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Sample records for amastigotes enhance hiv-1

  1. Citron kinase enhances ubiquitination of HIV-1 Gag protein and intracellular HIV-1 budding.

    PubMed

    Ding, Jiwei; Zhao, Jianyuan; Sun, Lei; Mi, Zeyun; Cen, Shan

    2016-09-01

    Assembly and budding of human immunodeficiency virus type 1 (HIV-1) particles is a complex process involving a number of host proteins. We have previously reported that the RhoA effector citron kinase enhances HIV-1 production. However, the underlying mechanism is not clear. In this study, we found that citron kinase interacted with HIV-1 Gag protein via its zinc finger and leucine zipper domains. Electron microscopy analysis revealed that citron kinase induced viral particle assembly in multivesicular bodies (MVBs). Citron kinase enhanced ubiquitination of HIV-1 Gag protein. Knockdown of Nedd4L, a member of the HECT ubiquitin E3 ligase family, partly decreased the ability of citron kinase to enhance HIV-1 production and reduced ubiquitination of HIV-1 Gag. Interestingly, the function of citron kinase to promote HIV-1 budding was severely impaired when endogenous ALIX was knocked down. Overexpression of the AAA-type ATPase VPS4 eliminated citron-kinase-mediated enhancement of HIV-1 production. Our results suggest that citron kinase interacts with HIV-1 Gag and enhances HIV-1 production by promoting Gag ubiquitination and inducing viral release via the MVB pathway. PMID:27339686

  2. Enhanced HIV-1 neutralization by antibody heteroligation

    PubMed Central

    Mouquet, Hugo; Warncke, Malte; Scheid, Johannes F.; Seaman, Michael S.; Nussenzweig, Michel C.

    2012-01-01

    Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti–HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation. PMID:22219363

  3. 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. PMID:26774171

  4. Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein.

    PubMed

    Zhang, J L; Sharma, P L; Crumpacker, C S

    2000-03-15

    Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA-protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappaB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-kappaB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters. PMID:10704334

  5. APOBEC4 Enhances the Replication of HIV-1

    PubMed Central

    Hofmann, Henning; Hanschmann, Kay-Martin; Mühlebach, Michael D.; Schumann, Gerald G.; König, Renate; Cichutek, Klaus; Häussinger, Dieter; Münk, Carsten

    2016-01-01

    APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters. PMID:27249646

  6. No SEVI-mediated enhancement of rectal HIV-1 transmission of HIV-1 in two humanized mouse cohorts.

    PubMed

    Van Dis, Erik S; Moore, Tyler C; Lavender, Kerry J; Messer, Ronald J; Keppler, Oliver T; Verheyen, Jens; Dittmer, Ulf; Hasenkrug, Kim J

    2016-01-15

    Amyloid fibrils from semen-derived peptide (SEVI) enhance HIV-1 infectivity in vitro but the ability of SEVI to mediate enhancement of HIV infection in vivo has not been tested. In this study we used immunodeficient mice reconstituted with human immune systems to test for in vivo enhancement of HIV-1 transmission. This mouse model supports mucosal transmission of HIV-1 via the intrarectal route leading to productive infection. In separate experiments with humanized mouse cohorts reconstituted with two different donor immune systems, high dose HIV-1JR-CSF that had been incubated with SEVI amyloid fibrils at physiologically relevant concentrations did not show an increased incidence of infection compared to controls. In addition, SEVI failed to enhance rectal transmission with a reduced concentration of HIV-1. Although we confirmed potent SEVI-mediated enhancement of HIV infectivity in vitro, this model showed no evidence that it plays a role in the much more complex situation of in vivo transmission. PMID:26609939

  7. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    PubMed

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells. PMID:27199430

  8. Cocaine Enhances HIV-1 Transcription in Macrophages by Inducing p38 MAPK Phosphorylation

    PubMed Central

    Swepson, Chelsie; Ranjan, Alok; Balasubramaniam, Muthukumar; Pandhare, Jui; Dash, Chandravanu

