV-1 spread in different populations and in different areas of Hebei, a cross-sectional HIV-1 molecular epidemiological investigation was performed across the province. Blood samples of 154 newly diagnosed HIV-1 individuals were collected from ten prefectures in Hebei using stratified sampling. Partial gag and env genes were amplified and sequenced. HIV-1 genotypes were identified by phylogenetic tree analyses. Among the 139 subjects genotyped, six HIV-1 subtypes were identified successfully, including subtype B (41.0 %), CRF01_AE (40.3 %), CRF07_BC (11.5 %), CRF08_BC (4.3 %), unique recombinant forms (URFs) (1.4 %) and subtype C (1.4 %). Subtype B was identified as the most frequent subtype. Two URF recombination patterns were the same as CRF01_AE/B. HIV-1 genotype distribution showed a significant statistical difference in different demographic characteristics, such as source (P < 0.05), occupation (P < 0.05) and ethnicity (P < 0.05). The distributions of subtype B (P < 0.05), CRF01_AE (P < 0.05), CRF07_BC (P < 0.05) and subtype C (P < 0.05) showed significant differences in all ten prefectures, and the distributions of all six subtypes were significantly different in Shijiazhuang (P < 0.05) and Xingtai (P < 0.05), but not in other prefectures (P > 0.05). The differences in HIV-1 genotype distribution were closely associated with transmission routes. Particularly, all six subtype strains were found in heterosexuals, showing that HIV-1 has spread from the high-risk populations to the general populations in Hebei, China. In addition, CRF01_AE instead of subtype B has become the major strain of HIV-1 infection among homosexuals. Our study revealed HIV-1 evolution and genotype distribution by

  14. Crystal structure of an HIV assembly and maturation switch

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wagner, Jonathan M.; Zadrozny, Kaneil K.; Chrustowicz, Jakub

    Virus assembly and maturation proceed through the programmed operation of molecular switches, which trigger both local and global structural rearrangements to produce infectious particles. HIV-1 contains an assembly and maturation switch that spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the precursor structural protein, Gag. The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn, and the stem comprises a 6-helix bundle. The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysismore » during maturation. This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses. Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate.« less

  15. Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.

    PubMed

    Barouch, Dan H; O'Brien, Kara L; Simmons, Nathaniel L; King, Sharon L; Abbink, Peter; Maxfield, Lori F; Sun, Ying-Hua; La Porte, Annalena; Riggs, Ambryice M; Lynch, Diana M; Clark, Sarah L; Backus, Katherine; Perry, James R; Seaman, Michael S; Carville, Angela; Mansfield, Keith G; Szinger, James J; Fischer, Will; Muldoon, Mark; Korber, Bette

    2010-03-01

    The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

  16. Phylogenetic Analysis of the Latvian HIV-1 Epidemic

    PubMed Central

    Balode, Dace; Skar, Helena; Mild, Mattias; Kolupajeva, Tatjana; Ferdats, Andris; Rozentale, Baiba; Leitner, Thomas

    2012-01-01

    Abstract The Latvian HIV-1 outbreak among intravenous drug users (IDUs) in 1997–1998 involved subtype A1. To obtain a more complete picture of the Latvian HIV-1 epidemic, 315 HIV-1-infected patients diagnosed in 1990–2005 representing different transmission groups and geographic regions were phylogenetically characterized using env V3 and gag p17 sequences. Subtypes A1 and B infections were found in 76% and 22% of the patients, respectively. The subtype A1 sequences formed one large cluster, which also included sequences from other parts of the former Soviet Union (FSU), whereas most subtype B sequences formed three distinct clusters. We estimated that subtype A1 was introduced from FSU around 1997 and initially spread explosively among IDUs in Riga. A recent increase of heterosexually infected persons did not form a separate subepidemic, but had multiple interactions with the IDU epidemic. Subtype B was introduced before the collapse of the Soviet Union and primarily has spread among men who have sex with men. PMID:22049908

  17. Immune-driven recombination and loss of control after HIV superinfection.

    PubMed

    Streeck, Hendrik; Li, Bin; Poon, Art F Y; Schneidewind, Arne; Gladden, Adrianne D; Power, Karen A; Daskalakis, Demetre; Bazner, Suzane; Zuniga, Rosario; Brander, Christian; Rosenberg, Eric S; Frost, Simon D W; Altfeld, Marcus; Allen, Todd M

    2008-08-04

    After acute HIV infection, CD8(+) T cells are able to control viral replication to a set point. This control is often lost after superinfection, although the mechanism behind this remains unclear. In this study, we illustrate in an HLA-B27(+) subject that loss of viral control after HIV superinfection coincides with rapid recombination events within two narrow regions of Gag and Env. Screening for CD8(+) T cell responses revealed that each of these recombination sites (approximately 50 aa) encompassed distinct regions containing two immunodominant CD8 epitopes (B27-KK10 in Gag and Cw1-CL9 in Env). Viral escape and the subsequent development of variant-specific de novo CD8(+) T cell responses against both epitopes were illustrative of the significant immune selection pressures exerted by both responses. Comprehensive analysis of the kinetics of CD8 responses and viral evolution indicated that the recombination events quickly facilitated viral escape from both dominant WT- and variant-specific responses. These data suggest that the ability of a superinfecting strain of HIV to overcome preexisting immune control may be related to its ability to rapidly recombine in critical regions under immune selection pressure. These data also support a role for cellular immune pressures in driving the selection of new recombinant forms of HIV.

  18. Structural And Functional Studies of ALIX Interactions With YPXnL Late Domains of HIV-1 And EIAV

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhai, Q.; Fisher, R.D.; Chung, H.-Y.

    2009-05-28

    Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPX{sub n}L late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPX{sub n}L late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalentmore » contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPX{sub n}L late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPX{sub n}L sequences with both n = 1 and n = 3.« less

  19. ESCRT-II's involvement in HIV-1 genomic RNA trafficking and assembly.

    PubMed

    Ghoujal, Bashar; Milev, Miroslav P; Ajamian, Lara; Abel, Karen; Mouland, Andrew J

    2012-12-01

    Several host proteins play crucial roles in the HIV-1 replication cycle. The endosomal sorting complex required for transport (ESCRT) exemplifies a large, multi-component host machinery that is required by HIV-1 for viral budding. ESCRT promotes the inward budding of vesicles from the membranes of late endosomes to generate multi-vesicular bodies. However, HIV-1 co-opts the ESCRT to enable outwards budding of virus particles from the plasma membrane, a phenomenon that is topologically similar to multi-vesicular body biogenesis. A role for ESCRTII in mRNA trafficking has been established in Drosophila in which the ESCRT-II components, Vps22 and Vps36, promote the localisation of the bicoid mRNA in the fertilised egg. This is achieved via specific interactions with the Staufen protein. In this work, we investigated a possible implication of ESCRT-II in the HIV-1 replication cycle. Co-immunoprecipitation analyses and live cell tri-molecular fluorescence complementation assays revealed that interactions between EAP30 and Gag and another between EAP30 and Staufen1 occur in mammalian cells. We then depleted EAP30 (the orthologue for Vps22) by siRNA to target ESCRT-II in HIV-1 expressing cells. This treatment disrupted ESCRT-II function and leads to the degradation of the two other ESCRT-II complex proteins, EAP45 and EAP20, as well as the associated Rab7-interacting lysosomal protein. The depletion of EAP30 led to dramatically reduced viral structural protein Gag and virus production levels, without any effect on viral RNA levels. On the contrary, the overexpression of EAP30 led to a several-fold increase in virus production. Unexpec-tedly, siRNA-mediated depletion of EAP30 led to a block to HIV-1 genomic RNA trafficking and resulted in the accumulation of genomic RNA in the nucleus and juxtanuclear domains. Our data provide the first evidence that the Staufen1-ESCRT-II interaction is evolutionarily conserved from lower to higher eukaryotes and reveal a novel role for

  20. Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

    PubMed

    Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

    2016-09-02

    HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

    NASA Astrophysics Data System (ADS)

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

    1997-04-01

    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

  2. Heparin (GAG-hed) inhibits LCR activity of human papillomavirus type 18 by decreasing AP1 binding.

    PubMed

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Angel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-08-31

    High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have antitumoral

  3. Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA.

    PubMed

    Bartels, Hanni; Luban, Jeremy

    2014-09-12

    All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto

  4. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

    PubMed Central

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E.; Gehring, Niels H.; Mouland, Andrew J.

    2015-01-01

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. PMID:26492277

  5. Early cytoplasmic uncoating is associated with infectivity of HIV-1

    PubMed Central

    Cianci, Gianguido C.; Anderson, Meegan R.; Hope, Thomas J.

    2017-01-01

    After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection. PMID:28784755

  6. The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr.

    PubMed

    Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

    2005-04-01

    We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

  7. The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nuclear localization and particle formation.

    PubMed

    Kim, Min-Kyung; Claiborn, Kathryn C; Levin, Henry L

    2005-08-01

    Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.

  8. Innate immunity and HIV-1 infection.

    PubMed

    Lehner, T; Wang, Y; Whittall, T; Seidl, T

    2011-04-01

    HIV-1 is predominantly transmitted through mucosal tissues, targeting CD4(+)CCR5(+) T cells, 50% of which are destroyed within 2 weeks of infection. Conventional vaccination strategies have so far failed to prevent HIV-1 infection. Neither antibodies nor cytotoxic lymphocytes are capable of mounting a sufficiently rapid immune response to prevent early destruction of these cells. However, innate immunity is an early-response system, largely independent of prior encounter with a pathogen. Innate immunity can be classified into cellular, extracellular, and intracellular components, each of which is exemplified in this review by γδ T cells, CC chemokines, and APOBEC3G, respectively. First, γδ T cells are found predominantly in mucosal tissues and produce cytokines, CC chemokines, and antiviral factors. Second, the CC chemokines CCL-3, CCL-4, and CCL-5 can be upregulated by immunization of macaques with SIVgp120 and gag p27, and these can bind and downmodulate CCR5, thereby inhibiting HIV-1 entry into the host cells. Third, APOBEC3G is generated and maintained following rectal mucosal immunization in rhesus macaques for over 17 weeks, and the innate anti-SIV factor is generated by CD4(+)CD95(+)CCR7(-) effector memory T cells. Thus, innate anti-HIV-1 or SIV immunity can be linked with immune memory, mediated by CD4(+) T cells generating APOBEC3G. The multiple innate functions may mount an early anti-HIV-1 response and either prevent viral transmission or contain the virus until an effective adaptive immune response develops.

  9. Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

    PubMed

    Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

    2017-08-01

    We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

  10. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

    PubMed

    Boyer, J D; Ugen, K E; Wang, B; Agadjanyan, M; Gilbert, L; Bagarazzi, M L; Chattergoon, M; Frost, P; Javadian, A; Williams, W V; Refaeli, Y; Ciccarelli, R B; McCallus, D; Coney, L; Weiner, D B

    1997-05-01

    Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

  11. HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

    PubMed Central

    Wu, Guoxin; Swanson, Michael; Talla, Aarthi; Graham, Donald; Strizki, Julie; Gorman, Daniel; Barnard, Richard J.O.; Blair, Wade; Søgaard, Ole S.; Tolstrup, Martin; Østergaard, Lars; Rasmussen, Thomas A.; Sekaly, Rafick-Pierre; Archin, Nancie M.; Hazuda, Daria J.; Howell, Bonnie J.

    2017-01-01

    Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3 bispecific antibody. These findings extend beyond classical nucleic acid endpoints, which are confounded by the predominance of mutated, defective proviruses and, of paramount importance, enable assessment of cells making HIV protein that can now be targeted by immunological approaches. PMID:28814661

  12. HIV-specific antibodies but not t-cell responses are associated with protection in seronegative partners of HIV-1-infected individuals in Cambodia.

    PubMed

    Nguyen, Marie; Pean, Polidy; Lopalco, Lucia; Nouhin, Janin; Phoung, Viseth; Ly, Nary; Vermisse, Pierre; Henin, Yvette; Barré-Sinoussi, Françoise; Burastero, Samuele E; Reynes, Jean-Marc; Carcelain, Guislaine; Pancino, Gianfranco

    2006-08-01

    To study biological factors related to protection against HIV-1 infection in Cambodia, we recruited 48 partners of HIV-1-infected patients who remained uninfected (exposed uninfected individuals, EUs) despite unprotected sexual intercourse for more than 1 year and 49 unexposed controls (UCs). HIV-1-specific antibodies (IgA anti-gp41 and IgG anti-CD4-gp120 complex), T-cell responses, and cellular factors that may be involved in protection (peripheral blood mononuclear cell [PBMC] resistance to HIV-1 infection and beta-chemokine production) were evaluated. Anti-HIV-1 antibodies were higher in EUs than those in UCs (P = 0.01 and P = 0.04 for anti-gp41 and anti-CD4-gp120, respectively). We observed a decreased susceptibility to a primary Cambodian isolate, HIV-1KH019, in EU PBMCs as compared with UC PBMCs (P = 0.03). A weak T-cell response to one pool of HIV-1 Gag peptides was found by ELISpot in 1 of 19 EUs. Whereas T-cell specific immunity was not associated to protection, our results suggest that HIV-specific humoral immunity and reduced cell susceptibility to infection may contribute to protection against HIV-1 infection in Cambodian EUs.

  13. Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

    PubMed Central

    Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2012-01-01

    Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

  14. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

    NASA Astrophysics Data System (ADS)

    Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.

    2004-05-01

    Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

  15. The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human Nup153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 VprV⃞

    PubMed Central

    Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

    2005-01-01

    We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro–translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124pAA264–454 and Nup153AA448–634 that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124+) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved. PMID:15659641

  16. Impact of HLA-DRB1 allele polymorphisms on control of HIV infection in a Peruvian MSM cohort.

    PubMed

    Oriol-Tordera, B; Llano, A; Ganoza, C; Cate, S; Hildebrand, W; Sanchez, J; Calle, M L; Brander, C; Olvera, A

    2017-10-01

    Associations between HLA class II polymorphisms and HIV control were assessed in a Peruvian MSM cohort. Among 233 treatment naïve HIV+ individuals, DRB1*13:02 was linked to elevated viral loads (P = .044) while DRB1*12:01 showed significantly lower viral set points (P = .015) and restricted a dominant T cell response to HIV Gag p24 (P = .038). The present work contributes to a better knowledge of the Peruvian immunogenetics and supports the important role of HLA class II restricted T cells in HIV control. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

    PubMed

    Müller, Barbara; Anders, Maria; Reinstein, Jochen

    2014-01-01

    Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

  18. A robust measure of HIV-1 population turnover within chronically infected individuals.

    PubMed

    Achaz, G; Palmer, S; Kearney, M; Maldarelli, F; Mellors, J W; Coffin, J M; Wakeley, J

    2004-10-01

    A simple nonparameteric test for population structure was applied to temporally spaced samples of HIV-1 sequences from the gag-pol region within two chronically infected individuals. The results show that temporal structure can be detected for samples separated by about 22 months or more. The performance of the method, which was originally proposed to detect geographic structure, was tested for temporally spaced samples using neutral coalescent simulations. Simulations showed that the method is robust to variation in samples sizes and mutation rates, to the presence/absence of recombination, and that the power to detect temporal structure is high. By comparing levels of temporal structure in simulations to the levels observed in real data, we estimate the effective intra-individual population size of HIV-1 to be between 10(3) and 10(4) viruses, which is in agreement with some previous estimates. Using this estimate and a simple measure of sequence diversity, we estimate an effective neutral mutation rate of about 5 x 10(-6) per site per generation in the gag-pol region. The definition and interpretation of estimates of such "effective" population parameters are discussed.

  19. HIV-1 vaccine-specific responses induced by Listeria vector vaccines are maintained in mice subsequently infected with a model helminth parasite, Schistosoma mansoni.

    PubMed

    Shollenberger, Lisa M; Bui, Cac T; Paterson, Yvonne; Nyhoff, Lindsay; Harn, Donald A

    2013-11-19

    In areas co-endemic for helminth parasites and HIV/AIDS, infants are often administered vaccines prior to infection with immune modulatory helminth parasites. Systemic Th2 biasing and immune suppression caused by helminth infection reduces cell-mediated responses to vaccines such as tetanus toxoid and BCG. Therefore, we asked if infection with helminthes post-vaccination, alters already established vaccine induced immune responses. In our model, mice are vaccinated against HIV-1 Gag using a Listeria vaccine vector (Lm-Gag) in a prime-boost manner, then infected with the human helminth parasite Schistosoma mansoni. This allows us to determine if established vaccine responses are maintained or altered after helminth infection. Our second objective asked if helminth infection post-vaccination alters the recipient's ability to respond to a second boost. Here we compared responses between uninfected mice, schistosome infected mice, and infected mice that were given an anthelminthic, which occurred coincident with the boost or four weeks prior, as well as comparing to un-boosted mice. We report that HIV-1 vaccine-specific responses generated by Listeria vector HIV-1 vaccines are maintained following subsequent chronic schistosome infection, providing further evidence that Listeria vector vaccines induce potent vaccine-specific responses that can withstand helminth infection. We also were able to demonstrate that administration of a second Listeria boost, which markedly enhanced the immune response, was minimally impacted by schistosome infection, or anthelminthic therapy. Surprisingly, we also observed enhanced antibody responses to HIV Gag in vaccinated mice subsequently infected with schistosomes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. The Use of an Alternative Extraoral Periapical Technique for Patients with Severe Gag Reflex

    PubMed Central

    e Silva, Mauro Henrique Chagas; Santos, Mariane Floriano Lopes; de Lima, Carolina Oliveira; Campos, Celso Neiva

    2016-01-01

    Gag reflex is a physiologic mechanism that promotes contraction of the muscles of the tongue and pharyngeal walls. Different factors, including intraoral radiographic films and sensors, may trigger this reflex. Patients with severe gag reflex may not be able to tolerate the presence of intraoral radiographic films or sensors during root canal therapy (RCT). This factor may prevent an appropriate intraoral radiograph, which is important in RCT. Different approaches have been used to facilitate dental procedures in patients suffering from severe gag reflex. The use of an extraoral radiographic technique is an alternative method to obtain working length confirmation in patients with severe gag reflex. In this report of 2 cases, the use of an extraoral radiographic technique as an alternative approach during RCT in patients with severe gag reflex associated with phobic behavior and trismus was successfully demonstrated. PMID:27547474

  1. The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

    PubMed

    Atwood, A; Lin, J H; Levin, H L

    1996-01-01

    The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

  2. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.

    PubMed

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E; Gehring, Niels H; Mouland, Andrew J

    2015-10-20

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

  3. GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

    PubMed Central

    Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

    2012-01-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

  4. Frequent and Variable Cytotoxic-T-Lymphocyte Escape-Associated Fitness Costs in the Human Immunodeficiency Virus Type 1 Subtype B Gag Proteins

    PubMed Central

    Boutwell, Christian L.; Carlson, Jonathan M.; Lin, Tien-Ho; Seese, Aaron; Power, Karen A.; Peng, Jian; Tang, Yanhua; Brumme, Zabrina L.; Heckerman, David; Schneidewind, Arne

    2013-01-01

    Cytotoxic-T-lymphocyte (CTL) escape mutations undermine the durability of effective human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cell responses. The rate of CTL escape from a given response is largely governed by the net of all escape-associated viral fitness costs and benefits. The observation that CTL escape mutations can carry an associated fitness cost in terms of reduced virus replication capacity (RC) suggests a fitness cost-benefit trade-off that could delay CTL escape and thereby prolong CD8 response effectiveness. However, our understanding of this potential fitness trade-off is limited by the small number of CTL escape mutations for which a fitness cost has been quantified. Here, we quantified the fitness cost of the 29 most common HIV-1B Gag CTL escape mutations using an in vitro RC assay. The majority (20/29) of mutations reduced RC by more than the benchmark M184V antiretroviral drug resistance mutation, with impacts ranging from 8% to 69%. Notably, the reduction in RC was significantly greater for CTL escape mutations associated with protective HLA class I alleles than for those associated with nonprotective alleles. To speed the future evaluation of CTL escape costs, we also developed an in silico approach for inferring the relative impact of a mutation on RC based on its computed impact on protein thermodynamic stability. These data illustrate that the magnitude of CTL escape-associated fitness costs, and thus the barrier to CTL escape, varies widely even in the conserved Gag proteins and suggest that differential escape costs may contribute to the relative efficacy of CD8 responses. PMID:23365420

  5. Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques▿

    PubMed Central

    Queen, Suzanne E.; Mears, Brian M.; Kelly, Kathleen M.; Dorsey, Jamie L.; Liao, Zhaohao; Dinoso, Jason B.; Gama, Lucio; Adams, Robert J.; Zink, M. Christine; Clements, Janice E.; Kent, Stephen J.; Mankowski, Joseph L.

    2011-01-01

    In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8+ T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8+ T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4+ T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8+ T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted. PMID:21715484

  6. Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

    PubMed

    Kim, Seong U; Batule, Bhagwan S; Mun, Hyoyoung; Byun, Ju-Young; Shim, Won-Bo; Kim, Min-Gon

    2018-02-07

    We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H 2 O 2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

  7. Incorporation of chimeric gag protein into retroviral particles.

    PubMed Central

    Weldon, R A; Erdie, C R; Oliver, M G; Wills, J W

    1990-01-01

    The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions. Images PMID:2166812

  8. Circulation of HIV-1 Multiple Complexity Recombinant Forms Among Female Sex Workers Recently Infected with HIV-1 in Thailand.

    PubMed

    Saeng-Aroon, Siriphan; Loket, Ruangchai; Plipat, Tanarak; Lumyai, Suttiwat; Chu, Pei-Yu; Sangkitporn, Somchai; Nakayama, Emi E; Takeda, Naokazu; Shioda, Tatsuo; Motomura, Kazushi

    2016-07-01

    The circulating subtype distribution of HIV-1 has not been well characterized in female sex worker (FSW) populations in Thailand. To understand the mechanisms and interrelationships of epidemics involving FSWs in Thailand, we performed a large molecular epidemiological study of FSWs aged 25 years with recently acquired HIV-1 infections. The samples were collected in 2005, 2007, 2009, and 2011 in 38 provinces, representing every region of Thailand. After gag (p24), pol (pro-RT), and env (C2/V3) were sequenced, comprehensive genome analysis was performed. Genetic subtypes were determined in 159 plasma samples. The percentage of circulating recombinant forms (CRFs) CRF01_AE (90.6%) predominated, while subtype B (1.3%), other CRFs (1.9%), and unique recombinant forms (URFs) (6.2%) were identified as minor populations. Interestingly, the unique recombinant nature of these HIV-1 strains was verified in 10 specimens, indicating the presence of new forms of HIV-1 intersubtypes G/A, C/B, AE/B/C, and AE/B with different recombination breakpoints. Subtype B has contributed to these new generations of unique CRF01/B recombinants, especially in the pol (RT) gene, in which the template switching of the RT genomes occurred during reverse transcription. These results imply that the several unique recombinant viruses circulating in Thailand were probably generated in the population or introduced from neighboring countries. Our study helps clarify the patterns of viral transmission and define transmission pathways in Thailand.

  9. Genotypic and Functional Impact of HIV-1 Adaptation to Its Host Population during the North American Epidemic

    PubMed Central

    Carlson, Jonathan M.; Chan, Benjamin; Chopera, Denis R.; Brumme, Chanson J.; Markle, Tristan J.; Martin, Eric; Shahid, Aniqa; Anmole, Gursev; Mwimanzi, Philip; Nassab, Pauline; Penney, Kali A.; Rahman, Manal A.; Milloy, M.-J.; Schechter, Martin T.; Markowitz, Martin; Carrington, Mary; Walker, Bruce D.; Wagner, Theresa; Buchbinder, Susan; Fuchs, Jonathan; Koblin, Beryl; Mayer, Kenneth H.; Harrigan, P. Richard; Brockman, Mark A.; Poon, Art F. Y.; Brumme, Zabrina L.

    2014-01-01

    HLA-restricted immune escape mutations that persist following HIV transmission could gradually spread through the viral population, thereby compromising host antiviral immunity as the epidemic progresses. To assess the extent and phenotypic impact of this phenomenon in an immunogenetically diverse population, we genotypically and functionally compared linked HLA and HIV (Gag/Nef) sequences from 358 historic (1979–1989) and 382 modern (2000–2011) specimens from four key cities in the North American epidemic (New York, Boston, San Francisco, Vancouver). Inferred HIV phylogenies were star-like, with approximately two-fold greater mean pairwise distances in modern versus historic sequences. The reconstructed epidemic ancestral (founder) HIV sequence was essentially identical to the North American subtype B consensus. Consistent with gradual diversification of a “consensus-like” founder virus, the median “background” frequencies of individual HLA-associated polymorphisms in HIV (in individuals lacking the restricting HLA[s]) were ∼2-fold higher in modern versus historic HIV sequences, though these remained notably low overall (e.g. in Gag, medians were 3.7% in the 2000s versus 2.0% in the 1980s). HIV polymorphisms exhibiting the greatest relative spread were those restricted by protective HLAs. Despite these increases, when HIV sequences were analyzed as a whole, their total average burden of polymorphisms that were “pre-adapted” to the average host HLA profile was only ∼2% greater in modern versus historic eras. Furthermore, HLA-associated polymorphisms identified in historic HIV sequences were consistent with those detectable today, with none identified that could explain the few HIV codons where the inferred epidemic ancestor differed from the modern consensus. Results are therefore consistent with slow HIV adaptation to HLA, but at a rate unlikely to yield imminent negative implications for cellular immunity, at least in North America

  10. Virological and immunological outcome of treatment interruption in HIV-1-infected subjects vaccinated with MVA-B

    PubMed Central

    Noguera-Julian, Marc; Bellido, Rocío; Puertas, Maria C.; Carrillo, Jorge; Rodriguez, C.; Perez-Alvarez, Núria; Cobarsí, Patricia; Gomez, Carmen E.; Esteban, Mariano; Jímenez, Jose Luis; García, Felipe; Blanco, Julià; Martinez-Picado, Javier; Paredes, Roger

    2017-01-01

    The most relevant endpoint in therapeutic HIV vaccination is the assessment of time to viral rebound or duration of sustained control of low-level viremia upon cART treatment cessation. Structured treatment interruptions (STI) are however not without risk to the patient and reliable predictors of viral rebound/control after therapeutic HIV-1 vaccination are urgently needed to ensure patient safety and guide therapeutic vaccine development. Here, we integrated immunological and virological parameters together with viral rebound dynamics after STI in a phase I therapeutic vaccine trial of a polyvalent MVA-B vaccine candidate to define predictors of viral control. Clinical parameters, proviral DNA, host HLA genetics and measures of humoral and cellular immunity were evaluated. A sieve effect analysis was conducted comparing pre-treatment viral sequences to breakthrough viruses after STI. Our results show that a reduced proviral HIV-1 DNA at study entry was independently associated with two virological parameters, delayed HIV-1 RNA rebound (p = 0.029) and lower peak viremia after treatment cessation (p = 0.019). Reduced peak viremia was also positively correlated with a decreased number of HLA class I allele associated polymorphisms in Gag sequences in the rebounding virus population (p = 0.012). Our findings suggest that proviral DNA levels and the number of HLA-associated Gag polymorphisms may have an impact on the clinical outcome of STI. Incorporation of these parameters in future therapeutic vaccine trials may guide refined immunogen design and help conduct safer STI approaches. PMID:28953921

  11. The Greek version of the Gagging Assessment Scale in children and adolescents: psychometric properties, prevalence of gagging, and the association between gagging and dental fear.