    2016-01-01

    Cocaine is a commonly used illicit drug among HIV-1 infected individuals and is known to increase HIV-1 replication in permissive cells including PBMCs, CD4+ T cells, and macrophages. Cocaine’s potentiating effects on HIV-1 replication in macrophages- the primary targets of the virus in the central nervous system, has been suggested to play an important role in HIV-1 neuro-pathogenesis. However, the mechanism by which cocaine enhances HIV-1 replication in macrophages remain poorly understood. Here, we report the identification of cocaine-induced signaling events that lead to enhanced HIV-1 transcription in macrophages. Treatment of physiologically relevant concentrations of cocaine enhanced HIV-1 transcription in a dose-dependent manner in infected THP-1 monocyte-derived macrophages (THP-1macs) and primary monocyte-derived macrophages (MDMs). Toward decoding the underlying mechanism, results presented in this report demonstrate that cocaine induces the phosphorylation of p38 mitogen activated protein kinase (p38 MAPK), a known activator of HIV-1 transcription. We also present data suggesting that the p38 MAPK-driven HIV-1 transcription is dependent on the induction of mitogen- and stress-activated protein kinase 1 (MSK1). Consequently, MSK1 mediates the phosphorylation of serine 10 residue of histone 3 (H3 Ser10), which is known to activate transcription of genes including that of HIV-1 in macrophages. Importantly, our results show that inhibition of p38 MAPK/MSK1 signaling by specific pharmacological inhibitors abrogated the positive effect of cocaine on HIV-1 transcription. These results validate the functional link between cocaine and p38 MAPK/MSK1 pathways. Together, our results demonstrate for the first time that the p38 MAPK/MSK1 signaling pathway plays a critical role in the cocaine-induced potentiating effects on HIV-1 infection, thus providing new insights into the interplay between cocaine abuse and HIV-1 neuro-pathogenesis. PMID:27375565

  9. Glycosomal bromodomain factor 1 from Trypanosoma cruzi enhances trypomastigote cell infection and intracellular amastigote growth.

    PubMed

    Ritagliati, Carla; Villanova, Gabriela Vanina; Alonso, Victoria Lucia; Zuma, Aline Araujo; Cribb, Pamela; Motta, María Cristina Machado; Serra, Esteban Carlos

    2016-01-01

    Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication. PMID:26500280

  10. Nef enhances HIV-1 infectivity via association with the virus assembly complex

    SciTech Connect

    Qi Mingli; Aiken, Christopher

    2008-04-10

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.

  11. Nef Enhances HIV-1 Infectivity via Association with the Virus Assembly Complex

    PubMed Central

    Qi, Mingli; Aiken, Christopher

    2008-01-01

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle. PMID:18191978

  12. 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. PMID:26733199

  13. Mechanism of multivalent nanoparticle encounter with HIV-1 for potency enhancement of peptide triazole virus inactivation.

    PubMed

    Rosemary Bastian, Arangassery; Nangarlia, Aakansha; Bailey, Lauren D; Holmes, Andrew; Kalyana Sundaram, R Venkat; Ang, Charles; Moreira, Diogo R M; Freedman, Kevin; Duffy, Caitlin; Contarino, Mark; Abrams, Cameron; Root, Michael; Chaiken, Irwin

    2015-01-01

    Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally. PMID:25371202

  14. Enhanced Heterosexual Transmission Hypothesis for the Origin of Pandemic HIV-1

    PubMed Central

    de Sousa, João Dinis; Alvarez, Carolina; Vandamme, Anne-Mieke; Müller, Viktor

    2012-01-01

    HIV-1 M originated from SIVcpz endemic in chimpanzees from southeast Cameroon or neighboring areas, and it started to spread in the early 20th century. Here we examine the factors that may have contributed to simian-to-human transmission, local transmission between humans, and export to a city. The region had intense ape hunting, social disruption, commercial sex work, STDs, and traffic to/from Kinshasa in the period 1899–1923. Injection treatments increased sharply around 1930; however, their frequency among local patients was far lower than among modern groups experiencing parenteral HIV-1 outbreaks. Recent molecular datings of HIV-1 M fit better the period of maximal resource exploitation and trade links than the period of high injection intensity. We conclude that although local parenteral outbreaks might have occurred, these are unlikely to have caused massive transmission. World War I led to additional, and hitherto unrecognized, risks of HIV-1 emergence. We propose an Enhanced Heterosexual Transmission Hypothesis for the origin of HIV-1 M, featuring at the time and place of its origin a coincidence of favorable co-factors (ape hunting, social disruption, STDs, and mobility) for both cross-species transmission and heterosexual spread. Our hypothesis does not exclude a role for parenteral transmission in the initial viral adaptation. PMID:23202448

  15. Crystal Structure of NFAT Bound to the HIV-1 LTR Tandem κB Enhancer Element

    SciTech Connect

    Bates, Darren L.; Barthel, Kristen K.B.; Wu, Yongqing; Kalhor, Reza; Stroud, James C.; Giffin, Michael J.; Chen, Lin

    2008-05-27

    Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem {kappa}B enhancer element of 3.05 {angstrom} resolution. NFAT binds as a dimer to the upstream {kappa}B site (Core II), but as a monomer to the 3' end of the downstream {kappa}B site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for futher investigating the functional mechanism of NFAT in HIV-1 transcription and replication.

  16. A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection

    PubMed Central

    Chen, Jinquan; Ren, Ruxia; Tan, Suiyi; Zhang, Wanyue; Zhang, Xuanxuan; Yu, Fei; Xun, Tianrong; Jiang, Shibo; Liu, Shuwen; Li, Lin

    2015-01-01

    Background Semen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP) fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils. Methodology and Principal Findings Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2), can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT) and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM), in the presence or absence of EP2. Circular dichroism (CD) spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells. Conclusions and Significance Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection. PMID:26656730

  17. HIV-1 Transgenic Rat Prefrontal Cortex Hyper-Excitability is Enhanced by Cocaine Self-Administration.

    PubMed

    Wayman, Wesley N; Chen, Lihua; Hu, Xiu-Ti; Napier, T Celeste

    2016-07-01

    The medial prefrontal cortex (mPFC) is dysregulated in HIV-1-infected humans and the dysregulation is enhanced by cocaine abuse. Understanding mPFC pathophysiology in this comorbid state has been hampered by the dearth of relevant animal models. To help fill this knowledge gap, electrophysiological assessments were made of mPFC pyramidal neurons (PN) from adult male HIV-1 transgenic (Tg) F344 rats (which express seven of the nine HIV-1 toxic proteins) and non-Tg F344 rats that self-administered cocaine for 14 days (COC-SA), as well as saline-yoked controls (SAL-Yoked) and experimentally naive Tg and non-Tg rats. Forebrain slices were harvested and prepared for whole-cell patch-clamp recording, and in treated rats, this occurred after 14-18 days of forced abstinence. Aged-matched rats were used for immunohistochemical detection of the L-channel protein, Cav1.2-α1c. We determined that: (i) the two genotypes acquired the operant task and maintained similar levels of COC-SA, (ii) forced abstinence from COC-SA enhanced mPFC PN excitability in both genotypes, and neurons from Tg rats exhibited the greatest pathophysiology, (iii) neurons from SAL-Yoked Tg rats were more excitable than those from SAL-Yoked non-Tg rats, and in Tg rats (iv) blockade of L-type Ca(2+) channels reduced the enhanced excitability, and (v) Cav1.2-immunoreactivity was increased. These findings provide the first assessment of the mPFC pathophysiology in a rodent model of HIV-1-mediated neuropathology with and without cocaine self-administration. Outcomes reveal an enhanced cortical excitability during chronic exposure to HIV-1 proteins that is excessively exacerbated with cocaine abuse. Such neuropathophysiology may underlie the cognitive dysregulation reported for comorbid humans. PMID:26677947

  18. Bispecific Anti-HIV-1 Antibodies with Enhanced Breadth and Potency.

    PubMed

    Bournazos, Stylianos; Gazumyan, Anna; Seaman, Michael S; Nussenzweig, Michel C; Ravetch, Jeffrey V

    2016-06-16

    Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) suppress viremia in animal models of HIV-1 and humans. To achieve potent activity without the emergence of viral escape mutants, co-administration of different bNAbs is necessary to target distinct epitopes essential for viral fitness. Here, we report the development of bispecific anti-Env neutralizing antibodies (biNAbs) with potent activity. Synergistic activity of biNAbs was achieved by combining an engineered hinge domain of IgG3 to increase Fab domain flexibility necessary for hetero-bivalent binding to the Env trimer while retaining the functional properties of the IgG1-Fc. Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1-infected humanized mice. These findings suggest innovative strategies for generating biNAbs with enhanced neutralization breadth and potency, representing ideal candidate molecules for the control of HIV-1 infection. PMID:27315478

  19. Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

    PubMed

    Kennedy, Edward M; Bogerd, Hal P; Kornepati, Anand V R; Kang, Dong; Ghoshal, Delta; Marshall, Joy B; Poling, Brigid C; Tsai, Kevin; Gokhale, Nandan S; Horner, Stacy M; Cullen, Bryan R

    2016-05-11

    Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression. PMID:27117054

  20. A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

    PubMed Central

    MCLAREN, MEREDITH; ASAI, KENGO; COCHRANE, ALAN

    2004-01-01

    Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3′ splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3′ end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3′ end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3′ end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression. PMID:15208447

  1. Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes.