    PubMed

    Katsouda, Maria; Provatenou, Efthymia; Arapostathis, Konstantinos; Coolidge, Trilby; Kotsanos, Nikolaos

    2017-03-01

    No studies assessing the association between gagging and dental fear are available in pediatric samples. To assess the psychometric properties of the Greek version of the Gagging Assessment Scale (GAS), to explore the prevalence of gagging, and to evaluate the relationship between gagging and dental fear in a pediatric sample. A total of 849 8- and 14-year-old children filled out a questionnaire consisting of demographic items, the Greek version of the GAS, and the Greek Children's Fear Survey Schedule Dental Subscale (CFSS-DS); the older children also completed the Greek version of the Modified Dental Anxiety Scale (MDAS). The short form of dentist part of the Gagging Problem Assessment (GPA-de-c/SF) was used to objectively assess gagging. A total of 51 children (6.0%) demonstrated gagging on the GPA-de-c/SF. Children rated as gaggers on the GPA-de-c/SF had significantly higher GAS scores. There were no relationships between GPA-de-c/SF and the CFSS-DS or MDAS. The GAS ratings were significantly correlated with the CFSS-DS (rho = 0.420, P < 0.001) and MDAS (rho = 0.429, P < 0.001). The internal consistency was good (Cronbach's alpha = 0.697). The GAS demonstrated good psychometric properties. Dental fear was correlated with the self-report gagging assessment, but not with the objective gagging assessment. © 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

    PubMed

    Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

    2012-05-01

    Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

    PubMed Central

    Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

    2012-01-01

    Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

  14. Heroin use is associated with lower levels of restriction factors and type I interferon expression and facilitates HIV-1 replication.

    PubMed

    Zhu, Jia-Wu; Liu, Feng-Liang; Mu, Dan; Deng, De-Yao; Zheng, Yong-Tang

    Heroin use is associated with increased incidence of infectious diseases such as HIV-1 infection, as a result of immunosuppression to a certain extent. Host restriction factors are recently identified cellular proteins with potent antiviral activities. Whether heroin use impacts on the in vivo expression of restriction factors that result in facilitating HIV-1 replication is poorly understood. Here we recruited 432 intravenous drug users (IDUs) and 164 non-IDUs at high-risk behaviors. Based on serological tests, significantly higher prevalence of HIV-1 infection was observed among IDUs compared with non-IDUs. We included those IDUs and non-IDUs without HIV-1 infection, and found IDUs had significantly lower levels of TRIM5α, TRIM22, APOBEC3G, and IFN-α, -β expression than did non-IDUs. We also directly examined plasma viral load in HIV-1 mono-infected IDUs and non-IDUs and found HIV-1 mono-infected IDUs had significantly higher plasma viral load than did non-IDUs. Moreover, intrinsically positive correlation between type I interferon and TRIM5α or TRIM22 was observed, however, which was dysregulated following heroin use. Collectively, heroin use benefits HIV-1 replication that may be partly due to suppression of host restriction factors and type I interferon expression. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. Addressing Unmet Need for HIV Testing in Emergency Care Settings: A Role for Computer-facilitated Rapid HIV Testing?

    PubMed Central

    Kurth, Ann E.; Severynen, Anneleen; Spielberg, Freya

    2014-01-01

    HIV testing in emergency departments (EDs) remains underutilized. We evaluated a computer tool to facilitate rapid HIV testing in an urban ED. Randomly assigned non-acute adult ED patients to computer tool (‘CARE’) and rapid HIV testing before standard visit (n=258) or to standard visit (n=259) with chart access. Assessed intervention acceptability and compared noted HIV risks. Participants were 56% non-white, 58% male; median age 37 years. In the CARE arm nearly all (251/258) completed the session and received HIV results; 4 declined test consent. HIV risks were reported by 54% of users and there was one confirmed HIV-positive and 2 false-positives (seroprevalence 0.4%, 95% CI 0.01–2.2%). Half (55%) preferred computerized, over face-to-face, counseling for future HIV testing. In standard arm, one HIV test and 2 referrals for testing occurred. Computer-facilitated HIV testing appears acceptable to ED patients. Future research should assess cost-effectiveness compared with staff-delivered approaches. PMID:23837807

  16. Analysis of HIV Diversity in HIV-Infected Black Men Who Have Sex with Men (HPTN 061)

    PubMed Central

    Chen, Iris; Chau, Gordon; Wang, Jing; Clarke, William; Marzinke, Mark A.; Cummings, Vanessa; Breaud, Autumn; Laeyendecker, Oliver; Fields, Sheldon D.; Griffith, Sam; Scott, Hyman M.; Shoptaw, Steven; del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P.; Mayer, Kenneth H.; Koblin, Beryl A.; Eshleman, Susan H.

    2016-01-01

    Background HIV populations often diversify in response to selective pressures, such as the immune response and antiretroviral drug use. We analyzed HIV diversity in Black men who have sex with men who were enrolled in the HIV Prevention Trials Network 061 study. Methods A high resolution melting (HRM) diversity assay was used to measure diversity in six regions of the HIV genome: two in gag, one in pol, and three in env. HIV diversity was analyzed for 146 men who were HIV infected at study enrollment, including three with acute infection and 13 with recent infection (identified using a multi-assay algorithm), and for 21 men who seroconverted during the study. HIV diversification was analyzed in a paired analysis for 62 HIV-infected men using plasma samples from the enrollment and 12-month (end of study) visits. Results Men with acute or recent infection at enrollment and seroconverters had lower median HRM scores (lower HIV diversity) than men with non-recent infection in all six regions analyzed. In univariate analyses, younger age, higher CD4 cell count, and HIV drug resistance were associated with lower median HRM scores in multiple regions; ARV drug detection was marginally associated with lower diversity in the pol region. In multivariate analysis, acute or recent infection (all six regions) and HIV drug resistance (both gag regions) were associated with lower median HRM scores. Diversification in the pol region over 12 months was greater for men with acute or recent infection, higher CD4 cell count, and lower HIV viral load at study enrollment. Conclusions HIV diversity was significantly associated with duration of HIV infection, and lower gag diversity was observed in men who had HIV drug resistance. HIV pol diversification was more pronounced in men with acute or recent infection, higher CD4 cell count, and lower HIV viral load. PMID:27936098

  17. Analysis of HIV Diversity in HIV-Infected Black Men Who Have Sex with Men (HPTN 061).

    PubMed

    Chen, Iris; Chau, Gordon; Wang, Jing; Clarke, William; Marzinke, Mark A; Cummings, Vanessa; Breaud, Autumn; Laeyendecker, Oliver; Fields, Sheldon D; Griffith, Sam; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

    2016-01-01

    HIV populations often diversify in response to selective pressures, such as the immune response and antiretroviral drug use. We analyzed HIV diversity in Black men who have sex with men who were enrolled in the HIV Prevention Trials Network 061 study. A high resolution melting (HRM) diversity assay was used to measure diversity in six regions of the HIV genome: two in gag, one in pol, and three in env. HIV diversity was analyzed for 146 men who were HIV infected at study enrollment, including three with acute infection and 13 with recent infection (identified using a multi-assay algorithm), and for 21 men who seroconverted during the study. HIV diversification was analyzed in a paired analysis for 62 HIV-infected men using plasma samples from the enrollment and 12-month (end of study) visits. Men with acute or recent infection at enrollment and seroconverters had lower median HRM scores (lower HIV diversity) than men with non-recent infection in all six regions analyzed. In univariate analyses, younger age, higher CD4 cell count, and HIV drug resistance were associated with lower median HRM scores in multiple regions; ARV drug detection was marginally associated with lower diversity in the pol region. In multivariate analysis, acute or recent infection (all six regions) and HIV drug resistance (both gag regions) were associated with lower median HRM scores. Diversification in the pol region over 12 months was greater for men with acute or recent infection, higher CD4 cell count, and lower HIV viral load at study enrollment. HIV diversity was significantly associated with duration of HIV infection, and lower gag diversity was observed in men who had HIV drug resistance. HIV pol diversification was more pronounced in men with acute or recent infection, higher CD4 cell count, and lower HIV viral load.

  18. Appearance of drug resistance mutations among the dominant HIV-1 subtype, CRF01_AE in Maumere, Indonesia.

    PubMed

    Indriati, Dwi Wahyu; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Witaningrum, Adiana Mutamsari; Matondang, Muhammad Qushai Yunifiar; Ueda, Shuhei; Nasronudin; Purnama, Asep; Kurniawan, Dwi; Kameoka, Masanori

    2018-05-01

    Human Immunodeficiency Virus (HIV) is still a major health issue in Indonesia. In recent years, the appearance of drug resistance-associated mutations has reduced the effectiveness of antiretroviral therapy (ART). We conducted genotypic studies, including the detection of drug resistance-associated mutations (from first-line regimen drugs), on HIV-1 genes derived from infected individuals in Maumere, West Nusa Tenggara. Maumere, a transit city in West Nusa Tenggara, which has a high HIV-1 transmission rate. We collected 60 peripheral blood samples from 53 ART-experienced and 7 ART-naive individuals at TC Hillers Hospital, Maumere between 2014 and 2015. The amplification and a sequencing analysis of pol genes encoding protease (the PR gene) and reverse transcriptase (the RT gene) as well as the viral env and gag genes were performed. HIV-1 subtyping and the detection of drug resistance-associated mutations were then conducted. Among 60 samples, 46 PR, 31 RT, 30 env, and 20 gag genes were successfully sequenced. The dominant HIV-1 subtype circulating in Maumere was CRF01_AE. Subtype B and recombinant viruses containing gene fragments of CRF01_AE, subtypes A, B, C, and/or G were also identified as minor populations. The major drug resistance-associated mutations, M184V, K103N, Y188L, and M230I, were found in the RT genes. However, no major drug resistance-associated mutations were detected in the PR genes. CRF01_AE was the major HIV-1 subtype prevalent in Maumere. The appearance of drug resistance-associated mutations found in the present study supports the necessity of monitoring the effectiveness of ART in Maumere. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium.

    PubMed

    Peifer, Andrew C; Maxwell, Patrick H

    2018-03-21

    Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1 , and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons.

  20. Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium

    PubMed Central

    Peifer, Andrew C.

    2018-01-01

    Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1, and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons. PMID:29562219

  1. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

    2016-01-01

    HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

  2. Anti-HERV-K (HML-2) capsid antibody responses in HIV elite controllers.

    PubMed

    de Mulder, Miguel; SenGupta, Devi; Deeks, Steven G; Martin, Jeffrey N; Pilcher, Christopher D; Hecht, Frederick M; Sacha, Jonah B; Nixon, Douglas F; Michaud, Henri-Alexandre

    2017-08-22

    Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome and while the majority are transcriptionally silent, the most recently integrated HERV, HERV-K (HML-2), remains active. During HIV infection, HERV-K (HML-2) specific mRNA transcripts and viral proteins can be detected. In this study, we aimed to understand the antibody response against HERV-K (HML-2) Gag in the context of HIV-1 infection. We developed an ELISA assay using either recombinant protein or 164 redundant "15mer" HERV-K (HML-2) Gag peptides to test sera for antibody reactivity. We identified a total of eight potential HERV-K (HML-2) Gag immunogenic domains: two on the matrix (peptides 16 and 31), one on p15 (peptide 85), three on the capsid (peptides 81, 97 and 117), one on the nucleocapsid (peptide 137) and one on the QP1 protein (peptide 157). Four epitopes (peptides 16, 31, 85 and 137) were highly immunogenic. No significant differences in antibody responses were found between HIV infected participants (n = 40) and uninfected donors (n = 40) for 6 out of the 8 epitopes tested. The antibody response against nucleocapsid (peptide 137) was significantly lower (p < 0.001), and the response to QP1 (peptide 157) significantly higher (p < 0.05) in HIV-infected adults compared to uninfected individuals. Among those with HIV infection, the level of response against p15 protein (peptide 85) was significantly lower in untreated individuals controlling HIV ("elite" controllers) compared to untreated non-controllers (p < 0.05) and uninfected donors (p < 0.05). In contrast, the response against the capsid protein (epitopes 81 and 117) was significantly higher in controllers compared to uninfected donors (p < 0.001 and <0.05 respectively) and non-controllers (p < 0.01 and <0.05). Peripheral blood mononuclear cells (PBMCs) from study participants were tested for responses against HERV-K (HML-2) capsid recombinant peptide in gamma interferon (IFN-γ) enzyme immunospot

  3. Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

    PubMed Central

    Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

    1984-01-01

    The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

  4. Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection

    PubMed Central

    Sunshine, Justine E.; Larsen, Brendan B.; Maust, Brandon; Casey, Ellie; Deng, Wenje; Chen, Lennie; Westfall, Dylan H.; Kim, Moon; Zhao, Hong; Ghorai, Suvankar; Lanxon-Cookson, Erinn; Rolland, Morgane; Collier, Ann C.; Maenza, Janine; Mullins, James I.

    2015-01-01

    ABSTRACT To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used

  5. Novel HIV IL-4R antagonist vaccine strategy can induce both high avidity CD8 T and B cell immunity with greater protective efficacy.

    PubMed

    Jackson, Ronald J; Worley, Matthew; Trivedi, Shubhanshi; Ranasinghe, Charani

    2014-09-29

    We have established that the efficacy of a heterologous poxvirus vectored HIV vaccine, fowlpox virus (FPV)-HIV gag/pol prime followed by attenuated vaccinia virus (VV)-HIV gag/pol booster immunisation, is strongly influenced by the cytokine milieu at the priming vaccination site, with endogenous IL-13 detrimental to the quality of the HIV specific CD8+ T cell response induced. We have now developed a novel HIV vaccine that co-expresses a C-terminal deletion mutant of the mouse IL-4, deleted for the essential tyrosine (Y119) required for signalling. In our vaccine system, the mutant IL-4C118 can bind to IL-4 type I and II receptors with high affinity, and transiently prevent the signalling of both IL-4 and IL-13 at the vaccination site. When this IL-4C118 adjuvanted vaccine was used in an intranasal rFPV/intramuscular rVV prime-boost immunisation strategy, greatly enhanced mucosal/systemic HIV specific CD8+ T cells with higher functional avidity, expressing IFN-γ, TNF-α and IL-2 and greater protective efficacy were detected. Surprisingly, the IL-4C118 adjuvanted vaccines also induced robust long-lived HIV gag-specific serum antibody responses, specifically IgG1 and IgG2a. The p55-gag IgG2a responses induced were of a higher magnitude relative to the IL-13Rα2 adjuvant vaccine. More interestingly, our recently tested IL-13Rα2 adjuvanted vaccine which only inhibited IL-13 activity, even though induced excellent high avidity HIV-specific CD8+ T cells, had a detrimental impact on the induction of gag-specific IgG2a antibody immunity. Our observations suggest that (i) IL-4 cell-signalling in the absence of IL-13 retarded gag-specific antibody isotype class switching, or (ii) IL-13Rα2 signalling was involved in inducing good gag-specific B cell immunity. Thus, we believe our novel IL-4R antagonist adjuvant strategy offers great promise not only for HIV-1 vaccines, but also against a range of chronic infections where sustained high quality mucosal and systemic T and B

  6. A newly emerging HIV-1 recombinant lineage (CRF58_01B) disseminating among people who inject drugs in Malaysia.

    PubMed

    Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2014-01-01

    The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B-D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes--subtype B (including subtype B', the Thai variant of subtype B), CRF01_AE, and CRF33_01B--have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B' and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention.

  7. A Newly Emerging HIV-1 Recombinant Lineage (CRF58_01B) Disseminating among People Who Inject Drugs in Malaysia

    PubMed Central

    Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2014-01-01

    The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B–D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes—subtype B (including subtype B′, the Thai variant of subtype B), CRF01_AE, and CRF33_01B—have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B′ and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention. PMID:24465513

  8. N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.

    Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

  9. N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

    DOE PAGES

    Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.; ...

    2015-10-23

    Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

  10. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

    PubMed Central

    2011-01-01

    Background The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C

  11. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

    PubMed

    George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

    2011-05-09

    The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and

  12. tRNAs promote nuclear import of HIV-1 intracellular reverse transcription complexes.

    PubMed

    Zaitseva, Lyubov; Myers, Richard; Fassati, Ariberto

    2006-10-01

    Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3' CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV gag did not incorporate tRNA species capable of inducing HIV-1 RTC nuclear import and failed to infect cell cycle-arrested cells. Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import.

  13. Uncommon Pathways of Immune Escape Attenuate HIV-1 Integrase Replication Capacity

    PubMed Central

    Chopera, Denis R.; Olvera, Alex; Brumme, Chanson J.; Sela, Jennifer; Markle, Tristan J.; Martin, Eric; Carlson, Jonathan M.; Le, Anh Q.; McGovern, Rachel; Cheung, Peter K.; Kelleher, Anthony D.; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P. Richard; Heckerman, David; Brander, Christian; Walker, Bruce D.; Brumme, Zabrina L.

    2012-01-01

    An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies. PMID:22496233

  14. Uncommon pathways of immune escape attenuate HIV-1 integrase replication capacity.

    PubMed

    Brockman, Mark A; Chopera, Denis R; Olvera, Alex; Brumme, Chanson J; Sela, Jennifer; Markle, Tristan J; Martin, Eric; Carlson, Jonathan M; Le, Anh Q; McGovern, Rachel; Cheung, Peter K; Kelleher, Anthony D; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P Richard; Heckerman, David; Brander, Christian; Walker, Bruce D; Brumme, Zabrina L

    2012-06-01

    An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.

  15. HIV-1 adaptation studies reveal a novel Env-mediated homeostasis mechanism for evading lethal hypermutation by APOBEC3G

    PubMed Central

    Ikeda, Terumasa; Albin, John S.; Li, Ming; Thali, Markus

    2018-01-01

    HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs. PMID:29677220

  16. Barriers and Facilitators to HIV Testing Among Zambian Female Sex Workers in Three Transit Hubs.

    PubMed

    Chanda, Michael M; Perez-Brumer, Amaya G; Ortblad, Katrina F; Mwale, Magdalene; Chongo, Steven; Kamungoma, Nyambe; Kanchele, Catherine; Fullem, Andrew; Barresi, Leah; Bärnighausen, Till; Oldenburg, Catherine E

    2017-07-01

    Zambia has a generalized HIV epidemic, and HIV is concentrated along transit routes. Female sex workers (FSWs) are disproportionately affected by the epidemic. HIV testing is the crucial first step for engagement in HIV care and HIV prevention activities. However, to date little work has been done with FSWs in Zambia, and little is known about barriers and facilitators to HIV testing in this population. FSW peer educators were recruited through existing sex worker organizations for participation in a trial related to HIV testing among FSWs. We conducted five focus groups with FSW peer educators (N = 40) in three transit towns in Zambia (Livingstone, Chirundu, and Kapiri Mposhi) to elicit community norms related to HIV testing. Emerging themes demonstrated barriers and facilitators to HIV testing occurring at multiple levels, including individual, social network, and structural. Stigma and discrimination, including healthcare provider stigma, were a particularly salient barrier. Improving knowledge, social support, and acknowledgment of FSWs and women's role in society emerged as facilitators to testing. Interventions to improve HIV testing among FSWs in Zambia will need to address barriers and facilitators at multiple levels to be maximally effective.

  17. Exosomes from uninfected cells activate transcription of latent HIV-1.

    PubMed

    Barclay, Robert A; Schwab, Angela; DeMarino, Catherine; Akpamagbo, Yao; Lepene, Benjamin; Kassaye, Seble; Iordanskiy, Sergey; Kashanchi, Fatah

    2017-07-14

    HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR- gag , can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo , especially in the presence of combination antiretroviral therapy.

  18. Population dynamics of HIV-1 inferred from gene sequences.

    PubMed Central

    Grassly, N C; Harvey, P H; Holmes, E C

    1999-01-01

    A method for the estimation of population dynamic history from sequence data is described and used to investigate the past population dynamics of HIV-1 subtypes A and B. Using both gag and env gene alignments the effective population size of each subtype is estimated and found to be surprisingly small. This may be a result of the selective sweep of mutations through the population, or may indicate an important role of genetic drift in the fixation of mutations. The implications of these results for the spread of drug-resistant mutations and transmission dynamics, and also the roles of selection and recombination in shaping HIV-1 genetic diversity, are discussed. A larger estimated effective population size for subtype A may be the result of differences in time of origin, transmission dynamics, and/or population structure. To investigate the importance of population structure a model of population subdivision was fitted to each subtype, although the improvement in likelihood was found to be nonsignificant. PMID:9927440

  19. Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial

    PubMed Central

    Saxena, Deepti; Spino, Michael; Tricta, Fernando; Connelly, John; Cracchiolo, Bernadette M.; Hanauske, Axel-Rainer; D’Alliessi Gandolfi, Darlene; Mathews, Michael B.; Karn, Jonathan; Holland, Bart; Park, Myung Hee; Pe’ery, Tsafi; Palumbo, Paul E.; Hanauske-Abel, Hartmut M.

    2016-01-01

    cluster (‘polyproline’)-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound. Trial Registration: ClinicalTrial.gov NCT02191657 PMID:27191165

  20. Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

    PubMed Central

    Mboumba Bouassa, Ralph-Sydney; Jenabian, Mohammad-Ali; Wolyec, Serge Tonen; Robin, Leman; Matta, Mathieu; Longo, Jean de Dieu; Grésenguet, Gérard; Andreoletti, Laurent; Bélec, Laurent

    2016-01-01

    Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France), combining automated station extraction (Amplix station 16 Dx) and real-time PCR (Amplix NG), for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL), across wide dynamic range (1.4–10 log copies/mL), 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche), with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa. PMID:28050283

  1. Addressing unmet need for HIV testing in emergency care settings: a role for computer-facilitated rapid HIV testing?

    PubMed

    Kurth, Ann E; Severynen, Anneleen; Spielberg, Freya

    2013-08-01

    HIV testing in emergency departments (EDs) remains underutilized. The authors evaluated a computer tool to facilitate rapid HIV testing in an urban ED. Randomly assigned nonacute adult ED patients were randomly assigned to a computer tool (CARE) and rapid HIV testing before a standard visit (n = 258) or to a standard visit (n = 259) with chart access. The authors assessed intervention acceptability and compared noted HIV risks. Participants were 56% nonWhite and 58% male; median age was 37 years. In the CARE arm, nearly all (251/258) of the patients completed the session and received HIV results; four declined to consent to the test. HIV risks were reported by 54% of users; one participant was confirmed HIV-positive, and two were confirmed false-positive (seroprevalence 0.4%, 95% CI [0.01, 2.2]). Half (55%) of the patients preferred computerized rather than face-to-face counseling for future HIV testing. In the standard arm, one HIV test and two referrals for testing occurred. Computer-facilitated HIV testing appears acceptable to ED patients. Future research should assess cost-effectiveness compared with staff-delivered approaches.

  2. HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication.

    PubMed

    Duchon, Alice A; St Gelais, Corine; Titkemeier, Nathan; Hatterschide, Joshua; Wu, Li; Musier-Forsyth, Karin

    2017-11-01

    A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNA Lys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNA Lys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication. IMPORTANCE Human tRNA Lys3 , the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNA Lys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNA Lys3 and Lys

  3. Characterization of a New HIV-1 CRF01_AE/ CRF07_BC recombinant virus in Tianjin, China.

    PubMed

    Zhou, Zhehua; Ma, Ping; Feng, Yi; Ou, Weidong; Qian, Jing; Gao, Liying; Zhang, Defa; Shao, Yiming; Wei, Min

    2018-05-04

    Human immunodeficiency virus (HIV) is notorious for its rapid evolving since its transmissions from money to human. Currently, HIV contains multiple subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Here, from an HIV-positive mother and her child in Tianjin, China, we identified a novel HIV-1 second-generation recombinant virus (TJ20170316 and TJ20170317) between CRF01_AE and CRF07_BC. Near full-length genomes were obtained from both samples, and they shared very close sequences, except some point mutations. Phylogenetic analyses of the near full-length genomes showed that they consist of CRF01_AE backbone and part CRF07_BC sequences. Recombinant Identification Program (RIP) and Simplot software identified four breakpoints in gag, pol, vif, tat genes in TJ20170316, totally different from other reported CRFs and URFs. The emergence of such URF in Tianjin, China, highlights the complexity of HIV-1 epidemic and more measures should be taken to prevent HIV transmissions.

  4. Balance between transmitted HLA preadapted and nonassociated polymorphisms is a major determinant of HIV-1 disease progression.

    PubMed

    Mónaco, Daniela C; Dilernia, Dario A; Fiore-Gartland, Andrew; Yu, Tianwei; Prince, Jessica L; Dennis, Kristine K; Qin, Kai; Schaefer, Malinda; Claiborne, Daniel T; Kilembe, William; Tang, Jianming; Price, Matt A; Farmer, Paul; Gilmour, Jill; Bansal, Anju; Allen, Susan; Goepfert, Paul; Hunter, Eric

    2016-09-19

    HIV-1 adapts to a new host through mutations that facilitate immune escape. Here, we evaluate the impact on viral control and disease progression of transmitted polymorphisms that were either preadapted to or nonassociated with the new host's HLA. In a cohort of 169 Zambian heterosexual transmission pairs, we found that almost one-third of possible HLA-linked target sites in the transmitted virus Gag protein are already adapted, and that this transmitted preadaptation significantly reduced early immune recognition of epitopes. Transmitted preadapted and nonassociated polymorphisms showed opposing effects on set-point VL and the balance between the two was significantly associated with higher set-point VLs in a multivariable model including other risk factors. Transmitted preadaptation was also significantly associated with faster CD4 decline (<350 cells/µl) and this association was stronger after accounting for nonassociated polymorphisms, which were linked with slower CD4 decline. Overall, the relative ratio of the two classes of polymorphisms was found to be the major determinant of CD4 decline in a multivariable model including other risk factors. This study reveals that, even before an immune response is mounted in the new host, the balance of these opposing factors can significantly influence the outcome of HIV-1 infection. © 2016 Mónaco et al.

  5. Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

    PubMed Central

    Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2016-01-01

    The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem–loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR–cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59. PMID:26826129

  6. Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

    PubMed Central

    Hajek, Kathryn L.; Friesen, Paul D.

    1998-01-01

    TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

  7. Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

    PubMed

    Hajek, K L; Friesen, P D

    1998-11-01

    TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

  8. Recombination elevates the effective evolutionary rate and facilitates the establishment of HIV-1 infection in infants after mother-to-child transmission

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sanborn, Keri B.; Somasundaran, Mohan; Luzuriaga, Katherine

    Some previous studies have demonstrated that single HIV-1 genotypes are commonly transmitted from mother to child, but such analyses primarily used single samples from mother and child. It is possible that in a single sample, obtained early after infection, only the most replication competent virus is detected even when other forms may have been transmitted. Such forms may have advantages later in infection, and may thus be detected in follow-up samples. Furthermore, because HIV-1 frequently recombines, phylogenetic analyses that ignore recombination may miss transmission of multiple forms if they recombine after transmission. Moreover, recombination may facilitate adaptation, thus providing anmore » advantage in establishing infection. The effect of recombination on viral evolution in HIV-1 infected children has not been well defined.« less

  9. Recombination elevates the effective evolutionary rate and facilitates the establishment of HIV-1 infection in infants after mother-to-child transmission

    DOE PAGES

    Sanborn, Keri B.; Somasundaran, Mohan; Luzuriaga, Katherine; ...