    PubMed

    Williams, Rachel; Dhillon, Navneet K; Hegde, Sonia T; Yao, Honghong; Peng, Fuwang; Callen, Shannon; Chebloune, Yahia; Davis, Randall L; Buch, Shilpa J

    2009-05-01

    HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-gamma inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN-gamma and TNF-alpha, are also markedly increased in CNS tissues during HIV-1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-kappaB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine-mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD. PMID:18985732

  2. 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

    PubMed

    Rawson, Jonathan M O; Daly, Michele B; Xie, Jiashu; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-04-01

    5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses. PMID:26833151

  3. Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues

    PubMed Central

    Macura, Sherrill L.; Lathrop, Melissa J.; Gui, Jiang; Doncel, Gustavo F.; Rollenhagen, Christiane

    2016-01-01

    Objectives: The interferon-gamma–induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti–HIV-1 activity of prophylactic antiretrovirals. Design: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4+ T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. Methods: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4+ T cells was quantified using fluorescence-activated cell sorting. Results: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4+ T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. Conclusions: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals. PMID:26545124

  4. Cocaine enhances HIV-1 gp120-induced lymphatic endothelial dysfunction in the lung

    PubMed Central

    Zhang, Xuefeng; Jiang, Susan; Yu, Jinlong; Kuzontkoski, Paula M; Groopman, Jerome E

    2015-01-01

    Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV+ donors were significantly elevated as compared to HIV− donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population. PMID:26311830

  5. Cocaine enhances HIV-1 gp120-induced lymphatic endothelial dysfunction in the lung.

    PubMed

    Zhang, Xuefeng; Jiang, Susan; Yu, Jinlong; Kuzontkoski, Paula M; Groopman, Jerome E

    2015-08-01

    Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV(+) donors were significantly elevated as compared to HIV(-) donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population. PMID:26311830

  6. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  7. Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

    SciTech Connect

    Meyuhas, Ronit; Noy, Hava; Fishman, Sigal; Margalit, Alon; Montefiori, David C.; Gross, Gideon

    2009-08-21

    HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.

  8. Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment.

    PubMed

    Parker, Zahra F; Rux, Ann H; Riblett, Amber M; Lee, Fang-Hua; Rauova, Lubica; Cines, Douglas B; Poncz, Mortimer; Sachais, Bruce S; Doms, Robert W

    2016-07-01

    Platelet factor 4 (PF4) has been recently shown to inhibit infection by a broad range of human immunodeficiency virus type 1 (HIV-1) isolates in vitro. We found that the inhibitory effects of PF4 are limited to a defined concentration range where PF4 exists largely in a monomeric state. Under these conditions, PF4 bound the HIV-1 envelope protein and inhibited HIV-1 attachment to the cell surface. However, as concentrations increased to the point where PF4 exists largely in tetrameric or higher-order forms, viral infection in vitro was enhanced. Enhancement could be inhibited by mutations in PF4 that shift the oligomeric equilibrium toward the monomeric state, or by using soluble glycosaminoglycans (GAGs) to which tetrameric PF4 avidly binds. We conclude that at physiologically relevant concentrations, oligomeric PF4 enhances infection by HIV-1 by interacting with the viral envelope protein as well as cell surface GAGs, enhancing virus attachment to the cell surface. This effect was not specific to HIV-1, as enhancement was seen with some but not all other viruses tested. The biphasic effects of PF4 on HIV-1 infection suggest that native PF4 will not be a useful antiviral agent and that PF4 could contribute to the hematologic abnormalities commonly seen in HIV-infected individuals by enhancing virus infection in the bone marrow. PMID:26847431

  9. The miRNA miR-34a enhances HIV-1 replication by targeting PNUTS/PPP1R10, which negatively regulates HIV-1 transcriptional complex formation.