    2015-11-16

    Some previous studies have demonstrated that single HIV-1 genotypes are commonly transmitted from mother to child, but such analyses primarily used single samples from mother and child. It is possible that in a single sample, obtained early after infection, only the most replication competent virus is detected even when other forms may have been transmitted. Such forms may have advantages later in infection, and may thus be detected in follow-up samples. Furthermore, because HIV-1 frequently recombines, phylogenetic analyses that ignore recombination may miss transmission of multiple forms if they recombine after transmission. Moreover, recombination may facilitate adaptation, thus providing anmore » advantage in establishing infection. The effect of recombination on viral evolution in HIV-1 infected children has not been well defined.« less

  10. Facilitating and supporting HIV+ parenthood: Lessons for developing the advocate role of voluntary HIV support services workers.

    PubMed

    Cane, Tam Pheona Chipawe

    2018-06-01

    Increasingly as people living with HIV (PLWHIV) aim to become parents, they engage with HIV voluntary services for support through either fertility or adoption services. Yet, little is known about the role of HIV support services workers in facilitating access to fertility treatment or child adoption. The purpose of this study was to explore the role of HIV support workers based in HIV voluntary organisations who have a key role helping PLWHIV in navigating relevant fertility and adoption processes. This was an exploratory qualitative study which involved interviewing six HIV support workers, from across the UK. Interviews were conducted using face to face interviews, recorded and transcribed. Findings revealed that HIV services support workers provide practical support in advocating service provision, and emotional and social support along the journey. They also face challenges in their role from health care professionals including information sharing and gatekeeping. The role of HIV support workers is important in facilitating access to resources and complex systems. HIV support workers should be recognised and as they are often a trusted professional to address stigma, discrimination and barriers to services. The study contributes to research seeking to understand the emerging needs and support requirements for people living with HIV seeking fertility and adoption. Further work in this area is warranted. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein.

    PubMed

    Kelly, Brian N; Kyere, Sampson; Kinde, Isaac; Tang, Chun; Howard, Bruce R; Robinson, Howard; Sundquist, Wesley I; Summers, Michael F; Hill, Christopher P

    2007-10-19

    The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.

  12. Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar.

    PubMed

    Motomura, K; Kusagawa, S; Kato, K; Nohtomi, K; Lwin, H H; Tun, K M; Thwe, M; Oo, K Y; Lwin, S; Kyaw, O; Zaw, M; Nagai, Y; Takebe, Y

    2000-11-20

    We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.

  13. A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

    PubMed Central

    Eckhardt, Manon; Anders, Maria; Muranyi, Walter; Heilemann, Mike; Krijnse-Locker, Jacomine; Müller, Barbara

    2011-01-01

    Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIVSNAP, which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. PMID:21799764

  14. Reduction of the HIV-1 reservoir in resting CD4+ T-lymphocytes by high dosage intravenous immunoglobulin treatment: a proof-of-concept study.

    PubMed

    Lindkvist, Annica; Edén, Arvid; Norström, Melissa M; Gonzalez, Veronica D; Nilsson, Staffan; Svennerholm, Bo; Karlsson, Annika C; Sandberg, Johan K; Sönnerborg, Anders; Gisslén, Magnus

    2009-07-01

    The latency of HIV-1 in resting CD4+ T-lymphocytes constitutes a major obstacle for the eradication of virus in patients on antiretroviral therapy (ART). As yet, no approach to reduce this viral reservoir has proven effective. Nine subjects on effective ART were included in the study and treated with high dosage intravenous immunoglobulin (IVIG) for five consecutive days. Seven of those had detectable levels of replication-competent virus in the latent reservoir and were thus possible to evaluate. Highly purified resting memory CD4+ T-cells were activated and cells containing replication-competent HIV-1 were quantified. HIV-1 from plasma and activated memory CD4+ T-cells were compared with single genome sequencing (SGS) of the gag region. T-lymphocyte activation markers and serum interleukins were measured. The latent HIV-1 pool decreased with in median 68% after IVIG was added to effective ART. The reservoir decreased in five, whereas no decrease was found in two subjects with detectable virus. Plasma HIV-1 RNA >or= 2 copies/mL was detected in five of seven subjects at baseline, but in only one at follow-up after 8-12 weeks. The decrease of the latent HIV-1 pool and the residual plasma viremia was preceded by a transitory low-level increase in plasma HIV-1 RNA and serum interleukin 7 (IL-7) levels, and followed by an expansion of T regulatory cells. The magnitude of the viral increase in plasma correlated to the size of the latent HIV-1 pool and SGS of the gag region showed that viral clones from plasma clustered together with virus from activated memory T-cells, pointing to the latent reservoir as the source of HIV-1 RNA in plasma. The findings from this uncontrolled proof-of-concept study suggest that the reservoir became accessible by IVIG treatment through activation of HIV-1 gene expression in latently-infected resting CD4+ T-cells. We propose that IVIG should be further evaluated as an adjuvant to effective ART.

  15. The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

    PubMed

    Watanabe, Susan M; Simon, Viviana; Durham, Natasha D; Kemp, Brittney R; Machihara, Satoshi; Kemal, Kimdar Sherefa; Shi, Binshan; Foley, Brian; Li, Hongru; Chen, Benjamin K; Weiser, Barbara; Burger, Harold; Anastos, Kathryn; Chen, Chaoping; Carter, Carol A

    2016-09-06

    The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

  16. HIV-1 transmission networks in high risk fishing communities on the shores of Lake Victoria in Uganda: A phylogenetic and epidemiological approach

    PubMed Central

    Kiwuwa-Muyingo, Sylvia; Nazziwa, Jamirah; Ssemwanga, Deogratius; Ilmonen, Pauliina; Ndembi, Nicaise; Parry, Chris; Kitandwe, Paul Kato; Gershim, Asiki; Mpendo, Juliet; Neilsen, Leslie; Seeley, Janet; Seppälä, Heikki; Lyagoba, Fred; Kamali, Anatoli; Kaleebu, Pontiano

    2017-01-01

    Background Fishing communities around Lake Victoria in sub-Saharan Africa have been characterised as a population at high risk of HIV-infection. Methods Using data from a cohort of HIV-positive individuals aged 13–49 years, enrolled from 5 fishing communities on Lake Victoria between 2009–2011, we sought to identify factors contributing to the epidemic and to understand the underlying structure of HIV transmission networks. Clinical and socio-demographic data were combined with HIV-1 phylogenetic analyses. HIV-1 gag-p24 and env-gp-41 sub-genomic fragments were amplified and sequenced from 283 HIV-1-infected participants. Phylogenetic clusters with ≥2 highly related sequences were defined as transmission clusters. Logistic regression models were used to determine factors associated with clustering. Results Altogether, 24% (n = 67/283) of HIV positive individuals with sequences fell within 34 phylogenetically distinct clusters in at least one gene region (either gag or env). Of these, 83% occurred either within households or within community; 8/34 (24%) occurred within household partnerships, and 20/34 (59%) within community. 7/12 couples (58%) within households clustered together. Individuals in clusters with potential recent transmission (11/34) were more likely to be younger 71% (15/21) versus 46% (21/46) in un-clustered individuals and had recently become resident in the community 67% (14/21) vs 48% (22/46). Four of 11 (36%) potential transmission clusters included incident-incident transmissions. Independently, clustering was less likely in HIV subtype D (adjusted Odds Ratio, aOR = 0.51 [95% CI 0.26–1.00]) than A and more likely in those living with an HIV-infected individual in the household (aOR = 6.30 [95% CI 3.40–11.68]). Conclusions A large proportion of HIV sexual transmissions occur within house-holds and within communities even in this key mobile population. The findings suggest localized HIV transmissions and hence a potential benefit for the

  17. HIV-1 transmission networks in high risk fishing communities on the shores of Lake Victoria in Uganda: A phylogenetic and epidemiological approach.

    PubMed

    Kiwuwa-Muyingo, Sylvia; Nazziwa, Jamirah; Ssemwanga, Deogratius; Ilmonen, Pauliina; Njai, Harr; Ndembi, Nicaise; Parry, Chris; Kitandwe, Paul Kato; Gershim, Asiki; Mpendo, Juliet; Neilsen, Leslie; Seeley, Janet; Seppälä, Heikki; Lyagoba, Fred; Kamali, Anatoli; Kaleebu, Pontiano

    2017-01-01

    Fishing communities around Lake Victoria in sub-Saharan Africa have been characterised as a population at high risk of HIV-infection. Using data from a cohort of HIV-positive individuals aged 13-49 years, enrolled from 5 fishing communities on Lake Victoria between 2009-2011, we sought to identify factors contributing to the epidemic and to understand the underlying structure of HIV transmission networks. Clinical and socio-demographic data were combined with HIV-1 phylogenetic analyses. HIV-1 gag-p24 and env-gp-41 sub-genomic fragments were amplified and sequenced from 283 HIV-1-infected participants. Phylogenetic clusters with ≥2 highly related sequences were defined as transmission clusters. Logistic regression models were used to determine factors associated with clustering. Altogether, 24% (n = 67/283) of HIV positive individuals with sequences fell within 34 phylogenetically distinct clusters in at least one gene region (either gag or env). Of these, 83% occurred either within households or within community; 8/34 (24%) occurred within household partnerships, and 20/34 (59%) within community. 7/12 couples (58%) within households clustered together. Individuals in clusters with potential recent transmission (11/34) were more likely to be younger 71% (15/21) versus 46% (21/46) in un-clustered individuals and had recently become resident in the community 67% (14/21) vs 48% (22/46). Four of 11 (36%) potential transmission clusters included incident-incident transmissions. Independently, clustering was less likely in HIV subtype D (adjusted Odds Ratio, aOR = 0.51 [95% CI 0.26-1.00]) than A and more likely in those living with an HIV-infected individual in the household (aOR = 6.30 [95% CI 3.40-11.68]). A large proportion of HIV sexual transmissions occur within house-holds and within communities even in this key mobile population. The findings suggest localized HIV transmissions and hence a potential benefit for the test and treat approach even at a community

  18. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    PubMed

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  19. Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

    PubMed Central

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques

  20. Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

    PubMed

    Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

    2008-05-01

    The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

  1. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation

    PubMed Central

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth A.; Contreras, Nataly; Hertz, Marla I.; Olivares, Eduardo; Cáceres, C. Joaquín; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R.; López-Lastra, Marcelo

    2016-01-01

    The 5′leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work we examine the internal ribosome entry site (IRES) located in the 5′leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5′leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

  2. Disclosure of HIV results among discordant couples in Rakai, -Uganda: A facilitated couple counselling approach

    PubMed Central

    Kairania, Robert M.; Gray, Ronald H.; Kiwanuka, Noah; Makumbi, Fredrick; Sewankambo, Nelson K.; Serwadda, David; Nalugoda, Fred; Kigozi, Godfrey; Semanda, John; Wawer, Maria J.

    2010-01-01

    Disclosure of HIV seropositive results among HIV-discordant couples in sub-Saharan Africa is generally low. We describe a facilitated couple counselling approach to enhance disclosure among HIV-discordant couples. Using unique identifiers, 293 HIV-discordant couples were identified through retrospective linkage of married or cohabiting consenting adults individually enrolled into a cohort study and into two randomized trials of male circumcision in Rakai, Uganda. HIV discordant couples and a random sample of HIV-infected concordant and HIV-negative concordant couples (to mask HIV status) were invited to sensitization meetings to discuss the benefits of disclosure and couple counselling. HIV-infected partners were subsequently contacted to encourage HIV disclosure to their HIV uninfected partners. If the index positive partner agreed, the counsellor facilitated the disclosure of HIV results, and provided ongoing support. The proportion of disclosure was determined. 81% of HIV-positive partners in discordant relationships disclosed their status to their HIV-uninfected partners in the presence of the counsellor. The rates of disclosure were 81.3% in male HIV-positive and 80.2% in female HIV-positive discordant couples. Disclosure did not vary by age, education or occupation. In summary, disclosure of HIV-positive results in discordant couples using facilitated couple counselling approach is high, but requires a stepwise process of sensitization and agreement by the infected partner. PMID:20824557

  3. Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1.

    PubMed

    Van Baelen, Kurt; Salzwedel, Karl; Rondelez, Evelien; Van Eygen, Veerle; De Vos, Stephanie; Verheyen, Ann; Steegen, Kim; Verlinden, Yvan; Allaway, Graham P; Stuyver, Lieven J

    2009-05-01

    In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

  4. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process.

    PubMed

    Adamson, Catherine S

    2012-01-01

    Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR), which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs) have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM) the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

  5. Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

    PubMed

    Re, Maria Carla; Schiavone, Pasqua; Bon, Isabella; Vitone, Francesca; De Crignis, Elisa; Biagetti, Carlo; Gibellini, Davide

    2010-11-01

    To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection. Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  6. The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid.

    PubMed

    Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

    2004-06-29

    Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

  7. Understanding the HIV-1 protease nelfinavir resistance mutation D30N in subtypes B and C through molecular dynamics simulations.

    PubMed

    Soares, Rosemberg O; Batista, Paulo R; Costa, Mauricio G S; Dardenne, Laurent E; Pascutti, Pedro G; Soares, Marcelo A

    2010-09-01

    A major concern in the antiretroviral (ARV) treatment of HIV infections with protease inhibitors (PI) is the emergence of resistance, which results from the selection of distinct mutations within the viral protease (PR) gene. Among patients who do not respond to treatment with the PI nelfinavir (NFV), the D30N mutation is often observed. However, several reports have shown that D30N emerges with different frequencies in distinct HIV-1 genetic forms or subtypes. In the present work, we analyzed the binding of NFV and the Gag substrate CA/p2 to PR from HIV-1 subtypes B and C through molecular dynamics (MD) simulations. The wild-type and drug-resistant D30N mutants were investigated in both subtypes. The compensatory mutations N83T and N88D, observed in vitro and in vivo when subtype C acquires D30N, were also studied. D30N appears to facilitate conformational changes in subtype B PR, but not in that from subtype C, and this could be associated with disestablishment of an alpha-helical region of the PR. Furthermore, the total contact areas of NFV or the CA/p2 substrate with the mutant PR correlated with changes in the resistance patterns and replicative capacity. Finally, we observed in our MD simulations that mutant PR proteins show different patterns for hydrophobic/van der Waals contact. These findings suggest that different molecular mechanisms contribute to resistance, and we propose that a single mutation has distinct impacts on different HIV-1 subtypes. (c) 2010 Elsevier Inc. All rights reserved.

  8. Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr.

    PubMed

    Datta, Prasun K; Deshmane, Satish; Khalili, Kamel; Merali, Salim; Gordon, John C; Fecchio, Chiara; Barrero, Carlos A

    2016-09-01

    HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.

  9. Gag grouper larvae pathways on the West Florida Shelf

    NASA Astrophysics Data System (ADS)

    Weisberg, Robert H.; Zheng, Lianyuan; Peebles, Ernst

    2014-10-01

    A numerical circulation model, quantitatively assessed against in situ observations, is used to describe the circulation on the West Florida Continental Shelf during spring 2007 when pre-settlement gag (Mycteroperca microlepis) were present in the surf zone near Tampa Bay, Florida. The pre-settlement fish were found to be isotopically distinct from settled juveniles in the area, which is consistent with recent arrival at near shore nursery habitats from offshore spawning grounds. Simulated particle trajectories are employed to test hypotheses relating to either a surface or a near-bottom route of across-shelf transport. The surface-route hypothesis is rejected, whereas the bottom-route hypothesis is found to be consistent with the location of pre-settlement fish and their co-occurrence with macroalgae of offshore, hard-bottom origin. We conclude that gag larvae are transported to the near shore via the bottom Ekman layer and that such transport is facilitated by remote forcing associated with Gulf of Mexico Loop Current interactions with the shelf slope near the Dry Tortugas. Being that such remote forcing occurs inter-annually and not always in phase with the preferred spawning months (late winter through early spring), gag recruitment success should similarly vary with year and location.

  10. Perceptions of Barriers and Facilitators to Cervical Cancer Screening among Low-Income, HIV-Infected Women from an Integrated HIV Clinic

    PubMed Central

    Buchberg, Meredith; Schover, Leslie; Basen-Engquist, Karen; Kempf, Mirjam-Colette; Arduino, Roberto C.; Vidrine, Damon J.

    2014-01-01

    Significantly elevated rates of cervical cancer and low rates of Papanicolaou (Pap) smear screening have been documented among HIV-infected women. However, little is known about women’s perceptions of cervical cancer screening utilization. Hence, this study describes barriers and facilitators related to cervical cancer screening in a sample of HIV-infected women seeking care at an integrated HIV clinic in Houston, Texas. Using an inductive qualitative methodological approach, data were obtained from five focus group discussions with a total of 33, HIV-infected women. The majority of the study sample consisted of women who self-identified as Black (69.7%), and reported heterosexual contact as the mode of HIV acquisition (75.8%). Barriers to cervical cancer screening were described as pain and discomfort associated with receiving Pap smears and subsequent procedures; lack of awareness of cervical cancer as a preventable disease; limited transportation access; and systemic issues as it relates to scheduling gynecological appointments. Facilitators were described as awareness of HIV-infected women’s increased risk of cervical cancer and strong provider-patient relationships. To address disparities in cervical cancer screening among low-income HIV-infected women, programs should capitalize on the identified facilitators and alleviate modifiable barriers using multi-level strategies. PMID:24635664

  11. Ectopic expression of anti-HIV-1 shRNAs protects CD8{sup +} T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamata, Masakazu, E-mail: masa3k@ucla.edu; Kim, Patrick Y.; Ng, Hwee L.

    Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8{sup +} T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To testmore » this possibility, highly purified CD8{sup +} T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8{sup +} T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24{sup Gag} in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8{sup +} T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8{sup +} T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8{sup +} T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. - Highlights: • Ectopic expression of CD4ζ CAR in CD8{sup +} T cells renders them susceptible to HIV-1 infection. • Co-expression of two anti-HIV-1 shRNAs protects CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection. • Protecting CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection suppresses its cytopathic effect.« less

  12. Attenuated Listeria monocytogenes vectors overcome suppressive plasma factors during HIV infection to stimulate myeloid dendritic cells to promote adaptive immunity and reactivation of latent virus.

    PubMed

    Miller, Elizabeth A; Spadaccia, Meredith R; Norton, Thomas; Demmler, Morgan; Gopal, Ramya; O'Brien, Meagan; Landau, Nathaniel; Dubensky, Thomas W; Lauer, Peter; Brockstedt, Dirk G; Bhardwaj, Nina

    2015-01-01

    HIV-1 infection is characterized by myeloid dendritic cell (DC) dysfunction, which blunts the responsiveness to vaccine adjuvants. We previously showed that nonviral factors in HIV-seropositive plasma are partially responsible for mediating this immune suppression. In this study we investigated recombinant Listeria monocytogenes (Lm) vectors, which naturally infect and potently activate DCs from seronegative donors, as a means to overcome DC dysfunction associated with HIV infection. Monocyte-derived DCs were cocultured with plasma from HIV-infected donors (HIV-moDCs) to induce a dysregulated state and infected with an attenuated, nonreplicative vaccine strain of Lm expressing full length clade B consensus gag (KBMA Lm-gag). Lm infection stimulated cytokine secretion [interleukin (IL)-12p70, tumor necrosis factor (TNF)-α, and IL-6] and Th-1 skewing of allogeneic naive CD4 T cells by HIV-moDCs, in contrast to the suppressive effects observed by HIV plasma on moDCs on toll-like receptor ligand stimulation. Upon coculture of "killed" but metabolically active (KBMA) Lm-gag-infected moDCs from HIV-infected donors with autologous cells, expansion of polyfunctional, gag-specific CD8(+) T cells was observed. Reactivation of latent proviruses by moDCs following Lm infection was also observed in models of HIV latency in a TNF-α-dependent manner. These findings reveal the unique ability of Lm vectors to contend with dysregulation of HIV-moDCs, while simultaneously possessing the capacity to activate latent virus. Concurrent stimulation of innate and adaptive immunity and disruption of latency may be an approach to reduce the pool of latently infected cells during HIV infection. Further study of Lm vectors as part of therapeutic vaccination and eradication strategies may advance this evolving field.

  13. Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.

    PubMed

    Svarovskaia, Evguenia S; Xu, Hongzhan; Mbisa, Jean L; Barr, Rebekah; Gorelick, Robert J; Ono, Akira; Freed, Eric O; Hu, Wei-Shau; Pathak, Vinay K

    2004-08-20

    Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

  14. The discovery of a class of novel HIV-1 maturation inhibitors and their potential in the therapy of HIV.

    PubMed

    Yu, Donglei; Wild, Carl T; Martin, David E; Morris-Natschke, Susan L; Chen, Chin-Ho; Allaway, Graham P; Lee, Kuo-Hsiung

    2005-06-01

    Although HIV infection is now primarily treated with reverse transcriptase and protease inhibitors, HIV therapy must look toward new drugs with novel mechanism(s) of action to both improve efficacy and address the growing problem of drug resistance. Using natural products as a source of biologically active compounds, our drug discovery program has successfully optimised the natural product betulinic acid to the first-in-class maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB). DSB's unique viral target has been identified as a late step in Gag processing. Specifically, it inhibits the cleavage of the capsid precursor, CA-SP1, resulting in a block to the processing of mature capsid protein leading to a defect in viral core condensation. DSB represents a unique class of anti-HIV compounds that inhibit virus maturation and provide additional opportunities for anti-HIV therapy. In this review, the discovery of DSB and its mode of action are summarised. Anti-AIDS Agents part 64. For part 63 in the series, see YU D, LEE KH: Recent progress and prospects on plant-derived anti-HIV agents and analogs. In: Medicinal Chemistry of Bioactive Natural Products. XT Liang, WS Fang (Eds), Wiley, New York, USA (2005) (In Press).

  15. HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1–Infected Children Who Received Early Treatment

    PubMed Central

    Luzuriaga, Katherine; Tabak, Barbara; Garber, Manuel; Chen, Ya Hui; Ziemniak, Carrie; McManus, Margaret M.; Murray, Danielle; Strain, Matthew C.; Richman, Douglas D.; Chun, Tae-Wook; Cunningham, Coleen K.; Persaud, Deborah

    2014-01-01

    Background. Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)–infected infants controls HIV-1 replication and reduces mortality. Methods. Plasma viremia (lower limit of detection, <2 copies/mL), T-cell activation, HIV-1–specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1–infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth. Results. Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1–specific immune responses. Conclusions. Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies. PMID:24850788

  16. Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.

    HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different

  17. Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

    DOE PAGES

    Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; ...

    2015-12-22

    HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different

  18. Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

    PubMed Central

    Datta, Prasun K.; Deshmane, Satish; Khalili, Kamel; Merali, Salim; Gordon, John C.; Fecchio, Chiara; Barrero, Carlos A.

    2016-01-01

    ABSTRACT HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS. PMID:27245560

  19. Linking HIV-Negative Youth to Prevention Services in 12 U.S. Cities: Barriers and Facilitators to Implementing the HIV Prevention Continuum.

    PubMed

    Doll, Mimi; Fortenberry, J Dennis; Roseland, Denise; McAuliff, Kathleen; Wilson, Craig M; Boyer, Cherrie B

    2018-04-01

    Linkage of HIV-negative youth to prevention services is increasingly important with the development of effective pre-exposure prophylaxis that complements behavioral and other prevention-focused interventions. However, effective infrastructure for delivery of prevention services does not exist, leaving many programs to address HIV prevention without data to guide program development/implementation. The objective of this study was to provide a qualitative description of barriers and facilitators of linkage to prevention services among high-risk, HIV-negative youth. Thematic analysis of structured interviews with staff implementing linkage to prevention services programs for youth aged 12-24 years. Twelve adolescent medicine HIV primary care programs as part of larger testing research program focused on young sexual minority men of color. The study included staff implementing linkage to prevention services programs along with community-based HIV testing programs. The main outcomes of the study were key barriers/facilitators to linkage to prevention services. Eight themes summarized perspectives on linkage to prevention services: (1) relationships with community partners, (2) trust between providers and youth, (3) youth capacity to navigate prevention services, (4) pre-exposure prophylaxis specific issues, (5) privacy issues, (6) gaps in health records preventing tailored services, (7) confidentiality of care for youth accessing services through parents'/caretakers' insurance, and (8) need for health-care institutions to keep pace with models that prioritize HIV prevention among at-risk youth. Themes are discussed in the context of factors that facilitated/challenged linkage to prevention services. Several evidence-based HIV prevention tools are available; infrastructures for coordinated service delivery to high-risk youth have not been developed. Implementation of such infrastructures requires attention to community-, provider-, and youth-related issues. Copyright

  20. Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease.

    PubMed

    Jochmans, Dirk; Anders, Maria; Keuleers, Inge; Smeulders, Liesbeth; Kräusslich, Hans-Georg; Kraus, Günter; Müller, Barbara

    2010-10-15

    Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active

  1. Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

    PubMed Central

    2010-01-01

    Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of

  2. Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

    PubMed

    Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2016-04-01

    The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59. © 2016 Belfetmi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein*

    PubMed Central

    Vlach, Jiri; Samal, Alexandra B.; Saad, Jamil S.

    2014-01-01

    Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8–43 (MA-(8–43)). Here, we present the NMR structure of CaM bound to MA-(8–43). Our data revealed that MA-(8–43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8–43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8–43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication. PMID:24500712

  4. Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

    PubMed

    Dang, Mai T; Yokoi, Fumiaki; McNaught, Kevin St P; Jengelley, Toni-Ann; Jackson, Tehone; Li, Jianyong; Li, Yuqing

    2005-12-01

    A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.

  5. A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial

    PubMed Central

    Rolland, M.; Magaret, C.A.; Rademeyer, C.; Fiore-Gartland, A.; Edlefsen, P.T.; DeCamp, A.; Ahmed, H.; Ngandu, N.; Larsen, B.B.; Frahm, N.; Marais, J.; Thebus, R.; Geraghty, D.; Hural, J.; Corey, L.; Kublin, J.; Gray, G.; McElrath, M.J.; Mullins, J.I.; Gilbert, P.B.; Williamson, C.

    2016-01-01

    Introduction The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. Materials and methods A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. Results There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p = 0.045) and Nef (p = 0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p < 0.05) in Gag (n = 10), Pol (n = 7) and Nef (n = 10), although they did not remain significant after adjustment for multiple comparisons. We found the epitope sieve effect in Step was driven by HLA A*02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. Discussion This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes. PMID:27756485

  6. Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells.

    PubMed

    Vacharaksa, Anjalee; Asrani, Anil C; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Janoff, Edward N; Ross, Karen F; Herzberg, Mark C

    2008-07-17

    Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Deltaenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

  7. A correlate of HIV-1 control consisting of both innate and adaptive immune parameters best predicts viral load by multivariable analysis in HIV-1 infected viremic controllers and chronically-infected non-controllers.

    PubMed

    Tomescu, Costin; Liu, Qin; Ross, Brian N; Yin, Xiangfan; Lynn, Kenneth; Mounzer, Karam C; Kostman, Jay R; Montaner, Luis J

    2014-01-01

    HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4+ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8+ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8+ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8+ T cell responses) immune parameters best predicted viral load (R2 = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8+ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.

  8. Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.

    PubMed

    Jolly, Clare; Mitar, Ivonne; Sattentau, Quentin J

    2007-06-01

    Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

  9. Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

    PubMed

    Takahata, Tatsuro; Takeda, Eri; Tobiume, Minoru; Tokunaga, Kenzo; Yokoyama, Masaru; Huang, Yu-Lun; Hasegawa, Atsuhiko; Shioda, Tatsuo; Sato, Hironori; Kannagi, Mari; Masuda, Takao

    2017-01-01

    Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic

  10. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

    PubMed

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

    2016-07-01

    The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. © 2016 Federation of European Biochemical Societies.

  11. Barriers and Facilitators of HIV Care Engagement: Results of a Qualitative Study in St. Petersburg, Russia

    PubMed Central

    Kuznetsova, Anna V.; Meylakhs, Anastasia Y.; Amirkhanian, Yuri A.; Kelly, Jeffrey A.; Yakovlev, Alexey A.; Musatov, Vladimir B.; Amirkhanian, Anastasia G.

    2016-01-01

    Russia has a large HIV epidemic, but medical care engagement is low. Eighty HIV-positive persons in St. Petersburg completed in-depth interviews to identify barriers and facilitators of medical HIV care engagement. The most commonly-reported barriers involved difficulties accessing care providers, dissatisfaction with the quality of services, and negative attitudes of provider staff. Other barriers included not having illness symptoms, life stresses, low value placed on health, internalized stigma and wanting to hide one’s HIV status, fears of learning about one’s true health status, and substance abuse. Care facilitators were feeling responsible for one’s health and one’s family, care-related support from other HIV-positive persons, and the onset of health decline and fear of death. Substance use remission facilitated care engagement, as did good communication from providers and trust in one’s doctor. Interventions are needed in Russia to address HIV care infrastructural barriers and integrate HIV, substance abuse, care, and psychosocial services. PMID:26767534

  12. Barriers and Facilitators of HIV Care Engagement: Results of a Qualitative Study in St. Petersburg, Russia.

    PubMed

    Kuznetsova, Anna V; Meylakhs, Anastasia Y; Amirkhanian, Yuri A; Kelly, Jeffrey A; Yakovlev, Alexey A; Musatov, Vladimir B; Amirkhanian, Anastasia G

    2016-10-01

    Russia has a large HIV epidemic, but medical care engagement is low. Eighty HIV-positive persons in St. Petersburg completed in-depth interviews to identify barriers and facilitators of medical HIV care engagement. The most commonly-reported barriers involved difficulties accessing care providers, dissatisfaction with the quality of services, and negative attitudes of provider staff. Other barriers included not having illness symptoms, life stresses, low value placed on health, internalized stigma and wanting to hide one's HIV status, fears of learning about one's true health status, and substance abuse. Care facilitators were feeling responsible for one's health and one's family, care-related support from other HIV-positive persons, and the onset of health decline and fear of death. Substance use remission facilitated care engagement, as did good communication from providers and trust in one's doctor. Interventions are needed in Russia to address HIV care infrastructural barriers and integrate HIV, substance abuse, care, and psychosocial services.

  13. Longitudinal studies on maternal HIV-1 variants by biological phenotyping, sequence analysis and viral load.

    PubMed

    Renta, J Y; Cadilla, C L; Vega, M E; Hillyer, G V; Estrada, C; Jiménez, E; Abreu, E; Méndez, I; Gandía, J; Meléndez-Guerrero, L M

    1997-11-01

    In this study, the HIV-1 variant viruses from ten pregnant women and their infants were isolated and characterized longitudinally in order to determine the role that viral envelope (gp120-V3 loop) gene variation and viral tropism play in vertical transmission. Biological phenotyping of each HIV variant was accomplished by growth in MT-2, and macrophages from healthy and non-HIV-infected donors. Genetic characterization of the variants was accomplished by DNA sequence analysis. All the women enrolled in this study received ZDV therapy. Virus was cultured from eight out of ten env V3-PCR positive mothers. HIV-1 isolates were all non-syncitium inducing variants. None of the mothers were found to transmit HIV, as determined by DNA PCR and quantitative co-cultures on their infants which were seronegative for HIV-1 through one year after birth. Viral cultures from infant blood samples were negative and infants were all healthy. However, nested env V3-PCR detected proviral DNA in five out of ten infants. In contrast, conventional gag-PCR was negative in the same five infants. Sequences of the five maternal-infant pairs were different, suggesting unique infant HIV-1 variants. The three highest maternal viral load values corresponded to infants that were env V3-PCR positive. These results suggest that HIV-1 particles are transmitted from ZDV-treated mothers to infants. Infant follow up is recommended to determine if HIV-1 has been inhibited by the immune system of the infants.

  14. [Genetic subtype and epidemiological feature of HIV-1 circulating strains among recently infected patients in Fujian province].

    PubMed

    Deng, Yongyue; Zhang, Chunyang; Yan, Yansheng; Yan, Pingping; Wu, Shouli

    2014-06-01

    In order to evaluate the distribution of genetic subtypes and epidemiological feature of HIV-1 circulating strains in Fujian province. Blood samples and epidemiological data were collected from 104 newly infected patients who were distinguished by BED-CEIA methodology, during 2011-2012. Viral sequences(n = 81) of HIV-1 gag, env, and pol segments were amplified by nested PCR. Subtypes B and four Circulating Recombinant Forms, (CRF01_AE, CRF07_BC, CRF08_BC and CRF55_01B) were found in the samples, CRF01_AE(45.68%)and CRF07_BC(35.80%) were the two main HIV-1 strains in Fujian province. Compared with previous data, the proportion of CRF07_BC rose significantly while it gradually decreased in CRF01_AE. Heterosexual contact was still the principal transmission route in Fujian province, but the number of infection among men-who-have-sex-with- men grew rapidly. Results from this study suggested that different subtypes of HIV-1 strain existed in Fujian province. The distribution of subtypes and the mode of transmission were changing with the progress of epidemic. Dynamic monitoring of the molecular epidemiology trends of HIV-1 infection should be enhanced.

  15. The Spleen Is an HIV-1 Sanctuary During Combined Antiretroviral Therapy.

    PubMed

    Nolan, David J; Rose, Rebecca; Rodriguez, Patricia H; Salemi, Marco; Singer, Elyse J; Lamers, Susanna L; McGrath, Michael S

    2018-01-01

    Combined antiretroviral therapy (cART) does not eradicate HIV, which persists for years and can re-establish replication if treatment is stopped. The current challenge is identifying those tissues harboring virus through cART. Here, we used HIV env-nef single genome sequencing and HIV gag droplet digital PCR (ddPCR) to survey 50 tissues from five subjects on cART with no detectable plasma viral load at death. The spleen most consistently contained multiple proviral and expressed sequences (4/5 participants). Spleen-derived HIV demonstrated two distinct phylogenetic patterns: multiple identical sequences, often from different tissues, as well as diverse viral sequences on long terminal branches. Our results suggested that ddPCR may overestimate the size of the tissue-based viral reservoir. The spleen, a lymphatic organ at the intersection of the immune and circulatory systems, may play a key role in viral persistence.

  16. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    PubMed

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  17. The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

    PubMed Central

    Sette, Paola; Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Snyder, Greg; Smith, Patrick; Xiao, Tsan. Sam; Bouamr, Fadila

    2011-01-01

    Summary Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects due to interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical “boomerang” folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently mutation of Phe105 and surrounding residues at the tip of the loop compromises the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix. PMID:21889351

  18. The HIV hide and seek game: an immunogenomic analysis of the HIV epitope repertoire.

    PubMed

    Vider-Shalit, Tal; Almani, Michal; Sarid, Ronit; Louzoun, Yoram

    2009-07-17

    Viruses employ various means to evade immune detection. One common evasion strategy is the removal of CD8 cytotoxic T-lymphocyte (CTL) epitopes. Here, we use bioinformatic tools to compute the HIV CTL epitope repertoire presented by over 8000 HIV sequences in multiple Human Leukocyte Antigen alleles. We define the 'Size of Immune Repertoire' (SIR) score, which represents the ratio between the number of the predicted epitopes within a protein and their expected number within a scrambled version of the same protein. We show that HIV proteins present less epitopes than expected and that the number of epitopes gradually decreases from SIV to recent HIV sequences. The decrease of the SIR score of HIV is accompanied by a high frequency of replacement mutations within epitopes. The SIR score of the different HIV proteins is not uniform. The regulatory proteins, Tat and Rev, expressed early during cellular infection have a low SIR score, whereas virion-associated genes that are expressed later, such as Env, Pol and Gag, have a higher SIR score. Actually, the SIR score of Gag keeps increasing over time. We hypothesize that our results reflect an HIV immune evasion strategy. This involves the targeting of the CTL immune response to viral structural and enzyme proteins, allowing the virus a time interval to propagate before its host cells are destroyed by CTLs. An efficient anti-HIV CTL response against HIV should thus also target the regulatory genes that HIV seeks to hide from the immune system.

  19. Reduced evolutionary rates in HIV-1 reveal extensive latency periods among replicating lineages.

    PubMed

    Immonen, Taina T; Leitner, Thomas

    2014-10-16

    HIV-1 can persist for the duration of a patient's life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle. We developed a novel molecular clock-based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past. These results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis.

  20. Management of exaggerated gag reflex in dental patients using intravenous sedation with dexmedetomidine.

    PubMed

    Reshetnikov, Aleksei P; Kasatkin, Anton A; Urakov, Aleksandr L; Baimurzin, Dmitrii Y

    2017-01-01

    Pharmacological sedation is one of the effective ways of prevention of gag reflex development in patients experiencing anxiety and fright before dental treatment. We are reporting a case where we could successfully eliminate exaggerated gag reflex (intravenous [IV] Gagging Severity Index) in a dental patient using IV sedation with dexmedetomidine. IV administration of dexmedetomidine provided elimination of gag reflex at a depth of sedation for the patient with the Richmond Agitation-Sedation Scale score of -2 and -1. The patient received dexmedetomidine 1.0 μg/kg for 10 min and then a continuous infusion of dexmedetomidine 0.4 μg/kg/h. The use of dexmedetomidine for sedation may be an alternative to other pharmacological agents in patients with dental anxiety accompanied by exaggerated gag reflex.

  1. Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

    PubMed

    Scarborough, Robert J; Adams, Kelsey L; Daher, Aïcha; Gatignol, Anne

    2015-09-01

    We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

    PubMed

    Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

    2013-07-23

    The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

  3. Enhancement of Gag-specific but reduction of Env- and Pol-specific CD8+ T cell responses by simian immunodeficiency virus nonstructural proteins in mice.

    PubMed

    Zhang, Yinfeng; Sun, Caijun; Feng, Liqiang; Xiao, Lijun; Chen, Ling

    2012-04-01

    Accessory and regulatory proteins (nonstructural proteins) have received increasing attention as components in novel HIV/SIV vaccine design. However, the complicated interactions between nonstructural proteins and structural proteins remain poorly understood, especially their effects on immunogenicity. In this study, the immunogenicity of structural proteins in the presence and absence of nonstructural proteins was compared. First, a series of recombinant plasmids and adenoviral vectors carrying various SIVmac239 nonstructural and structural genes was constructed. Then mice were primed with DNA plasmids and boosted with corresponding Ad5 vectors of different combinations, and the resulting immune responses were measured. Our results demonstrated that when the individual Gag, Pol, or Env gene products were coimmunized with the whole repertoire of nonstructural proteins, the Gag-specific CD8(+) T response was greatly enhanced, while the Env- and Pol-specific CD8(+) T responses were significantly reduced. The same pattern was not observed in CD4(+) T cell responses. Antibody responses against both the Gag and Env proteins were elicited more effectively when these structural antigens were immunized together with nonstructural antigens. These findings may provide helpful insights into the development of novel HIV/SIV vaccines.

  4. Adjuvant Activity of the Catalytic A1 Domain of Cholera Toxin for Retroviral Antigens Delivered by GeneGun▿

    PubMed Central

    Bagley, Kenneth C.; Lewis, George K.; Fouts, Timothy R.

    2011-01-01

    Most DNA-encoded adjuvants enhance immune responses to DNA vaccines in small animals but are less effective in primates. Here, we characterize the adjuvant activity of the catalytic A1 domain of cholera toxin (CTA1) for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) antigens in mice and macaques delivered by GeneGun. The inclusion of CTA1 with SIVmac239 Gag dramatically enhanced anti-Gag antibody responses in mice. The adjuvant effects of CTA1 for the secreted antigen HIV gp120 were much less pronounced than those for Gag, as the responses to gp120 were high in the absence of an adjuvant. CTA1 was a stronger adjuvant for Gag than was granulocyte-macrophage colony-stimulating factor (GM-CSF), and it also displayed a wider dose range than GM-CSF in mice. In macaques, CTA1 modestly enhanced the antibody responses to SIV Gag but potently primed for a recombinant Gag protein boost. The results of this study show that CTA1 is a potent adjuvant for SIV Gag when delivered by GeneGun in mice and that CTA1 provides a potent GeneGun-mediated DNA prime for a heterologous protein boost in macaques. PMID:21508173

  5. Adjuvant activity of the catalytic A1 domain of cholera toxin for retroviral antigens delivered by GeneGun.

    PubMed

    Bagley, Kenneth C; Lewis, George K; Fouts, Timothy R

    2011-06-01

    Most DNA-encoded adjuvants enhance immune responses to DNA vaccines in small animals but are less effective in primates. Here, we characterize the adjuvant activity of the catalytic A1 domain of cholera toxin (CTA1) for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) antigens in mice and macaques delivered by GeneGun. The inclusion of CTA1 with SIVmac239 Gag dramatically enhanced anti-Gag antibody responses in mice. The adjuvant effects of CTA1 for the secreted antigen HIV gp120 were much less pronounced than those for Gag, as the responses to gp120 were high in the absence of an adjuvant. CTA1 was a stronger adjuvant for Gag than was granulocyte-macrophage colony-stimulating factor (GM-CSF), and it also displayed a wider dose range than GM-CSF in mice. In macaques, CTA1 modestly enhanced the antibody responses to SIV Gag but potently primed for a recombinant Gag protein boost. The results of this study show that CTA1 is a potent adjuvant for SIV Gag when delivered by GeneGun in mice and that CTA1 provides a potent GeneGun-mediated DNA prime for a heterologous protein boost in macaques.

  6. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcriptionmore » elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR.« less

  7. Immune Escape Mutations Detected within HIV-1 Epitopes Associated with Viral Control During Treatment Interruption

    PubMed Central

    Schweighardt, Becky; Wrin, Terri; Meiklejohn, Duncan A.; Spotts, Gerald; Petropoulos, Christos J.; Nixon, Douglas F.; Hecht, Frederick M.

    2010-01-01

    We analyzed immune responses in chronically HIV-infected individuals who took part in a treatment interruption (TI) trial designed for patients who initiated anti-retroviral therapy within 6 months of seroconversion. In the two subjects that exhibited the best viral control, we detected CD8+ T cell responses against 1-2 Gag epitopes during the early weeks of TI and a subsequent increase in the number of epitopes recognized by the later time points. Each of these subjects developed mutations within the epitopes targeted by the highest magnitude responses. In the subject with the worst viral control, we detected responses against two Gag epitopes throughout the entire TI and no Gag mutations. The magnitude of these responses increased dramatically with time, greatly exceeding those detected in the virologic controllers. The highest levels of contemporaneous autologous neutralizing antibody activity were detected in the virologic controllers, and a subsequent escape mutation developed within the envelope gene of one controller that abrogated the response. These data suggest that immune escape mutations are a sign of viral control during TI, and that the absence of immune escape mutations in the presence of high-levels of viral replication indicates the lack of an effective host immune response. PMID:19910798

  8. Molecular epidemiology is becoming complex under the dynamic HIV prevalence: The perspective from Harbin, China.

    PubMed

    Shao, Bing; Song, Bo; Cao, Lijun; Du, Juan; Sun, Dongying; Lin, Yuanlong; Wang, Binyou; Wang, Fuxiang; Wang, Sunran

    2016-05-01

    Unlike most areas of China, HIV transmission via men who have sex with men (MSM) is increasing rapidly, and has become the main route of HIV transmission in Harbin city. The purpose of the current study was to elaborate the molecular epidemiologic characteristics of the new HIV epidemic. Eighty-one HIV-1 gag gene sequences (HXB2:806-1861) from local HIV infections were isolated; CRF01_AE predominated among HIV infections (71.6%), followed by subtype B (16.5%), CRF07_BC (6.2%), and unique recombinant strains (URFs; 6.2%). URFs were most often identified in the MSM population, which consisted of a recombination of CRF01_AE with subtype B or CRF07_BC. Six clusters were formed in this analysis; clusters I and II mainly circulated in southwest China. Clusters III and IV mainly circulated in southwest, southeast, and central China. Clusters V and VI mainly circulated in north and northeast China. Clusters III and IV may facilitate the transmission of the CRF01_AE strain from the southwest to the north and northeast regions of China. HIV subtypes are becoming diverse with the persistent epidemic in this geographic region. In brief, our results indicate that the molecular epidemiology of HIV is trending to be more complex. Thus, timely molecular epidemiologic supervision of HIV is necessary, especially for the MSM population. © 2015 Wiley Periodicals, Inc.

  9. Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

    PubMed

    Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

    2017-11-07

    The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Bevirimat: a novel maturation inhibitor for the treatment of HIV-1 infection.

    PubMed

    Martin, David E; Salzwedel, Karl; Allaway, Graham P

    2008-01-01

    Existing antiretroviral treatments for HIV type-1 (HIV-1) disease are limited by problems of resistance and drug-drug interactions. Bevirimat is a novel HIV-1 maturation inhibitor with a mechanism of action that is distinct from other antiretroviral agents. Specific inhibition of the final rate-limiting step in Gag processing by bevirimat prevents release of mature capsid protein from its precursor (CA-SP1), resulting in the production of immature, non-infectious virus particles. Bevirimat inhibits replication of both wild-type and drug-resistant HIV-1 isolates in vitro, achieving similar 50% inhibitory concentration values with both categories. Serial drug passage studies have identified six single amino acid substitutions that independently confer bevirimat resistance. These resistance mutations occur at or near the CA-SP1 cleavage site, which is not a known target for resistance to other antiretroviral drugs. Bevirimat has demonstrated a consistent pharmacokinetic profile in healthy volunteers and HIV-infected patients, with peak plasma concentrations attained approximately 1-3 h after dosing. Plasma concentrations decrease in a log-linear manner with a mean plasma elimination halflife of 58-80 h, supporting once-daily dosing. Animal studies suggest that elimination of bevirimat is primarily by hepatic glucuronidation and hepatobiliary excretion. There is minimal renal elimination, with < 1% of the administered dose appearing in the urine. In responsive patients, bevirimat has demonstrated a robust dosedependent reduction in viral load (> 1.5 log10 copies/ml). Short-term administration (< or = 14 days) of bevirimat is well tolerated, even when used in combination with other antiretroviral agents. Further studies to evaluate the long-term efficacy and tolerability of bevirimat are currently underway.

  11. Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

    NASA Astrophysics Data System (ADS)

    Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

    2017-02-01

    Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.

  12. Recombinant modified vaccinia virus Ankara–simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

    PubMed Central

    Seth, Aruna; Ourmanov, Ilnour; Kuroda, Marcelo J.; Schmitz, Jörn E.; Carroll, Miles W.; Wyatt, Linda S.; Moss, Bernard; Forman, Meryl A.; Hirsch, Vanessa M.; Letvin, Norman L.

    1998-01-01

    The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans. PMID:9707609

  13. Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

    PubMed Central

    Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

    2007-01-01

    Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348

  14. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    PubMed Central

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  15. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover

    PubMed Central

    Almeida, Jorge R.; Price, David A.; Papagno, Laura; Arkoub, Zaïna Aït; Sauce, Delphine; Bornstein, Ethan; Asher, Tedi E.; Samri, Assia; Schnuriger, Aurélie; Theodorou, Ioannis; Costagliola, Dominique; Rouzioux, Christine; Agut, Henri; Marcelin, Anne-Geneviève; Douek, Daniel; Autran, Brigitte; Appay, Victor

    2007-01-01

    The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)–B27 patients. To understand further the nature of CD8+ T cell–mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27–restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10–specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines. PMID:17893201

  16. The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

    PubMed Central

    Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

  17. Enhancement of HIV-1 VLP production using gene inhibition strategies.

    PubMed

    Fuenmayor, Javier; Cervera, Laura; Rigau, Cristina; Gòdia, Francesc

    2018-05-01

    Gag polyprotein from HIV-1 is able to generate virus-like particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved by the use of different strategies for increasing product titers. One of them is the so-called extended gene expression (EGE), based on repeated medium exchanges and retransfections of the cell culture to prolong the production phase. Another approach is the media supplementation with gene expression enhancers such as valproic acid and caffeine, despite their detrimental effect on cell viability. Valproic acid is a histone deacetylase inhibitor while caffeine has a phosphodiesterase inhibition effect. Here, the combination of the EGE protocol with additive supplementation to maximize VLP production is first tested. As an alternative to the direct additive supplementation, the replacement of these chemical additives by iRNA for obtaining the same inhibition action is also tested. The combination of the EGE protocol with caffeine and valproic acid supplementation resulted in a 1.5-fold improvement in HIV-1 VLP production compared with the EGE protocol alone, representing an overall 18-fold improvement over conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.3 fold without any detrimental effect on cell viability (91.7%) compared with the batch cultivation (92.0%). Finally, the combination of shRNA with EGE resulted in more than 15.6-fold improvement compared with the batch standard protocol traditionally used. The methodology developed enables the production of high titers of HIV-1 VLPs avoiding the toxic effects of additives.

  18. Examining the Efficacy of a Computer Facilitated HIV Prevention Tool in Drug Court

    PubMed Central

    Festinger, David S.; Dugosh, Karen L.; Kurth, Ann E.; Metzger, David S.

    2017-01-01

    Background Although they have demonstrated efficacy in reducing substance use and criminal recidivism, competing priorities and limited resources may preclude drug court programs from formally addressing HIV risk. This study examined the efficacy of a brief, three-session, computer-facilitated HIV prevention intervention in reducing HIV risk among adult felony drug court participants. Methods Two hundred participants were randomly assigned to an HIV intervention (n = 101) or attention control (n = 99) group. All clients attended judicial status hearings approximately every six weeks. At the first three status hearings following study entry, clients in the intervention group completed the computerized, interactive HIV risk reduction sessions while those in the control group viewed a series of educational life-skill videos of matched length. Outcomes included the rate of independently obtained HIV testing, engagement in high risk HIV-related behaviors, and rate of condom procurement from the research site at each session. Results Results indicated that participants who received the HIV intervention were significantly more likely to report having obtained HIV testing at some point during the study period than those in the control condition, although the effect was marginally significant when examined in a longitudinal model. In addition, they had higher rates of condom procurement. No group differences were found on rates of high-risk sexual behavior, and the low rate of injection drug reported precluded examination of high-risk drug-related behavior. Conclusions The study provides support for the feasibility and utility of delivering HIV prevention services to drug court clients using an efficient computer-facilitated program. PMID:26971228

  19. Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation

    PubMed Central

    Goulder, P.J.R.; Sewell, A.K.; Lalloo, D.G.; Price, D.A.; Whelan, J.A.; Evans, J.; Taylor, G.P.; Luzzi, G.; Giangrande, P.; Phillips, R.E.; McMichael, A.J.

    1997-01-01

    Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response. PMID:9126923

  20. HIV transmission patterns among The Netherlands, Suriname, and The Netherlands Antilles: a molecular epidemiological study.

    PubMed

    Kramer, Merlijn A; Cornelissen, Marion; Paraskevis, Dimitrios; Prins, Maria; Coutinho, Roel A; van Sighem, Ard I; Sabajo, Lesley; Duits, Ashley J; Winkel, Cai N; Prins, Jan M; van der Ende, Marchina E; Kauffmann, Robert H; Op de Coul, Eline L

    2011-02-01

    We aimed to study patterns of HIV transmission among Suriname, The Netherlands Antilles, and The Netherlands. Fragments of env, gag, and pol genes of 55 HIV-infected Surinamese, Antillean, and Dutch heterosexuals living in The Netherlands and 72 HIV-infected heterosexuals living in Suriname and the Antilles were amplified and sequenced. We included 145 pol sequences of HIV-infected Surinamese, Antillean, and Dutch heterosexuals living in The Netherlands from an observational cohort. All sequences were phylogenetically analyzed by neighbor-joining. Additionally, HIV-1 mobility among ethnic groups was estimated. A phylogenetic tree of all pol sequences showed two Surinamese and three Antillean clusters of related strains, but no clustering between ethnic groups. Clusters included sequences of individuals living in Suriname and the Antilles as well as those who have migrated to The Netherlands. Similar clustering patterns were observed in env and gag. Analysis of HIV mobility among ethnic groups showed significantly lower migration between groups than expected under the hypothesis of panmixis, apart from higher HIV migration between Antilleans in The Netherlands and all other groups. Our study shows that HIV transmission mainly occurs within the ethnic group. This suggests that cultural factors could have a larger impact on HIV mobility than geographic distance.

  1. Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates

    PubMed Central

    Agwale, Simon M.; Forbi, Joseph C.; Notka, Frank; Wrin, Terri; Wild, Jens; Wagner, Ralf; Wolf, Hans

    2011-01-01

    Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G. PMID:21829720

  2. Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

    PubMed

    Agwale, Simon M; Forbi, Joseph C; Notka, Frank; Wrin, Terri; Wild, Jens; Wagner, Ralf; Wolf, Hans

    2011-01-01

    Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

  3. HIV-1 Genetic Variability in Cuba and Implications for Transmission and Clinical Progression.

    PubMed

    Blanco, Madeline; Machado, Liuber Y; Díaz, Héctor; Ruiz, Nancy; Romay, Dania; Silva, Eladio

    2015-10-01

    INTRODUCTION Serological and molecular HIV-1 studies in Cuba have shown very low prevalence of seropositivity, but an increasing genetic diversity attributable to introduction of many HIV-1 variants from different areas, exchange of such variants among HIV-positive people with several coinciding routes of infection and other epidemiologic risk factors in the seropositive population. The high HIV-1 genetic variability observed in Cuba has possible implications for transmission and clinical progression. OBJECTIVE Study genetic variability for the HIV-1 env, gag and pol structural genes in Cuba; determine the prevalence of B and non-B subtypes according to epidemiologic and behavioral variables and determine whether a relationship exists between genetic variability and transmissibility, and between genetic variability and clinical disease progression in people living with HIV/AIDS. METHODS Using two molecular assays (heteroduplex mobility assay and nucleic acid sequencing), structural genes were characterized in 590 people with HIV-1 (480 men and 110 women), accounting for 3.4% of seropositive individuals in Cuba as of December 31, 2013. Nonrandom sampling, proportional to HIV prevalence by province, was conducted. Relationships between molecular results and viral factors, host characteristics, and patients' clinical, epidemiologic and behavioral variables were studied for molecular epidemiology, transmission, and progression analyses. RESULTS Molecular analysis of the three HIV-1 structural genes classified 297 samples as subtype B (50.3%), 269 as non-B subtypes (45.6%) and 24 were not typeable. Subtype B prevailed overall and in men, mainly in those who have sex with men. Non-B subtypes were prevalent in women and heterosexual men, showing multiple circulating variants and recombinant forms. Sexual transmission was the predominant form of infection for all. B and non-B subtypes were encountered throughout Cuba. No association was found between subtypes and

  4. Therapeutic DNA vaccination of vertically HIV-infected children: report of the first pediatric randomised trial (PEDVAC).

    PubMed

    Palma, Paolo; Romiti, Maria Luisa; Montesano, Carla; Santilli, Veronica; Mora, Nadia; Aquilani, Angela; Dispinseri, Stefania; Tchidjou, Hyppolite K; Montano, Marco; Eriksson, Lars E; Baldassari, Stefania; Bernardi, Stefania; Scarlatti, Gabriella; Wahren, Britta; Rossi, Paolo

    2013-01-01

    Twenty vertically HIV-infected children, 6-16 years of age, with stable viral load control and CD4+ values above 400 cells/mm(3). Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. clinicaltrialsregister.eu _2007-002359-18IT.