    PubMed

    Kapoor, Richa; Arora, Sakshi; Ponia, Sanket S; Kumar, Binod; Maddika, Subbareddy; Banerjea, Akhil C

    2015-09-15

    HIV-1 relies heavily on the host cellular machinery for its replication. During infection, HIV-1 is known to modulate the host-cell miRNA profile. One of the miRNAs, miR-34a, is up-regulated by HIV-1 in T-cells as suggested by miRNA microarray studies. However, the functional consequences and the mechanism behind this phenomenon were not explored. The present study shows that HIV-1 enhances miR-34a in a time-dependent manner in T-cells. Our overexpression and knockdown-based experimental results suggest that miR-34a promotes HIV-1 replication in T-cells. Hence, there is a positive feedback loop between miR-34a and HIV-1 replication. We show that the mechanism of action of miR-34a in HIV-1 replication involves a cellular protein, the phosphatase 1 nuclear-targeting subunit (PNUTS). PNUTS expression levels decrease with the progression of HIV-1 infection in T-cells. Also, the overexpression of PNUTS potently inhibits HIV-1 replication in a dose-dependent manner. We report for the first time that PNUTS negatively regulates HIV-1 transcription by inhibiting the assembly of core components of the transcription elongation factor P-TEFb, i.e. cyclin T1 and CDK9. Thus, HIV-1 increases miR-34a expression in cells to overcome the inhibitory effect of PNUTS on HIV-1 transcription. So, the present study provides new mechanistic details with regard to our understanding of a complex interplay between miR-34a and the HIV-1 transcription machinery involving PNUTS. PMID:26188041

  10. HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

    PubMed Central

    Vivarini, Áislan de Carvalho; Santos Pereira, Renata de Meirelles; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C.; Saraiva, Elvira M.; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

    2015-01-01

    HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49–57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling. PMID:26608746

  11. Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b

    PubMed Central

    Mantri, Chinmay K.; Pandhare Dash, Jui; Mantri, Jyoti Velamarti; Dash, Chandravanu C. V.

    2012-01-01

    The main objective of this study was to examine effects of cocaine on HIV-1 replication in primary CD4+ T cells. Cocaine a commonly used drug among HIV-1 positive individuals serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS). Accumulating evidence suggest that cocaine increases HIV-1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. Intriguingly, there are no studies on cocaine-induced alterations in HIV-1 replication in primary CD4+ T cells that serve as the main targets for HIV-1 replication in vivo. In this report, we demonstrate cocaine-induced enhancement of HIV-1 replication in primary CD4+ T cells isolated from human PBMCs. To decipher a potential mechanism, we examined whether cocaine targets the innate antiviral immunity of CD4+ T cells mediated by cellular microRNAs (miRNAs). This is because recently a network of anti-HIV miRNAs in CD4+ T cells is highlighted to suppress viral replication. Our genome wide miRNA expression analysis indicated downregulation of several anti-HIV miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) in cocaine treated CD4+ T cells. However, our real-time quantitative PCR analysis revealed significant downregulation of miR-125b only. Our results illustrated that miR-125b knockdown enhances HIV-1 replication, whereas overexpression of miR-125b decreases HIV-1 replication in these cells. Therefore, we believe miR-125b is a key player for the cocaine induced enhancement of HIV-1 replication in CD4+ T cells. Since, miR-125b targets the 3′ UTR regions of HIV-1 transcripts and inhibits viral protein translation, our data suggest modulation of post entry steps of HIV-1 by cocaine. Given that a plethora of studies suggest that cocaine regulates HIV entry, our results implicate a potentially novel mechanism by which cocaine can increase viral replication in CD4+ T cells. PMID:23251514

  12. HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

    PubMed Central

    Park, In-Woo; Fan, Yan; Luo, Xiaoyu; Ryou, Myoung-Gwi; Liu, Jinfeng; Green, Linden; He, Johnny J.

    2014-01-01

    HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. PMID:24911518

  13. Enhancement of Probe Signal for Screening of HIV-1 Protease Inhibitors in Living Cells

    PubMed Central

    Yao, Huantong; Jin, Sha

    2012-01-01

    The global human immunodeficiency virus infection/acquired immuno-deficiency syndrome (HIV/AIDS) epidemic is one of the biggest threats to human life. Mutation of the virus and toxicity of the existing drugs necessitate the development of new drugs for effective AIDS treatment. Previously, we developed a molecular probe that utilizes the Förster resonance energy transfer (FRET) principle to visualize HIV-1 protease inhibition within living cells for drug screening. We explored using AcGFP1 (a fluorescent mutant of the wild-type green fluorescent protein) as a donor and mCherry (a mutant of red fluorescent protein) as an acceptor for FRET microscopy imaging measurement of HIV-1 protease activity within living cells and demonstrated that the molecular probe is suitable for the High-Content Screening (HCS) of anti-HIV drugs through an automated FRET microscopy imaging measurement. In this study, we genetically engineered a probe with a tandem acceptor protein structure to enhance the probe’s signal. Both in vitro and in vivo studies revealed that the novel structure of the molecular probe exhibits a significant enhancement of FRET signals, reaching a probe FRET efficiency of 34%, as measured by fluorescence lifetime imaging microscopy (FLIM) measurement. The probe developed herein would enable high-content screening of new anti-HIV agents. PMID:23223077

  14. Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies.