  5. Therapeutic DNA Vaccination of Vertically HIV-Infected Children: Report of the First Pediatric Randomised Trial (PEDVAC)

    PubMed Central

    Palma, Paolo; Romiti, Maria Luisa; Montesano, Carla; Santilli, Veronica; Mora, Nadia; Aquilani, Angela; Dispinseri, Stefania; Tchidjou, Hyppolite K.; Montano, Marco; Eriksson, Lars E.; Baldassari, Stefania; Bernardi, Stefania; Scarlatti, Gabriella

    2013-01-01

    Subjects Twenty vertically HIV-infected children, 6–16 years of age, with stable viral load control and CD4+ values above 400 cells/mm3. Intervention Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Results Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. Conclusions The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. Trial registration clinicaltrialsregister.eu _2007-002359-18 IT PMID:24312194

  6. What Makes Me Screen for HIV? Perceived Barriers and Facilitators to Conducting Recommended Routine HIV Testing among Primary care Physicians in the Southeastern United States

    PubMed Central

    White, Becky L.; Walsh, Joan; Rayasam, Swati; Pathman, Donald E.; Adimora, Adaora A.; Golin, Carol E.

    2015-01-01

    The Centers for Disease Control and Prevention have recommended routinely testing patients (aged 13–64) for HIV since 2006. However, many physicians do not routinely test. From January 2011- March 2012, we conducted 18 in-depth individual interviews and explored primary care physicians’ perceptions of barriers and facilitators to implementing routine HIV testing in North Carolina. Physicians’ comments were categorized thematically and fell into five groups: policy, community, practice, physician and patient. Lack of universal reimbursement was identified as the major policy barrier. Participants believed endorsement from the United States Preventive Services Tasks Force would facilitate adoption of routine HIV testing policies. Physicians reported HIV/AIDS stigma, socially conservative communities, lack of confidentiality, and rural geography as community barriers. Physicians believed public HIV testing campaigns would legitimize testing and decrease stigma in communities. Physicians cited time constraints and competing clinical priorities as physician barriers that could be overcome by delegating testing to nursing staff. HIV test refusal, low HIV risk perception, and stigma emerged as patient barriers. Physicians recommended adoption of routine HIV testing for all patients to facilitate and destigmatize testing. Physicians continue to experience a variety of barriers when implementing routine HIV testing in primary care settings. Our findings support multilevel approaches to enhance physician routine HIV testing in primary care settings. PMID:24643412

  7. Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes.

    PubMed

    Mancone, Carmine; Grimaldi, Alessio; Refolo, Giulia; Abbate, Isabella; Rozera, Gabriella; Benelli, Dario; Fimia, Gian Maria; Barnaba, Vincenzo; Tripodi, Marco; Piacentini, Mauro; Ciccosanti, Fabiola

    2017-01-01

    Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. RT-qPCR targeting the LTR region, gag , Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

  8. A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma.

    PubMed

    Hamatake, Makiko; Nishizawa, Masako; Yamamoto, Naoki; Kato, Shingo; Sugiura, Wataru

    2007-06-01

    An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.

  9. Plasma membrane signaling in HIV-1 infection.

    PubMed

    Abbas, Wasim; Herbein, Georges

    2014-04-01

    Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4+ T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

    NASA Technical Reports Server (NTRS)

    Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.; hide

    1997-01-01

    between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

  11. A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Donglai; Wang, Chu; Hora, Bhavna

    Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 andmore » fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. In conclusion, the rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.« less

  12. A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies

    DOE PAGES

    Liu, Donglai; Wang, Chu; Hora, Bhavna; ...

    2017-10-10

    Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 andmore » fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. In conclusion, the rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.« less

  13. Discrimination Between Human Leukocyte Antigen Class I-Bound and Co-Purified HIV-Derived Peptides in Immunopeptidomics Workflows

    PubMed Central

    Partridge, Thomas; Nicastri, Annalisa; Kliszczak, Anna E.; Yindom, Louis-Marie; Kessler, Benedikt M.; Ternette, Nicola; Borrow, Persephone

    2018-01-01

    Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of “irrelevant” membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated

  14. Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

    PubMed Central

    König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

    2008-01-01

    Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

  15. Dendritic Cell Immune Responses in HIV-1 Controllers.

    PubMed

    Martin-Gayo, Enrique; Yu, Xu G

    2017-02-01

    Robust HIV-1-specific CD8 T cell responses are currently regarded as the main correlate of immune defense in rare individuals who achieve natural, drug-free control of HIV-1; however, the mechanisms that support evolution of such powerful immune responses are not well understood. Dendritic cells (DCs) are specialized innate immune cells critical for immune recognition, immune regulation, and immune induction, but their possible contribution to HIV-1 immune defense in controllers remains ill-defined. Recent studies suggest that myeloid DCs from controllers have improved abilities to recognize HIV-1 through cytoplasmic immune sensors, resulting in more potent, cell-intrinsic type I interferon secretion in response to viral infection. This innate immune response may facilitate DC-mediated induction of highly potent antiviral HIV-1-specific T cells. Moreover, protective HLA class I isotypes restricting HIV-1-specific CD8 T cells may influence DC function through specific interactions with innate myelomonocytic MHC class I receptors from the leukocyte immunoglobulin-like receptor family. Bi-directional interactions between dendritic cells and HIV-1-specific T cells may contribute to natural HIV-1 immune control, highlighting the importance of a fine-tuned interplay between innate and adaptive immune activities for effective antiviral immune defense.

  16. Barriers and facilitators of linkage to HIV care among HIV-infected young Chinese men who have sex with men: a qualitative study.

    PubMed

    Li, Haochu; Wei, Chongyi; Tucker, Joseph; Kang, Dianmin; Liao, Meizhen; Holroyd, Eleanor; Zheng, Jietao; Qi, Qian; Ma, Wei

    2017-03-16

    The Four Free and One Care Policy (HIV/AIDS-related free services) has been in place in China since 2004. However, linkage to human immunodeficiency virus (HIV) care is not yet achieved very well among people living with HIV. We conducted a qualitative study to explore individual and contextual factors that may influence a linkage to HIV care from the perspective of young HIV-infected men who have sex with men (MSM) in a highly centralized HIV care context of China. Purposive sampling was used to recruit 21 HIV-infected MSM in Shandong Province, with in-depth interviews conducted between March and July 2015. Thematic content analysis was subsequently used for data analysis. Key barriers and facilitators related to a linkage to HIV care emerged from participants' narratives. The barriers included perceived healthy status, low health literacy, and stigma associated with receiving HIV care. The facilitators included an awareness of responsibility, knowledge associated with health literacy, social support, and trusting and relying on services provided by the Center for Disease Control and Prevention (CDC) and the government. These were related to the quality of current HIV counselling and testing, service promotion, and the cost and placement of these HIV services. In order to improve the MSM linkage to HIV care in China, it is imperative to improve the quality of the current on-going counselling and testing. Further critical linkage support includes increasing supportive services among local CDC systems, designated hospitals and community-based organizations (CBOs), and more financial support for HIV/AIDS related testing, medical checkups and treatments.

  17. Mathematical modeling of HIV-like particle assembly in vitro.

    PubMed

    Liu, Yuewu; Zou, Xiufen

    2017-06-01

    In vitro, the recombinant HIV-1 Gag protein can generate spherical particles with a diameter of 25-30 nm in a fully defined system. It has approximately 80 building blocks, and its intermediates for assembly are abundant in geometry. Accordingly, there are a large number of nonlinear equations in the classical model. Therefore, it is difficult to compute values of geometry parameters for intermediates and make the mathematical analysis using the model. In this work, we develop a new model of HIV-like particle assembly in vitro by using six-fold symmetry of HIV-like particle assembly to decrease the number of geometry parameters. This method will greatly reduce computational costs and facilitate the application of the model. Then, we prove the existence and uniqueness of the positive equilibrium solution for this model with 79 nonlinear equations. Based on this model, we derive the interesting result that concentrations of all intermediates at equilibrium are independent of three important parameters, including two microscopic on-rate constants and the size of nucleating structure. Before equilibrium, these three parameters influence the concentration variation rates of all intermediates. We also analyze the relationship between the initial concentration of building blocks and concentrations of all intermediates. Furthermore, the bounds of concentrations of free building blocks and HIV-like particles are estimated. These results will be helpful to guide HIV-like particle assembly experiments and improve our understanding of the assembly dynamics of HIV-like particles in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

    PubMed

    Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

    1990-12-05

    The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

  19. HLA-Driven Convergence of HIV-1 Viral Subtypes B and F Toward the Adaptation to Immune Responses in Human Populations

    PubMed Central

    Dilernia, Dario Alberto; Jones, Leandro; Rodriguez, Sabrina; Turk, Gabriela; Rubio, Andrea E.; Pampuro, Sandra; Gomez-Carrillo, Manuel; Bautista, Christian; Deluchi, Gabriel; Benetucci, Jorge; Lasala, María Beatriz; Lourtau, Leonardo; Losso, Marcelo Horacio; Perez, Héctor; Cahn, Pedro; Salomón, Horacio

    2008-01-01

    Background Cytotoxic T-Lymphocyte (CTL) response drives the evolution of HIV-1 at a host-level by selecting HLA-restricted escape mutations. Dissecting the dynamics of these escape mutations at a population-level would help to understand how HLA-mediated selection drives the evolution of HIV-1. Methodology/Principal Findings We undertook a study of the dynamics of HIV-1 CTL-escape mutations by analyzing through statistical approaches and phylogenetic methods the viral gene gag sequenced in plasma samples collected between the years 1987 and 2006 from 302 drug-naïve HIV-positive patients. By applying logistic regression models and after performing correction for multiple test, we identified 22 potential CTL-escape mutations (p-value<0.05; q-value<0.2); 10 of these associations were confirmed in samples biologically independent by a Bayesian Markov Chain Monte-Carlo method. Analyzing their prevalence back in time we found that escape mutations that are the consensus residue in samples collected after 2003 have actually significantly increased in time in one of either B or F subtype until becoming the most frequent residue, while dominating the other viral subtype. Their estimated prevalence in the viral subtype they did not dominate was lower than 30% for the majority of samples collected at the end of the 80's. In addition, when screening the entire viral region, we found that the 75% of positions significantly changing in time (p<0.05) were located within known CTL epitopes. Conclusions Across HIV Gag protein, the rise of polymorphisms from independent origin during the last twenty years of epidemic in our setting was related to an association with an HLA allele. The fact that these mutations accumulated in one of either B or F subtypes have also dominated the other subtype shows how this selection might be causing a convergence of viral subtypes to variants which are more likely to evade the immune response of the population where they circulate. PMID:18941505

  20. CBFβ enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G.

    PubMed

    Miyagi, Eri; Kao, Sandra; Yedavalli, Venkat; Strebel, Klaus

    2014-05-01

    Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. In this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature

  1. Facilitators and Barriers to Linkage to HIV Care among Female Sex Workers Receiving HIV Testing Services at a Community-Based Organization in Periurban Uganda: A Qualitative Study

    PubMed Central

    Kintu, Betty N.

    2016-01-01

    Introduction. While four in ten female sex workers (FSWs) in sub-Saharan Africa are infected with HIV, only a small proportion is enrolled in HIV care. We explored facilitators and barriers to linkage to HIV care among FSWs receiving HIV testing services at a community-based organization in periurban Uganda. Methods. The cross-sectional qualitative study was conducted among 28 HIV positive FSWs from May to July 2014. Key informant interviews were conducted with five project staff and eleven peer educators. Data were collected on facilitators for and barriers to linkage to HIV care and manually analyzed following a thematic framework approach. Results. Facilitators for linkage to HIV care included the perceived good quality of health services with same-day results and immediate initiation of treatment, community peer support systems, individual's need to remain healthy, and having alternative sources of income. Linkage barriers included perceived stigma, fear to be seen at outreach HIV clinics, fear and myths about antiretroviral therapy, lack of time to attend clinic, and financial constraints. Conclusion. Linkage to HIV care among FSWs is influenced by good quality friendly services and peer support. HIV service delivery programs for FSWs should focus on enhancing these and dealing with barriers stemming from stigma and misinformation. PMID:27493826

  2. Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396.

    PubMed

    Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K; Westwood, Nicholas J; Adamson, Catherine S

    2016-09-15

    HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they

  3. Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase*

    PubMed Central

    Ulmer, Jonathan E.; Vilén, Eric Morssing; Namburi, Ramesh Babu; Benjdia, Alhosna; Beneteau, Julie; Malleron, Annie; Bonnaffé, David; Driguez, Pierre-Alexandre; Descroix, Karine; Lassalle, Gilbert; Le Narvor, Christine; Sandström, Corine; Spillmann, Dorothe; Berteau, Olivier

    2014-01-01

    Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans. PMID:25002587

  4. Nuclear localization of foamy virus Gag precursor protein.

    PubMed Central

    Schliephake, A W; Rethwilm, A

    1994-01-01

    All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

  5. Barriers and facilitators to antiretroviral therapy adherence among Peruvian adolescents living with HIV: A qualitative study

    PubMed Central

    Wong, Milagros; Muñoz, Maribel; Valle, Emiliano; Leon, Segundo R.; Díaz Perez, Dayana; Kolevic, Lenka; Franke, Molly

    2018-01-01

    AIDS deaths among adolescents are increasing globally. This qualitative study investigated the barriers and facilitators to cART adherence among Peruvian adolescents living with HIV. Guided by a social ecological model, we analyzed transcripts from 24 psychosocial support groups for HIV-positive adolescents aged 13–17 years and 15 individual, in-depth interviews with cART providers and caregivers to identify the barriers and facilitators to cART adherence at the individual, family/caregiver and hospital levels. Most barriers and facilitators to cART adherence clustered at the individual and family/caregiver levels, centering on support provided to adolescents; history of declining health due to suboptimal cART adherence; side effects from antiretroviral drugs; and cART misinformation. Interventions to support adolescent HIV cART adherence should begin at the individual and family/caregiver levels and include an educational component. No adolescent living with HIV should die from AIDS in an era of accessible cART. PMID:29447226

  6. Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.

    PubMed

    Wang, Guihua; Wang, Zhenzhen; Zhuang, Pingping; Zhao, Xiaomin; Cheng, Ziqiang

    2017-11-01

    J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J. Copyright © 2017. Published by Elsevier Ltd.

  7. Dual-acting stapled peptides target both HIV-1 entry and assembly

    PubMed Central

    2013-01-01

    Background Previously, we reported the conversion of the 12-mer linear and cell-impermeable peptide CAI to a cell-penetrating peptide NYAD-1 by using an i,i + 4 hydrocarbon stapling technique and confirmed its binding to the C-terminal domain (CTD) of the HIV-1 capsid (CA) protein with an improved affinity (Kd ~ 1 μM) compared to CAI (Kd ~ 15 μM). NYAD-1 disrupts the formation of both immature- and mature-like virus particles in in vitro and cell-based assembly assays. In addition, it displays potent anti-HIV-1 activity in cell culture against a range of laboratory-adapted and primary HIV-1 isolates. Results In this report, we expanded the study to i,i + 7 hydrocarbon-stapled peptides to delineate their mechanism of action and antiviral activity. We identified three potent inhibitors, NYAD-36, -66 and -67, which showed strong binding to CA in NMR and isothermal titration calorimetry (ITC) studies and disrupted the formation of mature-like particles. They showed typical α-helical structures and penetrated cells; however, the cell penetration was not as efficient as observed with the i,i + 4 peptides. Unlike NYAD-1, the i,i + 7 peptides did not have any effect on virus release; however, they impaired Gag precursor processing. HIV-1 particles produced in the presence of these peptides displayed impaired infectivity. Consistent with an effect on virus entry, selection for viral resistance led to the emergence of two mutations in the gp120 subunit of the viral envelope (Env) glycoprotein, V120Q and A327P, located in the conserved region 1 (C1) and the base of the V3 loop, respectively. Conclusion The i,i + 7 stapled peptides derived from CAI unexpectedly target both CA and the V3 loop of gp120. This dual-targeted activity is dependent on their ability to penetrate cells as well as their net charge. This mechanistic revelation will be useful in further modifying these peptides as potent anti-HIV-1 agents. PMID:24237936

  8. Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries

    PubMed Central

    Chow, Wei Zhen; Bon, Abdul Hamid; Keating, Sheila; Anderios, Fread; Halim, Hazwan Abdul; Takebe, Yutaka; Kamarulzaman, Adeeba; Busch, Michael P.; Tee, Kok Keng

    2016-01-01

    Transfusion-transmissible infections including HIV-1 continue to pose major risks for unsafe blood transfusions due to both window phase infections and divergent viruses that may not be detected by donor screening assays. Given the recent emergence of several HIV-1 circulating recombinant forms (CRFs) in high-risk populations in the Southeast Asia region, we investigated the genetic diversity of HIV-1 among the blood donors in Kuala Lumpur, Malaysia. A total of 211 HIV-positive plasma samples detected among 730,188 donations to the National Blood Centre between 2013 and 2014 were provided (90.5% male, median age: 27.0 years old). Recent or long-term infection status at the time of donation was determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA). HIV-1 gag-pol genes were amplified and sequenced from residual plasma for 149 cases followed by genotype determination using phylogenetic and recombination analyses. Transmitted antiretroviral resistance mutations were not observed among the blood donors, among which 22.7% were classified as recent or incident infections. Major circulating HIV-1 genotypes determined by neighbour-joining phylogenetic inference included CRF01_AE at 40.9% (61/149), CRF33_01B at 21.5% (32/149), and subtype B at 10.1% (15/149). Newly-described CRFs including CRF54_01B circulated at 4.0%, CRF74_01B at 2.0%, and CRF53_01B and CRF48_01B at 0.7% each. Interestingly, unique HIV-1 genotypes including African subtype G (8.7%), CRF45_cpx (1.3%), CRF02_AG (0.7%) and CRF07_BC (0.7%) from China were detected for the first time in the country. A cluster of subtype G sequences formed a distinct founder sub-lineage within the African strains. In addition, 8.7% (13/149) of HIV-infected donors had unique recombinant forms (URFs) including CRF01_AE/B' (4.7%), B'/C (2.7%) and B'/G (1.3%) recombinants. Detailed analysis identified similar recombinant structures with shared parental strains among the B'/C and B'/G URFs, some of which

  9. Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries.

    PubMed

    Chow, Wei Zhen; Bon, Abdul Hamid; Keating, Sheila; Anderios, Fread; Halim, Hazwan Abdul; Takebe, Yutaka; Kamarulzaman, Adeeba; Busch, Michael P; Tee, Kok Keng

    2016-01-01

    Transfusion-transmissible infections including HIV-1 continue to pose major risks for unsafe blood transfusions due to both window phase infections and divergent viruses that may not be detected by donor screening assays. Given the recent emergence of several HIV-1 circulating recombinant forms (CRFs) in high-risk populations in the Southeast Asia region, we investigated the genetic diversity of HIV-1 among the blood donors in Kuala Lumpur, Malaysia. A total of 211 HIV-positive plasma samples detected among 730,188 donations to the National Blood Centre between 2013 and 2014 were provided (90.5% male, median age: 27.0 years old). Recent or long-term infection status at the time of donation was determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA). HIV-1 gag-pol genes were amplified and sequenced from residual plasma for 149 cases followed by genotype determination using phylogenetic and recombination analyses. Transmitted antiretroviral resistance mutations were not observed among the blood donors, among which 22.7% were classified as recent or incident infections. Major circulating HIV-1 genotypes determined by neighbour-joining phylogenetic inference included CRF01_AE at 40.9% (61/149), CRF33_01B at 21.5% (32/149), and subtype B at 10.1% (15/149). Newly-described CRFs including CRF54_01B circulated at 4.0%, CRF74_01B at 2.0%, and CRF53_01B and CRF48_01B at 0.7% each. Interestingly, unique HIV-1 genotypes including African subtype G (8.7%), CRF45_cpx (1.3%), CRF02_AG (0.7%) and CRF07_BC (0.7%) from China were detected for the first time in the country. A cluster of subtype G sequences formed a distinct founder sub-lineage within the African strains. In addition, 8.7% (13/149) of HIV-infected donors had unique recombinant forms (URFs) including CRF01_AE/B' (4.7%), B'/C (2.7%) and B'/G (1.3%) recombinants. Detailed analysis identified similar recombinant structures with shared parental strains among the B'/C and B'/G URFs, some of which

  10. The Vaginal Acquisition and Dissemination of HIV-1 Infection in a Novel Transgenic Mouse Model Is Facilitated by Coinfection with Herpes Simplex Virus 2 and Is Inhibited by Microbicide Treatment.

    PubMed

    Seay, Kieran; Khajoueinejad, Nazanin; Zheng, Jian Hua; Kiser, Patrick; Ochsenbauer, Christina; Kappes, John C; Herold, Betsy; Goldstein, Harris

    2015-09-01

    Epidemiological studies have demonstrated that herpes simplex virus 2 (HSV-2) infection significantly increases the risk of HIV-1 acquisition, thereby contributing to the expanding HIV-1 epidemic. To investigate whether HSV-2 infection directly facilitates mucosal HIV-1 acquisition, we used our transgenic hCD4/R5/cT1 mouse model which circumvents major entry and transcription blocks preventing murine HIV-1 infection by targeting transgenic expression of human CD4, CCR5, and cyclin T1 genes to CD4(+) T cells and myeloid-committed cells. Productive infection of mucosal leukocytes, predominantly CD4(+) T cells, was detected in all hCD4/R5/cT1 mice intravaginally challenged with an HIV-1 infectious molecular clone, HIV-Du151.2env-NLuc, which expresses an env gene (C.Du151.2) cloned from an acute heterosexually infected woman and a NanoLuc luciferase reporter gene. Lower genital tract HIV-1 infection after HIV-Du151.2env-NLuc intravaginal challenge was increased ~4-fold in hCD4/R5/cT1 mice coinfected with HSV-2. Furthermore, HIV-1 dissemination to draining lymph nodes was detected only in HSV-2-coinfected mice. HSV-2 infection stimulated local infiltration and activation of CD4(+) T cells and dendritic cells, likely contributing to the enhanced HIV-1 infection and dissemination in HSV-2-coinfected mice. We then used this model to demonstrate that a novel gel containing tenofovir disoproxil fumarate (TDF), the more potent prodrug of tenofovir (TFV), but not the TFV microbicide gel utilized in the recent CAPRISA 004, VOICE (Vaginal and Oral Interventions to Control the Epidemic), and FACTS 001 clinical trials, was effective as preexposure prophylaxis (PrEP) to completely prevent vaginal HIV-1 infection in almost half of HSV-2-coinfected mice. These results also support utilization of hCD4/R5/cT1 mice as a highly reproducible immunocompetent preclinical model to evaluate HIV-1 acquisition across the female genital tract. Multiple epidemiological studies have reported that

  11. HIV-1 early and late diagnosis in the Emilia Romagna Region (Italy): a three year study.

    PubMed

    Musumeci, Giuseppina; Magnani, Giacomo; Bon, Isabella; Longo, Serena; Bertoldi, Alessia; Degli Antoni, Anna Maria; Rossi, Maria Rita; Ruggeri, Alessandro; Sambri, Vittorio; Semprini, Simona; Sighinolfi, Laura; Ursitti, Maria Alessandra; Zerbini, Alessandro; Colangeli, Vincenzo; Calza, Leonardo; Finarelli, Alba Carola; Massimiliani, Erika; Re, Maria Carla

    2016-10-01

    It is crucial to establish the timing of infection and distinguish between early and long-lasting HIV-1 infections not only for partner notification and epidemiological surveillance, but also to offer early drug treatment and contain the spread of infection. This study analyzed serum and/or plasma samples with a first positive HIV antibody/antigen result coming from different Medical Centers in the Emilia Romagna Region, North East Italy, using the avidity assay, Western Blotting, RNA viral load, CD4 cell counts and genotyping assay. From May 2013 to May 2016, we certified 845 new HIV-1 infections, 18.7% of which were classified on the basis of avidity index as recent infections and 81.3% as long-lasting infections, with an estimated conversion time exceeding six months at the time of study. Western Blotting showed reactivity to only one or two HIV-1 proteins in recently infected patients (RIPs), while a complete pattern to gag, env and pol proteins was observed in most long-lasting infected patients (LLIPs). The median age, gender, nationality and risk transmission factors were comparable in RIPs and LLIPs. Phylogenetic analysis performed in available plasma disclosed B strains, non-B subtypes and circulating recombinant forms (CRFs) in both groups of patients, with a major presence of CRFs in non-Italian HIV subjects. The large number of patients unaware of their HIV status makes it crucial to discover hidden epidemics and implement appropriate targeted public health interventions.

  12. Immunotherapy with an HIV-DNA Vaccine in Children and Adults

    PubMed Central

    Palma, Paolo; Gudmundsdotter, Lindvi; Finocchi, Andrea; Eriksson, Lars E.; Mora, Nadia; Santilli, Veronica; Aquilani, Angela; Manno, Emma C.; Zangari, Paola; Romiti, Maria Luisa; Montesano, Carla; Grifoni, Alba; Brave, Andreas; Ljungberg, Karl; Blomberg, Pontus; Bernardi, Stefania; Sandström, Eric; Hejdeman, Bo; Rossi, Paolo; Wahren, Britta

    2014-01-01

    Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals’ immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children’s and adults’ responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4–16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation

  13. Barriers, Motivators, and Facilitators to Engagement in HIV Care Among HIV-Infected Ghanaian Men Who have Sex with Men (MSM).

    PubMed

    Ogunbajo, Adedotun; Kershaw, Trace; Kushwaha, Sameer; Boakye, Francis; Wallace-Atiapah, Nii-Dromo; Nelson, LaRon E

    2018-03-01

    In Ghana, men who have sex with men (MSM) bear a high burden of HIV. Identifying factors that influence engagement in HIV care among HIV-infected Ghanaian MSM is critical to devising novel interventions and strengthening existing programs aimed at improving outcomes across the HIV care continuum. Consequently, we conducted an exploratory qualitative research study with 30 HIV-infected Ghanaian MSM between May 2015 and July 2015. Common barriers were fear of being seen in HIV-related health facility, financial difficulties, and health system challenges. Major motivators for engagement in care included social support, fear of mortality from HIV, and knowledge of effectiveness of HIV treatment. Key facilitators were enrollment in health insurance, prior relationship and familiarity with hospital personnel, and positive experience in healthcare setting. Our findings highlight the need for new and innovative care delivery mediums, affirming and competent healthcare providers, and increased access to health insurance.

  14. Facilitators and barriers for HIV-testing in Zambia: A systematic review of multi-level factors.

    PubMed

    Qiao, Shan; Zhang, Yao; Li, Xiaoming; Menon, J Anitha

    2018-01-01

    It was estimated that 1.2 million people live with HIV/AIDS in Zambia by 2015. Zambia has developed and implemented diverse programs to reduce the prevalence in the country. HIV-testing is a critical step in HIV treatment and prevention, especially among all the key populations. However, there is no systematic review so far to demonstrate the trend of HIV-testing studies in Zambia since 1990s or synthesis the key factors that associated with HIV-testing practices in the country. Therefore, this study conducted a systematic review to search all English literature published prior to November 2016 in six electronic databases and retrieved 32 articles that meet our inclusion criteria. The results indicated that higher education was a common facilitator of HIV testing, while misconception of HIV testing and the fear of negative consequences were the major barriers for using the testing services. Other factors, such as demographic characteristics, marital dynamics, partner relationship, and relationship with the health care services, also greatly affects the participants' decision making. The findings indicated that 1) individualized strategies and comprehensive services are needed for diverse key population; 2) capacity building for healthcare providers is critical for effectively implementing the task-shifting strategy; 3) HIV testing services need to adapt to the social context of Zambia where HIV-related stigma and discrimination is still persistent and overwhelming; and 4) family-based education and intervention should involving improving gender equity.

  15. Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development.

    PubMed

    Franco, David; Li, Wenjing; Qing, Fang; Stoyanov, Cristina T; Moran, Thomas; Rice, Charles M; Ho, David D

    2010-08-09

    The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5' end of YF17D, in frame and upstream of the polyprotein (YF-5'/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8(+) IFN-gamma T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4(+) T cells expressing IFN-gamma and IL-2. A balanced CD4(+) and CD8(+) T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. Conformational Plasticity in Broadly Neutralizing HIV-1 Antibodies Triggers Polyreactivity.

    PubMed

    Prigent, Julie; Jarossay, Annaëlle; Planchais, Cyril; Eden, Caroline; Dufloo, Jérémy; Kök, Ayrin; Lorin, Valérie; Vratskikh, Oxana; Couderc, Thérèse; Bruel, Timothée; Schwartz, Olivier; Seaman, Michael S; Ohlenschläger, Oliver; Dimitrov, Jordan D; Mouquet, Hugo

    2018-05-29

    Human high-affinity antibodies to pathogens often recognize unrelated ligands. The molecular origin and the role of this polyreactivity are largely unknown. Here, we report that HIV-1 broadly neutralizing antibodies (bNAbs) are frequently polyreactive, cross-reacting with non-HIV-1 molecules, including self-antigens. Mutating bNAb genes to increase HIV-1 binding and neutralization also results in de novo polyreactivity. Unliganded paratopes of polyreactive bNAbs with improved HIV-1 neutralization exhibit a conformational flexibility, which contributes to enhanced affinity of bNAbs to various HIV-1 envelope glycoproteins and non-HIV antigens. Binding adaptation of polyreactive bNAbs to the divergent ligands mainly involves hydrophophic interactions. Plasticity of bNAbs' paratopes may, therefore, facilitate accommodating divergent viral variants, but it simultaneously triggers promiscuous binding to non-HIV-1 antigens. Thus, a certain level of polyreactivity can be a mark of adaptable antibodies displaying optimal pathogens' recognition. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

    NASA Astrophysics Data System (ADS)

    van Beuningen, Rinie; Marras, Salvatore A.; Kramer, Fred R.; Oosterlaken, Tom; Weusten, Jos; Borst, G.; van de Wiel, Paul

    2001-04-01

    HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1» hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

  18. Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication.

    PubMed

    Schwartzkopff, Franziska; Grimm, Tobias A; Lankford, Carla S R; Fields, Karen; Wang, Jiun; Brandt, Ernst; Clouse, Kathleen A

    2009-12-01

    Platelet factor 4 (CXCL4), a member of the CXC chemokine subfamily released in high amounts by activated platelets, has been identified as a monocyte survival factor that induces monocyte differentiation into macrophages. Although CXCL4 has been shown to have biological effects unique to chemokines, nothing is known about the role of CXCL4-derived human macrophages or CXCL4 in human immunodeficiency virus (HIV) disease. In this study, CXCL4-derived macrophages are compared with macrophage-colony stimulating factor (M-CSF)-derived macrophages for their ability to support HIV-1 replication. We show that CXCL4-derived macrophages can be infected with macrophage-tropic HIV-1 that uses either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) as a co-receptor for viral entry. We also find that M-CSF and the chemokines, monocyte chemoattractant protein 1 (MCP-1; CCL2) and macrophage-inflammatory-protein-1-alpha (MIP-1alpha; CCL3) are produced upon R5- and X4-tropic HIV-1 replication in both M-CSF- and CXCL4-derived human macrophages. In addition, CXCL4 added to M-CSF-derived macrophages after virus adsorption and maintained throughout the infection enhances HIV-1 replication. We thus propose a novel role for CXCL4 in HIV disease.

  19. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR.

    PubMed

    Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L; Karn, Jonathan; Hauser, Kurt F; Tyagi, Mudit

    2015-09-01

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

    PubMed Central

    Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit

    2015-01-01

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-κB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-κB at 276th serine residue. These modifications enhance the interaction of NF-κB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. PMID:25980739

  1. Engineering T Cells to Functionally Cure HIV-1 Infection.

    PubMed

    Leibman, Rachel S; Riley, James L

    2015-07-01

    Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients' lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1-resistant cells, redirecting HIV-1-specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1-specific lymphocytes within a patient's own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy-mediated functional cure.

  2. Pandemic HIV-1 Vpu overcomes intrinsic herd immunity mediated by tetherin.

    PubMed

    Iwami, Shingo; Sato, Kei; Morita, Satoru; Inaba, Hisashi; Kobayashi, Tomoko; Takeuchi, Junko S; Kimura, Yuichi; Misawa, Naoko; Ren, Fengrong; Iwasa, Yoh; Aihara, Kazuyuki; Koyanagi, Yoshio

    2015-07-17

    Among the four groups of HIV-1 (M, N, O, and P), HIV-1M alone is pandemic and has rapidly expanded across the world. However, why HIV-1M has caused a devastating pandemic while the other groups remain contained is unclear. Interestingly, only HIV-1M Vpu, a viral protein, can robustly counteract human tetherin, which tethers budding virions. Therefore, we hypothesize that this property of HIV-1M Vpu facilitates human-to-human viral transmission. Adopting a multilayered experimental-mathematical approach, we demonstrate that HIV-1M Vpu confers a 2.38-fold increase in the prevalence of HIV-1 transmission. When Vpu activity is lost, protected human populations emerge (i.e., intrinsic herd immunity develops) through the anti-viral effect of tetherin. We also reveal that all Vpus of transmitted/founder HIV-1M viruses maintain anti-tetherin activity. These findings indicate that tetherin plays the role of a host restriction factor, providing 'intrinsic herd immunity', whereas Vpu has evolved in HIV-1M as a tetherin antagonist.

  3. Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

    PubMed

    deCamp, Allan C; Rolland, Morgane; Edlefsen, Paul T; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A; Fiore-Gartland, Andrew J; Juraska, Michal; Carpp, Lindsay N; Karuna, Shelly T; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z; Gottardo, Raphael; Koup, Richard A; Bailer, Robert; Mascola, John R; Graham, Barney S; Roederer, Mario; O'Connell, Robert J; Michael, Nelson L; Robb, Merlin L; Adams, Elizabeth; D'Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E; Frahm, Nicole; Tomaras, Georgia D; McElrath, M Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E; Hammer, Scott M; Kim, Jerome H; Mullins, James I; Gilbert, Peter B

    2017-01-01

    Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

  4. Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120

    PubMed Central

    Edlefsen, Paul T.; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A.; Fiore-Gartland, Andrew J.; Juraska, Michal; Carpp, Lindsay N.; Karuna, Shelly T.; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z.; Gottardo, Raphael; Koup, Richard A.; Bailer, Robert; Mascola, John R.; Graham, Barney S.; Roederer, Mario; O’Connell, Robert J.; Michael, Nelson L.; Robb, Merlin L.; Adams, Elizabeth; D’Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E.; Frahm, Nicole; Tomaras, Georgia D.; McElrath, M. Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E.; Hammer, Scott M.; Kim, Jerome H.; Mullins, James I.; Gilbert, Peter B.

    2017-01-01

    Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector. PMID:29149197

  5. Facilitating HIV status disclosure for pregnant women and partners in rural Kenya: a qualitative study.

    PubMed

    Walcott, Melonie M; Hatcher, Abigail M; Kwena, Zachary; Turan, Janet M

    2013-12-02

    Women's ability to safely disclose their HIV-positive status to male partners is essential for uptake and continued use of prevention of mother-to-child transmission (PMTCT) services. However, little is known about the acceptability of potential approaches for facilitating partner disclosure. To lay the groundwork for developing an intervention, we conducted formative qualitative research to elicit feedback on three approaches for safe HIV disclosure for pregnant women and male partners in rural Kenya. This qualitative acceptability research included in-depth interviews with HIV-infected pregnant women (n = 20) and male partners of HIV-infected women (n = 20) as well as two focus groups with service providers (n = 16). The participants were recruited at health care facilities in two communities in rural Nyanza Province, Kenya, during the period June to November 2011. Data were managed in NVivo 9 and analyzed using a framework approach, drawing on grounded theory. We found that facilitating HIV disclosure is acceptable in this context, but that individual participants have varying expectations depending on their personal situation. Many participants displayed a strong preference for couples HIV counseling and testing (CHCT) with mutual disclosure facilitated by a trained health worker. Home-based approaches and programs in which pregnant women are asked to bring their partners to the healthcare facility were equally favored. Participants felt that home-based CHCT would be acceptable for this rural setting, but special attention must be paid to how this service is introduced in the community, training of the health workers who will conduct the home visits, and confidentiality. Pregnant couples should be given different options for assistance with HIV disclosure. Home-based CHCT could serve as an acceptable method to assist women and men with safe disclosure of HIV status. These findings can inform the design and implementation of programs geared at promoting HIV

  6. Facilitating HIV status disclosure for pregnant women and partners in rural Kenya: a qualitative study

    PubMed Central

    2013-01-01

    Background Women’s ability to safely disclose their HIV-positive status to male partners is essential for uptake and continued use of prevention of mother-to-child transmission (PMTCT) services. However, little is known about the acceptability of potential approaches for facilitating partner disclosure. To lay the groundwork for developing an intervention, we conducted formative qualitative research to elicit feedback on three approaches for safe HIV disclosure for pregnant women and male partners in rural Kenya. Methods This qualitative acceptability research included in-depth interviews with HIV-infected pregnant women (n = 20) and male partners of HIV-infected women (n = 20) as well as two focus groups with service providers (n = 16). The participants were recruited at health care facilities in two communities in rural Nyanza Province, Kenya, during the period June to November 2011. Data were managed in NVivo 9 and analyzed using a framework approach, drawing on grounded theory. Results We found that facilitating HIV disclosure is acceptable in this context, but that individual participants have varying expectations depending on their personal situation. Many participants displayed a strong preference for couples HIV counseling and testing (CHCT) with mutual disclosure facilitated by a trained health worker. Home-based approaches and programs in which pregnant women are asked to bring their partners to the healthcare facility were equally favored. Participants felt that home-based CHCT would be acceptable for this rural setting, but special attention must be paid to how this service is introduced in the community, training of the health workers who will conduct the home visits, and confidentiality. Conclusion Pregnant couples should be given different options for assistance with HIV disclosure. Home-based CHCT could serve as an acceptable method to assist women and men with safe disclosure of HIV status. These findings can inform the design and

  7. Barriers and Facilitators for Clinical Care Engagement Among HIV-Positive African American and Latino Men Who Have Sex with Men.

    PubMed

    Carey, James W; Carnes, Neal; Schoua-Glusberg, Alisú; Kenward, Katherine; Gelaude, Deborah; Denson, Damian; Gall, Elizabeth; Randall, Laura A; Frew, Paula M

    2018-05-01

    Achieving optimal health among people living with HIV (PLWH) requires linkage to clinical care upon diagnosis, followed by ongoing engagement in HIV clinical care. A disproportionate number of black/African American and Hispanic/Latino men who have sex with men (MSM) living with HIV do not, however, achieve ongoing care. We conducted semistructured interviews in 2014 with 84 urban black/African American and Hispanic/Latino MSM living with HIV to understand their barriers and facilitators to engagement. We classified men as care-engaged or not at the time of the interview, and conducted content analysis of the interview transcripts to identify barriers and facilitators to engagement. Respondent mean age was 42.4 years (range, 20-59). Over half (59.5%, n = 50) were black/African American. Slightly more than a third (38.1%, n = 32) reported not being continuously care-engaged since diagnosis, and 17.9% (n = 15) delayed entry, although they have subsequently entered and remained in care. Sustained engagement began with overcoming denial after diagnosis and having treatment plans, as well as having conveniently located care facilities. Engagement also was facilitated by services tailored to meet multiple patient needs, effective patient-provider communication, and providers who show empathy and respect for their patients. Respondents were less likely to be care-engaged when these factors were absent. It can be difficult for racial and ethnic minority MSM living with HIV to begin and sustain care engagement. To optimize care engagement, our findings underscore the value of (1) convenient multipurpose HIV care facilities that meet patient needs; (2) excellent provider-patient communication that reinforces respect, trust, and HIV treatment literacy; and (3) assisting PLWH to create personalized treatment plans and overcome possible challenges such as diagnosis denial.

  8. Patterns and rates of viral evolution in HIV-1 subtype B infected females and males.

    PubMed

    Dapp, Michael J; Kober, Kord M; Chen, Lennie; Westfall, Dylan H; Wong, Kim; Zhao, Hong; Hall, Breana M; Deng, Wenjie; Sibley, Thomas; Ghorai, Suvankar; Kim, Katie; Chen, Natalie; McHugh, Sarah; Au, Lily; Cohen, Mardge; Anastos, Kathryn; Mullins, James I

    2017-01-01

    Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6-11 years of infection from 8 Women's Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men.

  9. Retroviral Gag protein-RNA interactions: Implications for specific genomic RNA packaging and virion assembly.

    PubMed

    Olson, Erik D; Musier-Forsyth, Karin

    2018-03-31

    Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates

    PubMed Central

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan; Seaman, Michael; Montefiori, David C.; Labranche, Celia; Yates, Nicole L.; Shen, Xiaoying; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; McDermott, Adrian; Kao, Shing-Fen; Roederer, Mario; Hawkins, Natalie; Self, Steve; Yao, Jiansheng; Farrell, Patrick; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony; Weiss, Deborah; Lee, Carter; Kibler, Karen; Jacobs, Bert; Asbach, Benedikt; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

    2015-01-01

    ABSTRACT We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4+ T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8+ T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that

  11. Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

    PubMed Central

    Hansen, M S; Barklis, E

    1995-01-01

    We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

  12. Using nearly full-genome HIV sequence data improves phylogeny reconstruction in a simulated epidemic.

    PubMed

    Yebra, Gonzalo; Hodcroft, Emma B; Ragonnet-Cronin, Manon L; Pillay, Deenan; Brown, Andrew J Leigh

    2016-12-23

    HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to determine what gene(s) provide(s) the best approximation to the real phylogeny by analysing a simulated epidemic (created as part of the PANGEA_HIV project) with a known transmission tree. We sub-sampled a simulated dataset of 4662 sequences into different combinations of genes (gag-pol-env, gag-pol, gag, pol, env and partial pol) and sampling depths (100%, 60%, 20% and 5%), generating 100 replicates for each case. We built maximum-likelihood trees for each combination using RAxML (GTR + Γ), and compared their topologies to the corresponding true tree's using CompareTree. The accuracy of the trees was significantly proportional to the length of the sequences used, with the gag-pol-env datasets showing the best performance and gag and partial pol sequences showing the worst. The lowest sampling depths (20% and 5%) greatly reduced the accuracy of tree reconstruction and showed high variability among replicates, especially when using the shortest gene datasets. In conclusion, using longer sequences derived from nearly whole genomes will improve the reliability of phylogenetic reconstruction. With low sample coverage, results can be highly variable, particularly when based on short sequences.

  13. Barriers and Facilitators to Retaining and Reengaging HIV Clients in Care: A Case Study of North Carolina.

    PubMed

    Berger, Miriam B; Sullivan, Kristen A; Parnell, Heather E; Keller, Jennifer; Pollard, Alice; Cox, Mary E; Clymore, Jacquelyn M; Quinlivan, Evelyn Byrd

    2016-11-01

    Retention in HIV care is critical to decrease disease-related mortality and morbidity and achieve national benchmarks. However, a myriad of barriers and facilitators impact retention in care; these can be understood within the social-ecological model. To elucidate the unique factors that impact consistent HIV care engagement, a qualitative case study was conducted in North Carolina to examine the barriers and facilitators to retain and reengage HIV clients in care. HIV professionals (n = 21) from a variety of health care settings across the state participated in interviews that were transcribed and analyzed for emergent themes. Respondents described barriers to care at all levels within the HIV prevention and care system including intrapersonal, interpersonal, institutional, community, and public policy. Participants also described recent statewide initiatives with the potential to improve care engagement. Results from this study may assist other states with similar challenges to identify needed programs and priorities to optimize client retention in HIV care. © The Author(s) 2015.

  14. Molecular epidemiology demonstrated three emerging clusters of human immunodeficiency virus type 1 subtype B infection in Hong Kong.

    PubMed

    Leung, Tommy W C; Mak, Darwin; Wong, K H; Wang, Y; Song, Y H; Tsang, D N C; Wong, C; Shao, Y M; Lim, W L

    2008-07-01

    We conducted a molecular epidemiological study on newly diagnosed human immunodeficiency virus type 1 (HIV-1)-infected patients in Hong Kong to identify the epidemiological linkage of HIV-1 infection in the locality. Reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 was performed on newly diagnosed HIV-1-positive sera collected from January 2002 to December 2006. PCR products correspond to the env C2V3V4 region and gag p17/p24 junction of the HIV-1 genome were nucleotide sequenced. Phylogenetic analyses performed on the acquired nucleotide sequences revealed that CRF01_AE and subtype B were the two dominant HIV-1 subtypes. Analyses also demonstrated the presence of three emerging HIV-1 clusters among the subtype B sequences in Hong Kong. Individual cluster possesses a unique cluster-specific amino acid signature for identification. Data show that one of the clusters (Cluster I) is rapidly expanding. In addition to the unique cluster-specific amino acid signature, the majority of sequences in Cluster I harbor a 6-amino acid insertion at the gag p17/p24 junction in a region that is thought to be closely associated with HIV-1 infectivity.

  15. HIV-1 DNA predicts disease progression and post-treatment virological control

    PubMed Central

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

  16. Facilitators and barriers to effective scale-up of an evidence-based multilevel HIV prevention intervention.

    PubMed

    Kegeles, Susan M; Rebchook, Gregory; Tebbetts, Scott; Arnold, Emily

    2015-04-17

    Since the scale-up of HIV/AIDS prevention evidence-based interventions (EBIs) has not been simple, it is important to examine processes that occur in the translation of the EBIs into practice that affect successful implementation. The goal of this paper is to examine facilitators and barriers to effective implementation that arose among 72 community-based organizations as they moved into practice a multilevel HIV prevention intervention EBI, the Mpowerment Project, for young gay and bisexual men. CBOs that were implementing the Mpowerment Project participated in this study and were assessed at baseline, and 6-months, 1 year, and 2 years post-baseline. Semi-structured telephone interviews were conducted separately with individuals at each CBO. Study data came from 647 semi-structured interviews and extensive notes and commentaries from technical assistance providers. Framework Analysis guided the analytic process. Barriers and facilitators to implementation was the overarching thematic framework used across all the cases in our analysis. Thirteen themes emerged regarding factors that influence the successful implementation of the MP. These were organized into three overarching themes: HIV Prevention System Factors, Community Factors, and Intervention Factors. The entire HIV Prevention System, including coordinators, supervisors, executive directors, funders, and national HIV prevention policies, all influenced implementation success. Other Prevention System Factors that affected the effective translation of the EBI into practice include Knowledge About Intervention, Belief in the Efficacy of the Intervention, Desire to Change Existing Prevention Approach, Planning for Intervention Before Implementation, Accountability, Appropriateness of Individuals for Coordinator Positions, Evaluation of Intervention, and Organizational Stability. Community Factors included Geography and Sociopolitical Climate. Intervention Factors included Intervention Characteristics and

  17. Foamy virus reverse transcriptase is expressed independently from the Gag protein.

    PubMed Central

    Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

    1996-01-01

    In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

  18. Mapping of the self-interaction domains in the simian immunodeficiency virus Gag polyprotein.

    PubMed

    Rauddi, María L; Mac Donald, Cecilia L; Affranchino, José L; González, Silvia A

    2011-03-01

    To gain a better understanding of the assembly process in simian immunodeficiency virus (SIV), we first established the conditions under which recombinant SIV Gag lacking the C-terminal p6 domain (SIV GagΔp6) assembled in vitro into spherical particles. Based on the full multimerization capacity of SIV GagΔp6, and to identify the Gag sequences involved in homotypic interactions, we next developed a pull-down assay in which a panel of histidine-tagged SIV Gag truncation mutants was tested for its ability to associate in vitro with GST-SIVGagΔp6. Removal of the nucleocapsid (NC) domain from Gag impaired its ability to interact with GST-SIVGagΔp6. However, this Gag mutant consisting of the matrix (MA) and capsid (CA) domains still retained 50% of the wild-type binding activity. Truncation of SIV Gag from its N-terminus yielded markedly different results. The Gag region consisting of the CA and NC was significantly more efficient than wild-type Gag at interacting in vitro with GST-SIVGagΔp6. Notably, a small Gag subdomain containing the C-terminal third of the CA and the entire NC not only bound to GST-SIVGagΔp6 in vitro at wild-type levels, but also associated in vivo with full-length Gag and was recruited into extracellular particles. Interestingly, when the mature Gag products were analyzed, the MA and NC interacted with GST-SIVGagΔp6 with efficiencies representing 20% and 40%, respectively, of the wild-type value, whereas the CA failed to bind to GST-SIVGagΔp6, despite being capable of self-associating into multimeric complexes.

  19. Discovery of a novel and potent class of anti-HIV-1 maturation inhibitors with improved virology profile against gag polymorphisms.

    PubMed

    Tang, Jun; Jones, Stacey A; Jeffrey, Jerry L; Miranda, Sonia R; Galardi, Cristin M; Irlbeck, David M; Brown, Kevin W; McDanal, Charlene B; Johns, Brian A

    2017-06-15

    A new class of betulin-derived α-keto amides was identified as HIV-1 maturation inhibitors. Through lead optimization, GSK8999 was identified with IC 50 values of 17nM, 23nM, 25nM, and 8nM for wild type, Q369H, V370A, and T371A respectively. When tested in a panel of 62 HIV-1 isolates covering a diversity of CA-SP1 genotypes including A, AE, B, C, and G using a PBMC based assay, GSK8999 was potent against 57 of 62 isolates demonstrating an improvement over the first generation maturation inhibitor BVM. The data disclosed here also demonstrated that the new α-keto amide GSK8999 has a mechanism of action consistent with inhibition of the proteolytic cleavage of CA-SP1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial

    PubMed Central

    Buchbinder, Susan P.; Mehrotra, Devan V.; Duerr, Ann; Fitzgerald, Daniel W.; Mogg, Robin; Li, David; Gilbert, Peter B.; Lama, Javier R.; Marmor, Michael; Rio, Carlos del; McElrath, M. Juliana; Casimiro, Danilo R.; Gottesdiener, Keith M.; Chodakewitz, Jeffrey A.; Corey, Lawrence; Robertson, Michael N.

    2009-01-01

    Background Observational data and non-human primate challenge studies suggest that cell-mediated immune (CMI) responses may provide control of HIV replication. The Step Study is the first direct assessment of the efficacy of a CMI vaccine to protect against HIV infection or alter early plasma HIV levels in humans. Method HIV-seronegative participants (3000) were randomized (1:1) to receive 3 injections of MRKAd5 HIV-1 gag/pol/nef vaccine or placebo. Randomization was pre-stratified by gender, baseline adenovirus type 5 (Ad5) titer, and study site. Participants were tested ~every 6 months for HIV acquisition; early plasma HIV RNA was measured ~3 months post-HIV diagnosis. Findings The vaccine elicited IFN-γ ELISPOT responses in 75% of vaccinees. In a pre-specified interim analysis among participants with baseline Ad5 ≤200, 24 of 741 vaccinees became HIV infected, versus 21 of 762 placebo recipients. All but one infection occurred in men. The early geometric mean plasma HIV RNA was comparable in infected vaccine and placebo recipients. In exploratory multivariate analyses, HIV incidence was higher in vaccinees versus placebo recipients among Ad5 seropositive men (5.1% versus 2.2% per year, respectively) and uncircumcised men (5.2% versus 1.4% per year, respectively). HIV incidence was similar in vaccinees versus placebo recipients among Ad5 seronegative men and circumcised men. Interpretation This CMI vaccine did not prevent HIV infection or lower early viral level. Mechanisms for failure of the vaccine to protect and for the increased HIV infection rates in subgroups of vaccinees are being explored. Additional follow-up will determine if elevated HIV incidence in vaccinee subgroups persists. PMID:19012954

  1. A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

    PubMed

    Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

    2017-07-01

    Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Barriers and facilitators of HIV vaccine and prevention study participation among Young Black MSM and transwomen in New York City

    PubMed Central

    Koblin, Beryl; Vazquez, Tasha; Nandi, Vijay; Tieu, Hong-Van

    2017-01-01

    Background Black men who have sex with men (MSM), and Transwomen (TW) shoulder disproportionate burden of HIV. However, they are unrepresented in HIV vaccine trials. We investigated the perceptions of that factors associated with HIV vaccine trials participation among Black MSM and TW in New York. Methods Self-administered online questionnaires were administered to 18–29 years of NYC residents who identified as Black MSM and TW, assessing demographics, awareness and willingness to participate in HIV vaccine trials, barriers and facilitators associated with willingness, and sexual behaviors. Frequency summation was performed to determine barriers and facilitators, and logistic regression analysis was performed to determine factors association with expressed willingness. Results Black MSM and TW who reported engaging in risk behaviors had a 61% lower likelihood of participating in HIV vaccine trials when compared to those who did not report engaging in any risk behavior. Facilitators associated with trial participation were: cash compensation, confidentiality regarding participation, public transportation vouchers, gift cards, and food or grocery vouchers as potential facilitators for trial participation. Conversely, fear of side effects from the vaccine, concerns about testing positive on routine HIV testing due to an HIV vaccine, limited knowledge of research trials, and fear of being judged as HIV-positive were perceived as barriers. Conclusions These findings provided insights into the considerations and perceptions of Black MSM and TW towards HIV vaccine trials. However, further studies are needed to delineate the complex mechanisms underlying the decision-making process and establish approaches to increase study participation in this population. PMID:28723970

  3. Molecular Epidemiology of HIV-1 in Jilin Province, Northeastern China: Emergence of a New CRF07_BC Transmission Cluster and Intersubtype Recombinants

    PubMed Central

    Ning, Chuanyi; Feng, Yi; Xie, Cunxin; He, Xiang; Takebe, Yutaka; Sun, Liuyan; Guo, Qi; Xing, Hui; Kalish, Marcia L.; Shao, Yiming

    2014-01-01

    Objective To investigate the HIV-1 molecular epidemiology among newly diagnosed HIV-1 infected persons living in the Jilin province of northeastern China. Methods Plasma samples from 189 newly diagnosed HIV-1 infected patients were collected between June 2010 and August 2011 from all nine cities of Jilin province. HIV-1 nucleotide sequences of gag P17–P24 and env C2–C4 gene regions were amplified using a multiplex RT-PCR method and sequenced. Phylogenetic and recombination analyses were used to determine the HIV-1 genotypes. Results Based on all sequences generated, the subtype/CFR distribution was as follows: CRF01_AE (58.1%), CRF07_BC (13.2%), subtype B’ (13.2%), recombinant viruses (8.1%), subtype B (3.7%), CRF02_AG (2.9%), subtype C (0.7%). In addition to finding CRF01_AE strains from previously reported transmission clusters 1, 4 and 5, a new transmission cluster was described within the CRF07_BC radiation. Among 11 different recombinants identified, 10 contained portions of gene regions from the CRF01_AE lineage. CRF02_AG was found to form a transmission cluster of 4 in local Jilin residents. Conclusions Our study presents a molecular epidemiologic investigation describing the complex structure of HIV-1 strains co-circulating in Jilin province. The results highlight the critical importance of continuous monitoring of HIV-infections, along with detailed socio-demographic data, in order to design appropriate prevention measures to limit the spread of new HIV infections. PMID:25356726

  4. Genome-Wide Association Study Identifies Single Nucleotide Polymorphism in DYRK1A Associated with Replication of HIV-1 in Monocyte-Derived Macrophages

    PubMed Central

    Bol, Sebastiaan M.; Moerland, Perry D.; Limou, Sophie; van Remmerden, Yvonne; Coulonges, Cédric; van Manen, Daniëlle; Herbeck, Joshua T.; Fellay, Jacques; Sieberer, Margit; Sietzema, Jantine G.; van 't Slot, Ruben; Martinson, Jeremy; Zagury, Jean-François; Schuitemaker, Hanneke; van 't Wout, Angélique B.