    PubMed

    Gupta, Rupal; Lu, Manman; Hou, Guangjin; Caporini, Marc A; Rosay, Melanie; Maas, Werner; Struppe, Jochem; Suiter, Christopher; Ahn, Jinwoo; Byeon, In-Ja L; Franks, W Trent; Orwick-Rydmark, Marcella; Bertarello, Andrea; Oschkinat, Hartmut; Lesage, Anne; Pintacuda, Guido; Gronenborn, Angela M; Polenova, Tatyana

    2016-01-21

    Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies. PMID:26709853

  15. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

    PubMed Central

    Rudicell, Rebecca S.; Kwon, Young Do; Ko, Sung-Youl; Pegu, Amarendra; Louder, Mark K.; Georgiev, Ivelin S.; Wu, Xueling; Zhu, Jiang; Boyington, Jeffrey C.; Chen, Xuejun; Shi, Wei; Yang, Zhi-yong; Doria-Rose, Nicole A.; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D.; Chuang, Gwo-Yu; Druz, Aliaksandr; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Todd, John-Paul; Lloyd, Krissey E.; Eudailey, Joshua; Roberts, Kyle E.; Donald, Bruce R.; Bailer, Robert T.; Ledgerwood, Julie; Mullikin, James C.; Shapiro, Lawrence; Koup, Richard A.; Graham, Barney S.; Nason, Martha C.; Connors, Mark; Haynes, Barton F.; Rao, Srinivas S.; Roederer, Mario; Kwong, Peter D.

    2014-01-01

    ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope

  16. Morphine Enhances HIV-1SF162-Mediated Neuron Death and Delays Recovery of Injured Neurites

    PubMed Central

    Masvekar, Ruturaj R.; El-Hage, Nazira; Hauser, Kurt F.; Knapp, Pamela E.

    2014-01-01

    HIV-1 enters the CNS soon after initial systemic infection; within the CNS parenchyma infected and/or activated perivascular macrophages, microglia and astrocytes release viral and cellular toxins that drive secondary toxicity in neurons and other cell types. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162-infected differentiated-U937 cells (HIV+sup) was collected and p24 level was measured by ELISA to assess the infection. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. We therefore assessed HIV+sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+sup caused dose-dependent toxicity over a range of p24 levels (10–500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml), and GSK3β was implicated as a point of convergence. In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented, perhaps related to a decreased level of BDNF. Importantly, the arrest of neurite growth that occurred with exposure to HIV+sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. Opiate interactions observed in this HIV-infective environment were similar, though not entirely concordant, with Tat/gp120 interactions reported previously, suggesting unique interactions

  17. Natural polymorphism S119R of HIV-1 integrase enhances primary INSTI resistance.

    PubMed

    Hachiya, Atsuko; Ode, Hirotaka; Matsuda, Masakazu; Kito, Yumiko; Shigemi, Urara; Matsuoka, Kazuhiro; Imamura, Junji; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-07-01

    Integrase strand transfer inhibitors (INSTIs), which block proviral DNA integration into the host chromosome, are clinically effective against HIV-1 isolates exhibiting resistance to other classes of antiretroviral agents. Although naturally occurring amino acid variation has been less frequently observed in the integrase region, the functional constraints of this variation on primary INSTI resistance-associated mutations are not fully understood. In the present study, we focused on the S119G/R/P/T (S119X) polymorphisms, which are frequently observed in HIV-1 sequences derived from clinical specimens (naïve, n=458, 26%). The frequency of the S119X polymorphism together with Q148H/R (n=8, 63%) or N155H (n=12, 83%) was relatively high compared with that of naïve group. Our in vitro assays revealed that S119G/P/T alone exerted no effect on the susceptibility to INSTIs, whereas S119R enhanced the level of INSTI resistance induced by well-known INSTI resistance-associated mutations (Y143C, Q148H or N155H). Notably, the S119R polymorphism contributed to a significant (5.9-fold) increase in dolutegravir resistance caused by G140S/Q148H. Analysis of two cases of virological failure during raltegravir-based therapy showed that the accumulation and the rapid evolution of primary INSTI resistance-associated mutations coincided with the S119R mutation. These data highlight the role of the S119X polymorphism in INSTI resistance, and this polymorphism might be linked to the potential treatment outcome with INSTI-based therapy. PMID:25956162

  18. The MSHA strain of Pseudomonas aeruginosa activated TLR pathway and enhanced HIV-1 DNA vaccine immunoreactivity.

    PubMed

    Hou, Jue; Liu, Yong; Liu, Ying; Shao, Yiming

    2012-01-01

    The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells) and antigen presenting cells (APCs). Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR) pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs) following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells. PMID:23077664

  19. Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1.