    2011-01-01

    Background HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages. Methodology/Principal Findings Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16×10−5). While the association was not genome-wide significant (p<1×10−7), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84×10−6). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048). Conclusions/Significance These findings suggest that

  5. Host-Specific Adaptation of HIV-1 Subtype B in the Japanese Population

    PubMed Central

    Chikata, Takayuki; Carlson, Jonathan M.; Tamura, Yoshiko; Borghan, Mohamed Ali; Naruto, Takuya; Hashimoto, Masao; Murakoshi, Hayato; Le, Anh Q.; Mallal, Simon; John, Mina; Gatanaga, Hiroyuki; Oka, Shinichi; Brumme, Zabrina L.

    2014-01-01

    ABSTRACT The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However, HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naive Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol, and Nef using phylogenetically corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with the plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate the pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-APs identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways in Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures. IMPORTANCE Cytotoxic T lymphocyte (CTL) escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele

  6. Host-specific adaptation of HIV-1 subtype B in the Japanese population.

    PubMed

    Chikata, Takayuki; Carlson, Jonathan M; Tamura, Yoshiko; Borghan, Mohamed Ali; Naruto, Takuya; Hashimoto, Masao; Murakoshi, Hayato; Le, Anh Q; Mallal, Simon; John, Mina; Gatanaga, Hiroyuki; Oka, Shinichi; Brumme, Zabrina L; Takiguchi, Masafumi

    2014-05-01

    The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However, HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naive Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol, and Nef using phylogenetically corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with the plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate the pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-APs identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways in Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures. Cytotoxic T lymphocyte (CTL) escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele distributions differ among

  7. Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

    PubMed Central

    Schultz, A M; Rabin, E H; Oroszlan, S

    1979-01-01

    Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag. Images PMID:480454

  8. Weak anti-HIV CD8+ T-cell effector activity in HIV primary infection

    PubMed Central

    Dalod, Marc; Dupuis, Marion; Deschemin, Jean-Christophe; Goujard, Cécile; Deveau, Christiane; Meyer, Laurence; Ngo, Nicole; Rouzioux, Christine; Guillet, Jean-Gérard; Delfraissy, Jean-François; Sinet, Martine; Venet, Alain

    1999-01-01

    HIV-specific CD8+ T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-γ enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8+ T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0–6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1–13). The frequency of HIV-specific CD8+ T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8+CD28– terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination. J. Clin. Invest. 104:1431–1439 (1999). PMID:10562305

  9. Barriers to and Facilitators of Adherence to Exclusive Breastfeeding Practices Among HIV Infected and Non-Infected Women in Jos, Nigeria.

    PubMed

    Coetzee, Bronwynè; Tomlinson, Mark; Osawe, Sophia; Abimiku, Alash'le; Kagee, Ashraf

    2017-04-01

    Objectives In Nigeria adherence to exclusive breastfeeding (EBF) practices is currently suboptimal and a better understanding of the factors affecting adherence to EBF is needed. We sought to identify and delineate the barriers to and facilitators of adherence to EBF amongst HIV-infected and uninfected women in Nigeria. Methods We explored the barriers and facilitators to EBF amongst 37 (25 HIV-infected and 12 HIV-uninfected) pregnant women attending an antenatal clinic in Jos, Nigeria. In-depth interviews were conducted with each of the pregnant women in their third trimester of pregnancy and again 1 month after giving birth. Results The themes that emerged were mothers' feeding intentions, significant role players in the decision to breastfeed, perceived barriers (e.g. physiological issues, stigma, employment) and perceived facilitators (e.g. pleasure and enjoyment derived from breastfeeding, natural milk from God, disclosure and family support) associated with EBF. Conclusions Most women preferred EBF and offered it to their infants. However, more efforts are needed to improve support structures at home and at work to accommodate women who choose to do EBF.

  10. Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

    PubMed Central

    Aligeti, Mounavya; Behrens, Ryan T.; Pocock, Ginger M.; Schindelin, Johannes; Dietz, Christian; Eliceiri, Kevin W.; Swanson, Chad M.; Malim, Michael H.; Ahlquist, Paul

    2014-01-01

    ABSTRACT Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCE Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev

  11. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    PubMed

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  12. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

    PubMed Central

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-01-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

  13. In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid.

    PubMed Central

    Schultz, A M; Oroszlan, S

    1983-01-01

    It has recently been shown by mass spectral analysis (Henderson et al., Proc. Natl. Acad. Sci. U.S.A. 80:339-343, 1983) that the p15gag protein of murine leukemia viruses contains a novel post-translational modification, an amino-terminal myristyl (tetradecanoyl) amide. In this report we show that p15gag is the only structural protein to contain this fatty acid. In addition, the gag precursor polyproteins of type B, C, and D retroviruses have been examined for the presence of myristic acid by metabolic labeling and immunoprecipitation studies. In a panel of mammalian type C retroviruses we found that the precursor polyprotein Pr65gag homologs, but not the glycosylated forms (gPr80gag homologs), were specifically labeled after a 5-min incubation of infected cells with [3H]myristic acid. The gag precursor polyprotein was also labeled in mouse mammary tumor virus and in Mason-Pfizer monkey virus, but Pr76gag of Rous sarcoma virus failed to incorporate [3H]myristate. Under similar conditions, [3H]palmitate was not found to be incorporated into any viral gag proteins. Thus, myristylation appears to be a common feature of mammalian type B, C, and D retroviruses but not of avian retroviruses. Images PMID:6302307

  14. Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

    PubMed Central

    Paul, Sinu; Piontkivska, Helen

    2009-01-01

    Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms). The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape

  15. Using nearly full-genome HIV sequence data improves phylogeny reconstruction in a simulated epidemic

    PubMed Central

    Yebra, Gonzalo; Hodcroft, Emma B.; Ragonnet-Cronin, Manon L.; Pillay, Deenan; Brown, Andrew J. Leigh; Fraser, Christophe; Kellam, Paul; de Oliveira, Tulio; Dennis, Ann; Hoppe, Anne; Kityo, Cissy; Frampton, Dan; Ssemwanga, Deogratius; Tanser, Frank; Keshani, Jagoda; Lingappa, Jairam; Herbeck, Joshua; Wawer, Maria; Essex, Max; Cohen, Myron S.; Paton, Nicholas; Ratmann, Oliver; Kaleebu, Pontiano; Hayes, Richard; Fidler, Sarah; Quinn, Thomas; Novitsky, Vladimir; Haywards, Andrew; Nastouli, Eleni; Morris, Steven; Clark, Duncan; Kozlakidis, Zisis

    2016-01-01

    HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to determine what gene(s) provide(s) the best approximation to the real phylogeny by analysing a simulated epidemic (created as part of the PANGEA_HIV project) with a known transmission tree. We sub-sampled a simulated dataset of 4662 sequences into different combinations of genes (gag-pol-env, gag-pol, gag, pol, env and partial pol) and sampling depths (100%, 60%, 20% and 5%), generating 100 replicates for each case. We built maximum-likelihood trees for each combination using RAxML (GTR + Γ), and compared their topologies to the corresponding true tree’s using CompareTree. The accuracy of the trees was significantly proportional to the length of the sequences used, with the gag-pol-env datasets showing the best performance and gag and partial pol sequences showing the worst. The lowest sampling depths (20% and 5%) greatly reduced the accuracy of tree reconstruction and showed high variability among replicates, especially when using the shortest gene datasets. In conclusion, using longer sequences derived from nearly whole genomes will improve the reliability of phylogenetic reconstruction. With low sample coverage, results can be highly variable, particularly when based on short sequences. PMID:28008945

  16. Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

    PubMed Central

    Felsenstein, K M; Goff, S P

    1992-01-01

    The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

  17. Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

    PubMed Central

    Schultz, A M; Lockhart, S M; Rabin, E M; Oroszlan, S

    1981-01-01

    The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15. Images PMID:7241663

  18. PIP2: choreographer of actin-adaptor proteins in the HIV-1 dance

    PubMed Central

    Rocha-Perugini, Vera; Gordon-Alonso, Mónica; Sánchez-Madrid, Francisco

    2014-01-01

    The actin cytoskeleton plays a key role during the replication cycle of human immunodeficiency virus-1 (HIV-1). HIV-1 infection is affected by cellular proteins that influence the clustering of viral receptors or the subcortical actin cytoskeleton. Several of these actin-adaptor proteins are controlled by the second messenger phosphatidylinositol 4,5-biphosphate (PIP2), an important regulator of actin organization. PIP2 production is induced by HIV-1 attachment and facilitates viral infection. However, the importance of PIP2 in regulating cytoskeletal proteins and thus HIV-1 infection has been overlooked. This review examines recent reports describing the roles played by actin-adaptor proteins during HIV-1 infection of CD4+ T cells, highlighting the influence of the signaling lipid PIP2 in this process. PMID:24768560

  19. Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    PubMed

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-05-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle. IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein

  20. Genetic diversity in the feline leukemia virus gag gene.

    PubMed

    Kawamura, Maki; Watanabe, Shinya; Odahara, Yuka; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

    2015-06-02

    Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The maturation inhibitor bevirimat (PA-457) can be active in patients carrying HIV type-1 non-B subtypes and recombinants.

    PubMed

    Yebra, Gonzalo; Holguín, Africa

    2008-01-01

    Bevirimat (PA-457) is the first candidate of a new family of antiretroviral drugs, the maturation inhibitors. Its action is based on disruption of the protease cleavage of the Gag precursor region. Six resistance mutations have been described and analysed in virus from both treatment-naive and protease inhibitor (PI)-experienced patients, but only in the subtype B of HIV type-1 (HIV-1) virus. Thus, genotypic resistance in non-B subtypes still requires analysis. HIV-1 sequences of different subtypes (54 B, 81 non-B and recombinants) were analysed for the presence of resistance mutations to bevirimat, located within the capsid (CA) protein and spacer peptide 1 (SP1) cleavage site. No resistance mutations were found, although polymorphisms appeared in some CA-SP1 residues. The C-terminal CA protein and the N-terminal SP1 presented high conservation, whereas C-terminal SP1 was highly variable in sequence and length, with unknown influence in resistance acquisition. The results of the present study confirm an absolute conservation of the residues involved in bevirimat in vitro resistance in a large panel of HIV-1 subtypes and recombinants from both treatment-naive and PI-experienced patients. Treatment alone seemed to increase the polymorphisms account in CRF02_AG recombinant sequences; however, the influence of natural polymorphisms needs to be explored.

  2. Facilitating factors of self-care among HIV-positive young women in Iran: a qualitative study.

    PubMed

    Oskouie, Fatemeh; Kashefi, Farzaneh; Rafii, Forough; Gouya, Mohammad Mehdi; Vahid-Dastjerdi, Marzieh

    2018-02-05

    Background Providing care for chronic disease such as HIV is a growing challenge in the world. In order to address the challenges of linkage and care in chronic disease management, we need to identify factors that can influence people to get more involved in self-care. This study was part of an extensive qualitative study conducted in Tehran, Iran in 2016. Methods The data were collected through semi-structured interviews conducted on 25 women with HIV, and were analyzed using grounded theory. Four main themes were identified as facilitating self-care among participants: health system support, clinicians' support, family support and improved life expectancy. Sub-themes that emerged were free HIV tests; free medication; free membership in positive clubs; free psychological consultation; positive attitudes and friendly behavior from clinic staff; telephone follow up; support from husbands, mothers and peers; hope for recovery; hope for the future; and love for own children. Results Our results showed that, providing appropriate support and services, as well as a positive attitude of society towards HIV positive women, can contribute to adherence to self-care in young women with HIV. Conclusion Understanding the facilitating factors based on the patients' experiences can contribute to the development of new policies and procedures to improve the care of these patients.

  3. Novel host restriction factors implicated in HIV-1 replication.

    PubMed

    Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

    2018-04-01

    Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

  4. Deep sequencing of near full-length HIV-1 genomes from plasma identifies circulating subtype C and infrequent occurrence of AC recombinant form in Southern India

    PubMed Central

    Sampathkumar, Raghavan; Sivaraman, Karthi; U. K. J., Anto Jesuraj; Dhar, Chirag; D. Souza, George; Berry, Neil

    2017-01-01

    India has the third largest number of HIV-1-infected individuals accounting for approximately 2.1 million people, with a predominance of circulating subtype C strains and a low prevalence of subtype A and A1C and BC recombinant forms, identified over the past two decades. Recovery of near full-length HIV-1 genomes from a plasma source coupled with advances in next generation sequencing (NGS) technologies and development of universal methods for amplifying whole genomes of HIV-1 circulating in a target geography or population provides the opportunity for a detailed analysis of HIV-1 strain identification, evolution and dynamics. Here we describe the development and implementation of approaches for HIV-1 NGS analysis in a southern Indian cohort. Plasma samples (n = 20) were obtained from HIV-1-confirmed individuals living in and around the city of Bengaluru. Near full-length genome recovery was obtained for 9 Indian HIV-1 patients, with recovery of full-length gag and env genes for 10 and 2 additional subjects, respectively. Phylogenetic analyses indicate the majority of sequences to be represented by subtype C viruses branching within a monophyletic clade, comprising viruses from India, Nepal, Myanmar and China and closely related to a southern African cluster, with a low prevalence of the A1C recombinant form also present. Development of algorithms for bespoke recovery and analysis at a local level will further aid clinical management of HIV-1 infected Indian subjects and delineate the progress of the HIV-1 pandemic in this and other geographical regions. PMID:29220350

  5. Quantifying Ongoing HIV-1 Exposure in HIV-1–Serodiscordant Couples to Identify Individuals With Potential Host Resistance to HIV-1

    PubMed Central

    Mackelprang, Romel D.; Baeten, Jared M.; Donnell, Deborah; Celum, Connie; Farquhar, Carey; de Bruyn, Guy; Essex, Max; McElrath, M. Juliana; Nakku-Joloba, Edith; Lingappa, Jairam R.

    2012-01-01

    Background. Immunogenetic correlates of resistance to HIV-1 in HIV-1–exposed seronegative (HESN) individuals with consistently high exposure may inform HIV-1 prevention strategies. We developed a novel approach for quantifying HIV-1 exposure to identify individuals remaining HIV-1 uninfected despite persistent high exposure. Methods. We used longitudinal predictors of HIV-1 transmission in HIV-1 serodiscordant couples to score HIV-1 exposure and define HESN clusters with persistently high, low, and decreasing risk trajectories. The model was validated in an independent cohort of serodiscordant couples. We describe a statistical tool that can be applied to other HESN cohorts to identify individuals with high exposure to HIV-1. Results. HIV-1 exposure was best quantified by frequency of unprotected sex with, plasma HIV-1 RNA levels among, and presence of genital ulcer disease among HIV-1–infected partners and by age, pregnancy status, herpes simplex virus 2 serostatus, and male circumcision status among HESN participants. Overall, 14% of HESN individuals persistently had high HIV-1 exposure and exhibited a declining incidence of HIV-1 infection over time. Conclusions. A minority of HESN individuals from HIV-1–discordant couples had persistent high HIV-1 exposure over time. Decreasing incidence of infection in this group suggests these individuals were selected for resistance to HIV-1 and may be most appropriate for identifying biological correlates of natural host resistance to HIV-1 infection. PMID:22926009

  6. Fluidity of HIV-1-Specific T-Cell Responses during Acute and Early Subtype C HIV-1 Infection and Associations with Early Disease Progression ▿

    PubMed Central

    Mlotshwa, Mandla; Riou, Catherine; Chopera, Denis; de Assis Rosa, Debra; Ntale, Roman; Treunicht, Florette; Woodman, Zenda; Werner, Lise; van Loggerenberg, Francois; Mlisana, Koleka; Abdool Karim, Salim; Williamson, Carolyn; Gray, Clive M.

    2010-01-01

    Deciphering immune events during early stages of human immunodeficiency virus type 1 (HIV-1) infection is critical for understanding the course of disease. We characterized the hierarchy of HIV-1-specific T-cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses during acute subtype C infection in 53 individuals and associated temporal patterns of responses with disease progression in the first 12 months. There was a diverse pattern of T-cell recognition across the proteome, with the recognition of Nef being immunodominant as early as 3 weeks postinfection. Over the first 6 months, we found that there was a 23% chance of an increased response to Nef for every week postinfection (P = 0.0024), followed by a nonsignificant increase to Pol (4.6%) and Gag (3.2%). Responses to Env and regulatory proteins appeared to remain stable. Three temporal patterns of HIV-specific T-cell responses could be distinguished: persistent, lost, or new. The proportion of persistent T-cell responses was significantly lower (P = 0.0037) in individuals defined as rapid progressors than in those progressing slowly and who controlled viremia. Almost 90% of lost T-cell responses were coincidental with autologous viral epitope escape. Regression analysis between the time to fixed viral escape and lost T-cell responses (r = 0.61; P = 0.019) showed a mean delay of 14 weeks after viral escape. Collectively, T-cell epitope recognition is not a static event, and temporal patterns of IFN-γ-based responses exist. This is due partly to viral sequence variation but also to the recognition of invariant viral epitopes that leads to waves of persistent T-cell immunity, which appears to associate with slower disease progression in the first year of infection. PMID:20826686

  7. In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations

    PubMed Central

    Yang, Yi; Sun, Weilai; Guo, Jingjing; Zhao, Guangyu; Sun, Shihui; Yu, Hong; Guo, Yan; Li, Jungfeng; Jin, Xia; Du, Lanying; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-01-01

    The development of an HIV-1 vaccine that is capable of inducing effective and broadly cross-reactive humoral and cellular immune responses remains a challenging task because of the extensive diversity of HIV-1, the difference of virus subtypes (clades) in different geographical regions, and the polymorphism of human leukocyte antigens (HLA). We performed an in silico design of 3 DNA vaccines, designated pJW4303-MEG1, pJW4303-MEG2 and pJW4303-MEG3, encoding multi-epitopes that are highly conserved within the HIV-1 subtypes most prevalent in China and can be recognized through HLA alleles dominant in China. The pJW4303-MEG1-encoded protein consisted of one Th epitope in Env, and one, 2, and 6 epitopes in Pol, Env, and Gag proteins, respectively, with a GGGS linker sequence between epitopes. The pJW4303-MEG2-encoded protein contained similar epitopes in a different order, but with the same linker as pJW4303-MEG1. The pJW4303-MEG3-encoded protein contained the same epitopes in the same order as that of pJW4303-MEG2, but with a different linker sequence (AAY). To evaluate immunogenicity, mice were immunized intramuscularly with these DNA vaccines. Both pJW4303-MEG1 and pJW4303-MEG2 vaccines induced equally potent humoral and cellular immune responses in the vaccinated mice, while pJW4303-MEG3 did not induce immune responses. These results indicate that both epitope and linker sequences are important in designing effective epitope-based vaccines against HIV-1 and other viruses. PMID:25839222

  8. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    NASA Astrophysics Data System (ADS)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  9. [Physical restraints and gagging in unnatural death].

    PubMed

    Grellner, W; Madea, B

    1993-01-01

    Practices of binding and gagging can be found in different types of unnatural death; three case reports concerning fatal autoeroticism, suicide and homicide are presented. In such cases investigations are usually concentrated on the question whether binding and gagging could be carried out by the persons themselves. Loosely bound final loops, especially of the hands, missing local haemorrhages and the discovery of binding instructions support the assumption of self-binding. Accompanying injuries and the entire situation must be taken into special consideration.

  10. Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

    PubMed

    Abana, Chike O; Pilkinton, Mark A; Gaudieri, Silvana; Chopra, Abha; McDonnell, Wyatt J; Wanjalla, Celestine; Barnett, Louise; Gangula, Rama; Hager, Cindy; Jung, Dae K; Engelhardt, Brian G; Jagasia, Madan H; Klenerman, Paul; Phillips, Elizabeth J; Koelle, David M; Kalams, Spyros A; Mallal, Simon A

    2017-11-01

    Select CMV epitopes drive life-long CD8 + T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4 + T cells specific for human CMV (HCMV) are elevated in HIV + HCMV + subjects. To determine whether HCMV epitope-specific CD4 + T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4 + T cells in coinfected HLA-DR7 + long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4 + T cells were inflated among these HIV + subjects compared with those from an HIV - HCMV + HLA-DR7 + cohort or with HLA-DR7-restricted CD4 + T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4 + T cells consisted of effector memory or effector memory-RA + subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX 3 CR1, CD38, or HLA-DR but less often coexpressed CD38 + and HLA-DR + The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4 + T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  11. CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

    PubMed Central

    Carlson, Jonathan; Yan, Jiyu; Akinsiku, Olusimidele T.; Schaefer, Malinda; Sabbaj, Steffanie; Bet, Anne; Levy, David N.; Heath, Sonya; Tang, Jianming; Kaslow, Richard A.; Walker, Bruce D.; Ndung’u, Thumbi; Goulder, Philip J.; Heckerman, David; Hunter, Eric; Goepfert, Paul A.

    2010-01-01

    Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity. PMID:20065064

  12. Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

    PubMed Central

    Weldon, R A; Wills, J W

    1993-01-01

    Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25. Images PMID:8394460

  13. Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

    PubMed

    Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

    2010-10-15

    The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV

  14. Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    LeBlanc, Jason J.; Uddowla, Sabena; Abraham, Benjamin

    2007-07-05

    All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had amore » diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.« less

  15. Exploring barriers and facilitators to participation of male-to-female transgender persons in preventive HIV vaccine clinical trials.

    PubMed

    Andrasik, Michele Peake; Yoon, Ro; Mooney, Jessica; Broder, Gail; Bolton, Marcus; Votto, Teress; Davis-Vogel, Annet

    2014-06-01

    Observed seroincidence and prevalence rates in male-to-female (MTF) transgender individuals highlight the need for effective targeted HIV prevention strategies for this community. In order to develop an effective vaccine that can be used by transgender women, researchers must understand and address existing structural issues that present barriers to this group's participation in HIV vaccine clinical trials. Overcoming barriers to participation is important for ensuring HIV vaccine acceptability and efficacy for the MTF transgender community. To explore barriers and facilitators to MTF transgender participation in preventive HIV vaccine clinical trials, the HIV Vaccine Trials Network conducted focus groups among transgender women in four urban areas (Atlanta, Boston, Philadelphia, and San Francisco). Barriers and facilitators to engagement of transgender women in preventive HIV vaccine clinical trials led to the following recommendations: (a) transgender cultural competency training, (b) creating trans-friendly environments, (c) true partnerships with local trans-friendly organizations and health care providers, (d) protocols that focus on transgender specific issues, and (e) data collection and tracking of transgender individuals. These results have implications for the conduct of HIV vaccine trials, as well as engagement of transgender women in research programs in general.

  16. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia

    2006-09-01

    We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to themore » cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays.« less

  17. BARRIERS AND FACILITATORS TO THE IMPLEMENTATION OF A NATIONAL HIV LINKAGE, RE-ENGAGEMENT, AND RETENTION IN CARE PROGRAM

    PubMed Central

    Maulsby, Cathy; Sacamano, Paul; Jain, Kriti M.; Enobun, Blessing; Brantley, Meredith L.; Kim, Hae-Young; Riordan, Morey; Werner, Melissa; Holtgrave, David R.

    2018-01-01

    The 2020 National HIV AIDS Strategy (NHAS) sets a target of 90% of diagnosed people living with HIV (PLWH) retained in HIV care. Access to Care (A2C) was a national HIV linkage, re-engagement, and retention in care program funded by AIDS United with support from the Corporation for National and Community Service that aimed to link and retain the most vulnerable PLWH into high-quality HIV care. This study explores the barriers and facilitators of implementing the A2C program from the perspective of program staff. Ninety-eight qualitative interviews were conducted with staff at implementing organizations over the 5 years of the project. Barriers included challenges with recruiting and retaining participants, staffing and administration, harmonizing partnerships, and addressing the basic and psychosocial needs of participants. Facilitators included strong relationships with partner organizations, flexible program models, and the passion and dedication of staff. Findings will inform the development of future programs and policy. PMID:29068718

  18. HIV-1 Full-Genome Phylogenetics of Generalized Epidemics in Sub-Saharan Africa: Impact of Missing Nucleotide Characters in Next-Generation Sequences

    PubMed Central

    Wymant, Chris; Colijn, Caroline; Danaviah, Siva; Essex, Max; Frost, Simon; Gall, Astrid; Gaseitsiwe, Simani; Grabowski, Mary K.; Gray, Ronald; Guindon, Stephane; von Haeseler, Arndt; Kaleebu, Pontiano; Kendall, Michelle; Kozlov, Alexey; Manasa, Justen; Minh, Bui Quang; Moyo, Sikhulile; Novitsky, Vlad; Nsubuga, Rebecca; Pillay, Sureshnee; Quinn, Thomas C.; Serwadda, David; Ssemwanga, Deogratius; Stamatakis, Alexandros; Trifinopoulos, Jana; Wawer, Maria; Brown, Andy Leigh; de Oliveira, Tulio; Kellam, Paul; Pillay, Deenan; Fraser, Christophe

    2017-01-01

    Abstract To characterize HIV-1 transmission dynamics in regions where the burden of HIV-1 is greatest, the “Phylogenetics and Networks for Generalised HIV Epidemics in Africa” consortium (PANGEA-HIV) is sequencing full-genome viral isolates from across sub-Saharan Africa. We report the first 3,985 PANGEA-HIV consensus sequences from four cohort sites (Rakai Community Cohort Study, n = 2,833; MRC/UVRI Uganda, n = 701; Mochudi Prevention Project, n = 359; Africa Health Research Institute Resistance Cohort, n = 92). Next-generation sequencing success rates varied: more than 80% of the viral genome from the gag to the nef genes could be determined for all sequences from South Africa, 75% of sequences from Mochudi, 60% of sequences from MRC/UVRI Uganda, and 22% of sequences from Rakai. Partial sequencing failure was primarily associated with low viral load, increased for amplicons closer to the 3′ end of the genome, was not associated with subtype diversity except HIV-1 subtype D, and remained significantly associated with sampling location after controlling for other factors. We assessed the impact of the missing data patterns in PANGEA-HIV sequences on phylogeny reconstruction in simulations. We found a threshold in terms of taxon sampling below which the patchy distribution of missing characters in next-generation sequences (NGS) has an excess negative impact on the accuracy of HIV-1 phylogeny reconstruction, which is attributable to tree reconstruction artifacts that accumulate when branches in viral trees are long. The large number of PANGEA-HIV sequences provides unprecedented opportunities for evaluating HIV-1 transmission dynamics across sub-Saharan Africa and identifying prevention opportunities. Molecular epidemiological analyses of these data must proceed cautiously because sequence sampling remains below the identified threshold and a considerable negative impact of missing characters on phylogeny reconstruction is expected. PMID

  19. HIV-1 full-genome phylogenetics of generalized epidemics in sub-Saharan Africa: impact of missing nucleotide characters in next-generation sequences.