    PubMed

    Moriuchi, H; Moriuchi, M; Fauci, A S

    1998-05-18

    It remains controversial whether human T lymphotropic virus type I (HTLV-I) coinfection leads to more rapid progression of human immunodeficiency virus (HIV) disease in dually infected individuals. To investigate whether HTLV-I infection of certain cells can modulate HIV-1 infection of surrounding cells, primary CD4(+) T cells were treated with cell-free supernatants from HTLV-I-infected MT-2 cell cultures. The primary CD4+ T cells became resistant to macrophage (M)-tropic HIV-1 but highly susceptible to T cell (T)-tropic HIV-1. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in the MT-2 cell supernatants were identified as the major suppressive factors for M-tropic HIV-1 as well as the enhancers of T-tropic HIV-1 infection, whereas soluble Tax protein increased susceptibility to both M- and T-tropic HIV-1. The effect of Tax or CC chemokines on T-tropic HIV-1 was mediated, at least in part, by increasing HIV Env-mediated fusogenicity. Our data suggest that the net effect of HTLV-I coinfection in HIV-infected individuals favors the transition from M- to T-tropic HIV phenotype, which is generally indicative of progressive HIV disease. PMID:9584147

  20. Polyanionic Candidate Microbicides Accelerate the Formation of Semen-Derived Amyloid Fibrils to Enhance HIV-1 Infection

    PubMed Central

    Tan, Suiyi; Lu, Lu; Li, Lin; Liu, Jixiang; Oksov, Yelena; Lu, Hong; Jiang, Shibo; Liu, Shuwen

    2013-01-01

    Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides. PMID:23544097

  1. Cocaine enhances HIV-1-induced CD4(+) T-cell apoptosis: implications in disease progression in cocaine-abusing HIV-1 patients.

    PubMed

    Pandhare, Jui; Addai, Amma B; Mantri, Chinmay K; Hager, Cynthia; Smith, Rita M; Barnett, Louis; Villalta, Fernando; Kalams, Spyros A; Dash, Chandravanu

    2014-04-01

    Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers. PMID:24486327

  2. An AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection.

    PubMed

    Duverger, Alexandra; Wolschendorf, Frank; Zhang, Mingce; Wagner, Fredric; Hatcher, Brandon; Jones, Jennifer; Cron, Randall Q; van der Sluis, Renee M; Jeeninga, Rienk E; Berkhout, Ben; Kutsch, Olaf

    2013-02-01

    Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon. PMID:23236059

  3. An AP-1 Binding Site in the Enhancer/Core Element of the HIV-1 Promoter Controls the Ability of HIV-1 To Establish Latent Infection

    PubMed Central

    Duverger, Alexandra; Wolschendorf, Frank; Zhang, Mingce; Wagner, Fredric; Hatcher, Brandon; Jones, Jennifer; Cron, Randall Q.; van der Sluis, Renee M.; Jeeninga, Rienk E.; Berkhout, Ben

    2013-01-01

    Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon. PMID:23236059

  4. Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.

    PubMed

    LoRicco, Josephine G; Xu, Changmingzi Sherry; Neidleman, Jason; Bergkvist, Magnus; Greene, Warner C; Roan, Nadia R; Makhatadze, George I

    2016-07-01

    Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection. PMID:27226574

  5. A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin

    SciTech Connect

    Cheng, De-Chun; Zhong, Guo-Cai; Su, Ju-Xiang; Liu, Yan-Hong; Li, Yan; Wang, Jia-Ye; Hattori, Toshio; Ling, Hong; Zhang, Feng-Min

    2010-01-22

    To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.