    PubMed

    Ratmann, Oliver; Wymant, Chris; Colijn, Caroline; Danaviah, Siva; Essex, M; Frost, Simon D W; Gall, Astrid; Gaiseitsiwe, Simani; Grabowski, Mary; Gray, Ronald; Guindon, Stephane; von Haeseler, Arndt; Kaleebu, Pontiano; Kendall, Michelle; Kozlov, Alexey; Manasa, Justen; Minh, Bui Quang; Moyo, Sikhulile; Novitsky, Vladimir; Nsubuga, Rebecca; Pillay, Sureshnee; Quinn, Thomas C; Serwadda, David; Ssemwanga, Deogratius; Stamatakis, Alexandros; Trifinopoulos, Jana; Wawer, Maria; Leigh Brown, Andrew; de Oliveira, Tulio; Kellam, Paul; Pillay, Deenan; Fraser, Christophe

    2017-05-25

    To characterize HIV-1 transmission dynamics in regions where the burden of HIV-1 is greatest, the 'Phylogenetics and Networks for Generalised HIV Epidemics in Africa' consortium (PANGEA-HIV) is sequencing full-genome viral isolates from across sub-Saharan Africa. We report the first 3,985 PANGEA-HIV consensus sequences from four cohort sites (Rakai Community Cohort Study, n=2,833; MRC/UVRI Uganda, n=701; Mochudi Prevention Project, n=359; Africa Health Research Institute Resistance Cohort, n=92). Next-generation sequencing success rates varied: more than 80% of the viral genome from the gag to the nef genes could be determined for all sequences from South Africa, 75% of sequences from Mochudi, 60% of sequences from MRC/UVRI Uganda, and 22% of sequences from Rakai. Partial sequencing failure was primarily associated with low viral load, increased for amplicons closer to the 3' end of the genome, was not associated with subtype diversity except HIV-1 subtype D, and remained significantly associated with sampling location after controlling for other factors. We assessed the impact of the missing data patterns in PANGEA-HIV sequences on phylogeny reconstruction in simulations. We found a threshold in terms of taxon sampling below which the patchy distribution of missing characters in next-generation sequences has an excess negative impact on the accuracy of HIV-1 phylogeny reconstruction, which is attributable to tree reconstruction artifacts that accumulate when branches in viral trees are long. The large number of PANGEA-HIV sequences provides unprecedented opportunities for evaluating HIV-1 transmission dynamics across sub-Saharan Africa and identifying prevention opportunities. Molecular epidemiological analyses of these data must proceed cautiously because sequence sampling remains below the identified threshold and a considerable negative impact of missing characters on phylogeny reconstruction is expected.

  20. Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

    PubMed Central

    Gandapu, Upendhar; Chaitanya, R. K.; Kishore, Golla; Reddy, Raju C.; Kondapi, Anand K.

    2011-01-01

    Background Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research. Methodology/Principal Findings In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI50 = 15.6 µM) is twice more toxic than nano-curcumin (GI50 = 32.5 µM), nano-curcumin (IC50<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC50 = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis. Conclusion Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness. PMID:21887247

  1. HIITE: HIV-1 incidence and infection time estimator.

    PubMed

    Park, Sung Yong; Love, Tanzy M T; Kapoor, Shivankur; Lee, Ha Youn

    2018-06-15

    Around 2.1 million new HIV-1 infections were reported in 2015, alerting that the HIV-1 epidemic remains a significant global health challenge. Precise incidence assessment strengthens epidemic monitoring efforts and guides strategy optimization for prevention programs. Estimating the onset time of HIV-1 infection can facilitate optimal clinical management and identify key populations largely responsible for epidemic spread and thereby infer HIV-1 transmission chains. Our goal is to develop a genomic assay estimating the incidence and infection time in a single cross-sectional survey setting. We created a web-based platform, HIV-1 incidence and infection time estimator (HIITE), which processes envelope gene sequences using hierarchical clustering algorithms and informs the stage of infection, along with time since infection for incident cases. HIITE's performance was evaluated using 585 incident and 305 chronic specimens' envelope gene sequences collected from global cohorts including HIV-1 vaccine trial participants. HIITE precisely identified chronically infected individuals as being chronic with an error less than 1% and correctly classified 94% of recently infected individuals as being incident. Using a mixed-effect model, an incident specimen's time since infection was estimated from its single lineage diversity, showing 14% prediction error for time since infection. HIITE is the first algorithm to inform two key metrics from a single time point sequence sample. HIITE has the capacity for assessing not only population-level epidemic spread but also individual-level transmission events from a single survey, advancing HIV prevention and intervention programs. Web-based HIITE and source code of HIITE are available at http://www.hayounlee.org/software.html. Supplementary data are available at Bioinformatics online.

  2. Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

    PubMed Central

    Spearman, P; Horton, R; Ratner, L; Kuli-Zade, I

    1997-01-01

    The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction. PMID:9261380

  3. Weaker HLA Footprints on HIV in the Unique and Highly Genetically Admixed Host Population of Mexico

    PubMed Central

    2017-01-01

    ABSTRACT HIV circumvents HLA class I-restricted CD8+ T-cell responses through selection of escape mutations that leave characteristic mutational “footprints,” also known as HLA-associated polymorphisms (HAPs), on HIV sequences at the population level. While many HLA footprints are universal across HIV subtypes and human populations, others can be region specific as a result of the unique immunogenetic background of each host population. Using a published probabilistic phylogenetically informed model, we compared HAPs in HIV Gag and Pol (PR-RT) in 1,612 subtype B-infected, antiretroviral treatment-naive individuals from Mexico and 1,641 individuals from Canada/United States. A total of 252 HLA class I allele subtypes were represented, including 140 observed in both cohorts, 67 unique to Mexico, and 45 unique to Canada/United States. At the predefined statistical threshold of a q value of <0.2, 358 HAPs (201 in Gag, 157 in PR-RT) were identified in Mexico, while 905 (534 in Gag and 371 in PR-RT) were identified in Canada/United States. HAPs identified in Mexico included both canonical HLA-associated escape pathways and novel associations, in particular with HLA alleles enriched in Amerindian and mestizo populations. Remarkably, HLA footprints on HIV in Mexico were not only fewer but also, on average, significantly weaker than those in Canada/United States, although some exceptions were noted. Moreover, exploratory analyses suggested that the weaker HLA footprint on HIV in Mexico may be due, at least in part, to weaker and/or less reproducible HLA-mediated immune pressures on HIV in this population. The implications of these differences for natural and vaccine-induced anti-HIV immunity merit further investigation. IMPORTANCE HLA footprints on HIV identify viral regions under intense and consistent pressure by HLA-restricted immune responses and the common mutational pathways that HIV uses to evade them. In particular, HLA footprints can identify novel immunogenic

  4. Weaker HLA Footprints on HIV in the Unique and Highly Genetically Admixed Host Population of Mexico.

    PubMed

    Soto-Nava, Maribel; Avila-Ríos, Santiago; Valenzuela-Ponce, Humberto; García-Morales, Claudia; Carlson, Jonathan M; Tapia-Trejo, Daniela; Garrido-Rodriguez, Daniela; Alva-Hernández, Selma N; García-Tellez, Thalía A; Murakami-Ogasawara, Akio; Mallal, Simon A; John, Mina; Brockman, Mark A; Brumme, Chanson J; Brumme, Zabrina L; Reyes-Teran, Gustavo

    2018-01-15

    HIV circumvents HLA class I-restricted CD8 + T-cell responses through selection of escape mutations that leave characteristic mutational "footprints," also known as HLA-associated polymorphisms (HAPs), on HIV sequences at the population level. While many HLA footprints are universal across HIV subtypes and human populations, others can be region specific as a result of the unique immunogenetic background of each host population. Using a published probabilistic phylogenetically informed model, we compared HAPs in HIV Gag and Pol (PR-RT) in 1,612 subtype B-infected, antiretroviral treatment-naive individuals from Mexico and 1,641 individuals from Canada/United States. A total of 252 HLA class I allele subtypes were represented, including 140 observed in both cohorts, 67 unique to Mexico, and 45 unique to Canada/United States. At the predefined statistical threshold of a q value of <0.2, 358 HAPs (201 in Gag, 157 in PR-RT) were identified in Mexico, while 905 (534 in Gag and 371 in PR-RT) were identified in Canada/United States. HAPs identified in Mexico included both canonical HLA-associated escape pathways and novel associations, in particular with HLA alleles enriched in Amerindian and mestizo populations. Remarkably, HLA footprints on HIV in Mexico were not only fewer but also, on average, significantly weaker than those in Canada/United States, although some exceptions were noted. Moreover, exploratory analyses suggested that the weaker HLA footprint on HIV in Mexico may be due, at least in part, to weaker and/or less reproducible HLA-mediated immune pressures on HIV in this population. The implications of these differences for natural and vaccine-induced anti-HIV immunity merit further investigation. IMPORTANCE HLA footprints on HIV identify viral regions under intense and consistent pressure by HLA-restricted immune responses and the common mutational pathways that HIV uses to evade them. In particular, HLA footprints can identify novel immunogenic regions and

  5. Computer-facilitated rapid HIV testing in emergency care settings: provider and patient usability and acceptability.

    PubMed

    Spielberg, Freya; Kurth, Ann E; Severynen, Anneleen; Hsieh, Yu-Hsiang; Moring-Parris, Daniel; Mackenzie, Sara; Rothman, Richard

    2011-06-01

    Providers in emergency care settings (ECSs) often face barriers to expanded HIV testing. We undertook formative research to understand the potential utility of a computer tool, "CARE," to facilitate rapid HIV testing in ECSs. Computer tool usability and acceptability were assessed among 35 adult patients, and provider focus groups were held, in two ECSs in Washington State and Maryland. The computer tool was usable by patients of varying computer literacy. Patients appreciated the tool's privacy and lack of judgment and their ability to reflect on HIV risks and create risk reduction plans. Staff voiced concerns regarding ECS-based HIV testing generally, including resources for follow-up of newly diagnosed people. Computer-delivered HIV testing support was acceptable and usable among low-literacy populations in two ECSs. Such tools may help circumvent some practical barriers associated with routine HIV testing in busy settings though linkages to care will still be needed.

  6. Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome?

    PubMed Central

    Han, Weining; Li, Yuejin; Bagaya, Bernard S.; Tian, Meijuan; Chamanian, Mastooreh; Zhu, Chuanwu; Shen, Jie; Gao, Yong

    2016-01-01

    Although the process of reverse transcription is well elucidated, it remains unclear if viral core disruption provides a more cellular or viral milieu for HIV-1 reverse transcription. We have devised a method to require mixing of viral cores or core constituents to produce infectious progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each other and when together can complete viral reverse transcription. Only the heterodiploid virus containing both the nfl and cplt_R/U5/gag/Δpol sgRNAs can complete reverse transcription and propagate infectious virus upon de novo infection. Dual exposure of U87.CD4.CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/Δpol virus particles did not result in productive virus infection. On the other hand, in early endosomes, the HIV-1 sgRNAs released from viral cores can retain function and complete the reverse transcription and result in productive infection. These findings confirm the assumptions that, in natural infection, HIV-1 cores, and likely other retrovirus cores, remain largely intact and do not mix/fuse in the cytoplasm during the reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA (produced through transfection) could not help the complementary sgRNA in the viral core to complement the reverse transcription process. PMID:27239643

  7. The role of community conversations in facilitating local HIV competence: case study from rural Zimbabwe

    PubMed Central

    2013-01-01

    Background This paper examines the potential for community conversations to strengthen positive responses to HIV in resource-poor environments. Community conversations are an intervention method through which local people work with a facilitator to collectively identify local strengths and challenges and brainstorm potential strategies for solving local problems. Methods We conducted 18 community conversations (with six groups at three points in time) with a total of 77 participants in rural Zimbabwe (20% HIV positive). Participants were invited to reflect on how they were responding to the challenges of HIV, both as individuals and in community groups, and to think of ways to better support openness about HIV, kindness towards people living with HIV and greater community uptake of HIV prevention and treatment. Results Community conversations contributed to local HIV competence through (1) enabling participants to brainstorm concrete action plans for responding to HIV, (2) providing a forum to develop a sense of common purpose in relation to implementing these, (3) encouraging and challenging participants to overcome fear, denial and passivity, (4) providing an opportunity for participants to move from seeing themselves as passive recipients of information to active problem solvers, and (5) reducing silence and stigma surrounding HIV. Conclusions Our discussion cautions that community conversations, while holding great potential to help communities recognize their potential strengths and capacities for responding more effectively to HIV, are not a magic bullet. Poverty, poor harvests and political instability frustrated and limited many participants’ efforts to put their plans into action. On the other hand, support from outside the community, in this case the increasing availability of antiretroviral treatment, played a vital role in enabling communities to challenge stigma and envision new, more positive, ways of responding to the epidemic. PMID:23590640

  8. The role of community conversations in facilitating local HIV competence: case study from rural Zimbabwe.

    PubMed

    Campbell, Catherine; Nhamo, Mercy; Scott, Kerry; Madanhire, Claudius; Nyamukapa, Constance; Skovdal, Morten; Gregson, Simon

    2013-04-17

    This paper examines the potential for community conversations to strengthen positive responses to HIV in resource-poor environments. Community conversations are an intervention method through which local people work with a facilitator to collectively identify local strengths and challenges and brainstorm potential strategies for solving local problems. We conducted 18 community conversations (with six groups at three points in time) with a total of 77 participants in rural Zimbabwe (20% HIV positive). Participants were invited to reflect on how they were responding to the challenges of HIV, both as individuals and in community groups, and to think of ways to better support openness about HIV, kindness towards people living with HIV and greater community uptake of HIV prevention and treatment. Community conversations contributed to local HIV competence through (1) enabling participants to brainstorm concrete action plans for responding to HIV, (2) providing a forum to develop a sense of common purpose in relation to implementing these, (3) encouraging and challenging participants to overcome fear, denial and passivity, (4) providing an opportunity for participants to move from seeing themselves as passive recipients of information to active problem solvers, and (5) reducing silence and stigma surrounding HIV. Our discussion cautions that community conversations, while holding great potential to help communities recognize their potential strengths and capacities for responding more effectively to HIV, are not a magic bullet. Poverty, poor harvests and political instability frustrated and limited many participants' efforts to put their plans into action. On the other hand, support from outside the community, in this case the increasing availability of antiretroviral treatment, played a vital role in enabling communities to challenge stigma and envision new, more positive, ways of responding to the epidemic.

  9. PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1.

    PubMed

    Yoh, Sunnie M; Schneider, Monika; Seifried, Janna; Soonthornvacharin, Stephen; Akleh, Rana E; Olivieri, Kevin C; De Jesus, Paul D; Ruan, Chunhai; de Castro, Elisa; Ruiz, Pedro A; Germanaud, David; des Portes, Vincent; García-Sastre, Adolfo; König, Renate; Chanda, Sumit K

    2015-06-04

    Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. The Foreseeable Harms of Trump's Global Gag Rule.

    PubMed

    Bingenheimer, Jeffrey B; Skuster, Patty

    2017-09-01

    As one of his first acts as President of the United States, Donald Trump signed an executive order reinstating a version of the global gag rule. Under this rule, US grantees are barred from receiving global health funding if they engage in abortion-related work: not only abortion services, but also abortion referrals and counseling or advocacy for the liberalization of abortion laws. Critics of the Trump global gag rule generally raise three classes of objections: (1) that the rule fails to accomplish its presumed objective of reducing the number of abortions; (2) that it negatively affects the health and well-being of individuals and populations in affected countries; and (3) that it interferes with governments' ability to meet their international obligations. In this commentary, we examine the scientific and policy bases for these criticisms. © 2017 The Population Council, Inc.

  11. Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

    PubMed

    Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

    2016-03-01

    This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to

  12. Barriers and Facilitators to Engagement of Vulnerable Populations in HIV Primary Care in New York City

    PubMed Central

    Remien, Robert H.; Bauman, Laurie J.; Mantell, Joanne; Tsoi, Benjamin; Lopez-Rios, Javier; Chhabra, Rosy; DiCarlo, Abby; Watnick, Dana; Rivera, Angelic; Teitelman, Nehama; Cutler, Blayne; Warne, Patricia

    2015-01-01

    Background Engagement in HIV care helps to maximize viral suppression, which, in turn, reduces morbidity and mortality and prevents further HIV transmission. With more HIV cases than any other US city, New York City reported in 2012 that only 41% of all persons estimated to be living with HIV (PLWH) had a suppressed viral load, while nearly three-quarters of those in clinical care achieved viral suppression. Thus, retaining PLWH in HIV care addresses this central goal of both the US National HIV/AIDS Strategy and Governor Cuomo's plan to end the AIDS epidemic in New York State. Methods We conducted 80 in-depth qualitative interviews with PLWH in four NYC populations that were identified as being inconsistently engaged in HIV medical care: African immigrants, previously incarcerated adults, transgender women, and young men who have sex with men. Results Barriers to and facilitators of HIV care engagement fell into three domains: (1) system factors (e.g., patient-provider relationship, social service agencies, transitions between penal system and community); (2) social factors (e.g., family and other social support; stigma related to HIV, substance use, sexual orientation, gender identity, and incarceration); and (3) individual factors (e.g., mental illness, substance use, resilience). Similarities and differences in these themes across the four populations as well as research and public health implications were identified. Conclusions Engagement in care is maximized when the social challenges confronted by vulnerable groups are addressed; patient-provider communication is strong; and coordinated services are available, including housing, mental health and substance use treatment, and peer navigation. PMID:25867774

  13. A polyvalent Clade B virus-like particle HIV vaccine combined with partially protective oral preexposure prophylaxis prevents simian-human immunodeficiency virus Infection in macaques and primes for virus-amplified immunity.

    PubMed

    Ross, Ted M; Pereira, Lara E; Luckay, Amara; McNicholl, Janet M; García-Lerma, J Gerardo; Heneine, Walid; Eugene, Hermancia S; Pierce-Paul, Brooke R; Zhang, Jining; Hendry, R Michael; Smith, James M

    2014-11-01

    Vaccination and preexposure prophylaxis (PrEP) with antiretrovirals have shown only partial protection from HIV-1 infection in human trials. Oral Truvada (emtricitabine/tenofovir disoproxil fumarate) is FDA approved as PrEP but partial adherence reduces efficacy. If combined as biomedical preventions (CBP), an HIV vaccine could protect when PrEP adherence is low and PrEP could prevent vaccine breakthroughs. The efficacy of combining oral PrEP with an HIV vaccine has not been evaluated in humans. We determined the efficacy of combining a DNA/virus-like particle (VLP) vaccine with partially effective intermittent PrEP in Indian rhesus macaques (RM). Eight RM received intramuscular inoculations of five DNA plasmids encoding four HIV-1 Clade B primary isolate Envs and SIVmac239 Gag (at weeks 0 and 4), followed by intramuscular and intranasal inoculations of homologous Gag VLPs and four Env VLPs (at weeks 12, 16, and 53). At week 61, we initiated weekly rectal exposures with heterologous SHIV162p3 (10 TCID50) along with oral Truvada (TDF, 22 mg/kg; FTC 20 mg/kg) dosing 2 h before and 22 h after each exposure. This PrEP regimen previously demonstrated 50% efficacy. Five controls (no vaccine, no PrEP) received weekly SHIV162p3. All controls were infected after a median of four exposures; the mean peak plasma viral load (VL) was 3.9×10(7) vRNA copies/ml. CBP protected seven of eight (87.5%) RM. The one infected CBP RM had a reduced peak VL of 8.8×10(5) copies/ml. SHIV exposures during PrEP amplified Gag and Env antibody titers in protected RM. These results suggest that combining oral PrEP with HIV vaccines could enhance protection against HIV-1 infection.

  14. Barriers and facilitators to the implementation of SPNS interventions designed to engage and retain HIV positive women of color in medical care.

    PubMed

    Garcia, Iliana A; Blank, Arthur E; Eastwood, Elizabeth A; Karasz, Alison

    2015-04-01

    The use of evidence-based strategies to increase access to medical care and improve health outcomes for people living with HIV is a major public health priority in the United States. As part of a multi-site evaluation funded under the Health Resources and Services Administration (HRSA), a process evaluation was conducted with the goal of understanding barriers and facilitators to the implementation of eleven heterogeneous interventions designed to engage and retain HIV positive women of color (WoC) in medical care. Findings identified barriers and facilitators to program implementation at five levels: (1) program; (2) team; (3) agency; (4) partner network; and (5) the larger socio-ecological context. We conclude with a series of recommendations that may be useful for the implementation of similar interventions focused on recruitment and retention of WoC in HIV medical care.

  15. A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.

    PubMed

    Claireaux, M; Galperin, M; Benati, D; Nouël, A; Mukhopadhyay, M; Klingler, J; de Truchis, P; Zucman, D; Hendou, S; Boufassa, F; Moog, C; Lambotte, O; Chakrabarti, L A

    2018-05-08

    Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4 + T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels and compared them to treated patients with similarly low viral loads. HIV-specific cTfh (Tet + ), detected by Gag-major histocompatibility complex class II (MHC-II) tetramer labeling in the CD45RA - CXCR5 + CD4 + T cell population, proved more frequent in the controller group ( P = 0.002). The frequency of PD-1 expression in Tet + cTfh was increased in both groups (median, >75%) compared to total cTfh (<30%), but the intensity of PD-1 expression per cell remained higher in the treated patient group ( P = 0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet + cTfh correlated with HIV-specific IgG production ( R = 0.71 for Gag-specific and R = 0.79 for Env-specific IgG, respectively). Taken together, our findings indicate that key cTfh-B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control. IMPORTANCE The rare patients who spontaneously control HIV replication in the absence of therapy provide a unique model to identify determinants of an effective anti-HIV immune response. HIV controllers show signs of particularly efficient antiviral T cell responses, while their humoral response was until recently

  16. High prevalence of bevirimat resistance mutations in protease inhibitor-resistant HIV isolates.

    PubMed

    Verheyen, Jens; Verhofstede, Chris; Knops, Elena; Vandekerckhove, Linos; Fun, Axel; Brunen, Diede; Dauwe, Kenny; Wensing, Annemarie M J; Pfister, Herbert; Kaiser, Rolf; Nijhuis, Monique

    2010-03-13

    Bevirimat is the first drug of a new class of antivirals that hamper the maturation of HIV. The objective of this study was to evaluate the sequence variability of the gag region targeted by bevirimat in HIV subtype-B isolates. Of 484 HIV subtype-B isolates, the gag region comprising amino acids 357-382 was sequenced. Of the patients included, 270 were treatment naive and 214 were treatment experienced. In the latter group, 48 HIV isolates harboured mutations associated with reverse transcriptase inhibitor resistance only, and 166 HIV isolates carried mutations associated with protease inhibitor resistance. In the treatment-naive patient population, approximately 30% harboured an HIV isolate with at least one mutation associated with a reduced susceptibility to bevirimat (H358Y, L363M, Q369H, V370A/M/del and T371del). In HIV isolates with protease inhibitor resistance, the prevalence of bevirimat resistance mutations increased to 45%. Accumulation of mutations at four positions in the bevirimat target region, S368C, Q369H, V370A and S373P, was significantly observed. Mutations associated with bevirimat resistance were detected more frequently in HIV isolates with three or more protease inhibitor resistance mutations than in those with less than three protease inhibitor mutations. Reduced bevirimat activity can be expected in one-third of treatment-naive HIV subtype-B isolates and significantly more in protease inhibitor-resistant HIV. These data indicate that screening for bevirimat resistance mutations before administration of the drug is essential.

  17. X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feng, Youjun; Graduate School, Chinese Academy of Sciences, Beijing; Qi, Jianxun

    2006-01-01

    X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide. Simian immunodeficiency virus (SIV) in the rhesus macaque is regarded as a classic animal model, playing a crucial role in HIV vaccine strategies and therapeutics by characterizing various cytotoxic T-lymphocyte (CTL) responses in macaque monkeys. However, the availability of well documented structural reports focusing on rhesus macaque major histocompatibility complex class I (MHC I) molecules remains extremely limited. Here, a complex of the rhesus macaque MHC I molecule (Mamu-A*02) with human β{sub 2}m and an immunodominant SIV-Gag nonapeptide, GESNLKSLY (GY9), has been crystallized. The crystal diffractsmore » X-rays to 2.7 Å resolution and belongs to space group C2, with unit-cell parameters a = 124.11, b = 110.45, c = 100.06 Å, and contains two molecules in the asymmetric unit. The availability of the structure, which is being solved by molecular replacement, will provide new insights into rhesus macaque MHC I (Mamu-A*02) presenting pathogenic SIV peptides.« less

  18. Are religion and spirituality barriers or facilitators to treatment for HIV: a systematic review of the literature.

    PubMed

    Medved Kendrick, Haley

    2017-01-01

    Much attention has been given to the relationship between religion/spirituality (R/S) and HIV in recent years, but comparatively little has been explored in regard to R/S and HIV testing, retention in care, and adherence to medication. Religious views concerning HIV risk behavior pose challenges to communication and education about sexual health in religious communities and may serve as barriers to HIV treatment and care. Conversely, religious coping and spiritual well-being, as well as social support could serve as facilitators to HIV treatment and care. This study aims to fill a gap in the literature by addressing the following questions: (1) what dimensions of R/S have been found to be factors associated with HIV outcomes?; (2) which R/S factors function as barriers or facilitators to care among people living with HIV (PLWH)?; and (3) which R/S factors, if any, vary across socio-demographic groups? Thirty-three empirical articles were identified for systematic review. Of the 33 empirical studies included, 24 studies found that at least one measure of R/S was associated with better adherence and clinical health outcomes. Twelve studies found at least one measure of R/S to be associated with poorer adherence and clinical health outcomes. Seven of the studies found at least one R/S measure to have no significant association with outcomes. Though all of the studies included in this review focused on R/S experiences of PLWH, there was very little consistency in regard to measurement of R/S. Studies in this review included a wide range of R/S measures, including beliefs, religious/spiritual practices, R/S coping, organizational religion, and many more. Of the 33 studies reviewed, only 9 focused on unique populations such as women, people with histories of substance abuse, immigrants, etc. Findings from this review highlight opportunities for more studies in various populations using standardized R/S measures.

  19. An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice

    PubMed Central

    Dang, Mai T.; Yokoi, Fumiaki; Cheetham, Chad C.; Lu, Jun; Vo, Viet; Lovinger, David M.; Li, Yuqing

    2011-01-01

    DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia. PMID:21995941

  20. Facilitators and Barriers to HIV Activities in Religious Congregations: Perspectives of Clergy and Lay Leaders from a Diverse Urban Sample

    PubMed Central

    Derose, Kathryn Pitkin; Werber, Laura; Palar, Kartika; Kanouse, David E.; Mata, Michael

    2013-01-01

    This paper examines facilitators and barriers to HIV activities within religious congregations, the relative internal or external sources of these influences, and suggestive differences across congregational types. Results are based on in-depth interviews with clergy and lay leaders (n = 57) from 14 congregations in Los Angeles County, California, purposively selected to reflect diversity in racial-ethnic composition, denomination, size, and HIV activity level. Many common facilitators and barriers were related to norms and attitudes, only a few of which appeared overtly associated with theological orientations. Clergy support was a facilitator particularly prevalent among congregations having higher HIV activity levels, indicating its importance in sustaining and expanding HIV programs. Resource issues were also prominent, with material resource barriers more frequently mentioned by smaller congregations and human resource barriers more among larger congregations. Organizational structure issues were mostly centered on external linkages with various social service, public health, and faith-based entities. Analysis of internal versus external sources highlights the roles of different stakeholders within and outside congregations in promoting HIV activities. Potential differences across congregational types represent fruitful areas for future research. PMID:23990037