  6. Maturation of the viral core enhances the fusion of HIV-1 particles with primary human T cells and monocyte-derived macrophages

    SciTech Connect

    Jiang Jiyang; Aiken, Christopher . E-mail: chris.aiken@vanderbilt.edu

    2006-03-15

    HIV-1 infection requires fusion of viral and cellular membranes in a reaction catalyzed by the viral envelope proteins gp120 and gp41. We recently reported that efficient HIV-1 particle fusion with target cells is linked to maturation of the viral core by an activity of the gp41 cytoplasmic domain. Here, we show that maturation enhances the fusion of a variety of recombinant viruses bearing primary and laboratory-adapted Env proteins with primary human CD4{sup +} T cells. Overall, HIV-1 fusion was more dependent on maturation for viruses bearing X4-tropic envelope proteins than for R5-tropic viruses. Fusion of HIV-1 with monocyte-derived macrophages was also dependent on particle maturation. We conclude that the ability to couple fusion to particle maturation is a common feature of HIV-1 Env proteins and may play an important role during HIV-1 replication in vivo.

  7. CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

    PubMed Central

    Liu, Jun; Yu, Qigui; Stone, Geoffrey W.; Yue, Feng Yun; Ngai, Nicholas; Jones, R. Brad; Kornbluth, Richard S.; Ostrowski, Mario A.

    2011-01-01

    Summary Human immunodeficiency virus-1 (HIV-1) canarypox vaccines are safe but poorly immunogenic. CD40 ligand (CD40L), a member of the tumor necrosis factor superfamily (TNFSF), is a pivotal co-stimulatory molecule for immune responses. To explore whether CD40L can be used as an adjuvant for HIV-1 canarypox vaccine, we constructed recombinant canarypox viruses expressing CD40L. Co-immunization of mice with CD40L expressing canarypox and the canarypox vaccine expressing HIV-1 proteins, vCP1452, augmented HIV-1 specific cytotoxic T lymphocyte (CTL) responses in terms of frequency, polyfunctionality and interleukin (IL)-7 receptor α chain (IL-7Rα, CD127) expression. In addition, CD40L expressed from canarypox virus could significantly augment CD4+ T cell responses against HIV-1 in mice. CD40L expressed from canarypox virus matured human monocyte-derived dendritic cells (MDDCs) in a tumor necrosis factor α (TNF-α) independent manner, which underwent less apoptosis, and could expand ex vivo Epstein-Barr virus (EBV)-specific CTL responses from healthy human individuals and ex vivo HIV-1-specific CTL responses from HIV-1-infected individuals in the presence or absence of CD4+ T cells. Taken together, our results suggest that CD40L incorporation into poxvirus vectors could be used as a strategy to enhance their immunogenicity. PMID:18562053

  8. Manganese-Enhanced Magnetic Resonance Imaging Reflects Brain Pathology During Progressive HIV-1 Infection of Humanized Mice.

    PubMed

    Bade, Aditya N; Gorantla, Santhi; Dash, Prasanta K; Makarov, Edward; Sajja, Balasrinivasa R; Poluektova, Larisa Y; Luo, Jiangtao; Gendelman, Howard E; Boska, Michael D; Liu, Yutong

    2016-07-01

    Progressive human immunodeficiency viral (HIV) infection commonly leads to a constellation of cognitive, motor, and behavioral impairments. These are collectively termed HIV-associated neurocognitive disorders (HAND). While antiretroviral therapy (ART) reduces HAND severity, it does not affect disease prevalence. Despite decades of research, there remain no biomarkers for HAND and all potential comorbid conditions must first be excluded for a diagnosis to be made. To this end, we now report that manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) can reflect brain region-specific HIV-1-induced neuropathology in chronically virus-infected NOD/scid-IL-2Rγc(null) humanized mice. MEMRI diagnostics mirrors the abilities of Mn(2+) to enter and accumulate in affected neurons during disease. T1 relaxivity and its weighted signal intensity are proportional to Mn(2+) activities in neurons. In 16-week virus-infected humanized mice, altered MEMRI signal enhancement was easily observed in affected brain regions. These included, but were not limited to, the hippocampus, amygdala, thalamus, globus pallidus, caudoputamen, substantia nigra, and cerebellum. MEMRI signal was coordinated with levels of HIV-1 infection, neuroinflammation (astro- and micro-gliosis), and neuronal injury. MEMRI accurately demonstrates the complexities of HIV-1-associated neuropathology in rodents that reflects, in measure, the clinical manifestations of neuroAIDS as it is seen in a human host. PMID:26063593

  9. Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells

    PubMed Central

    Roesch, Ferdinand; Richard, Léa; Rua, Réjane; Porrot, Françoise; Casartelli, Nicoletta

    2015-01-01

    ABSTRACT The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo. IMPORTANCE The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation

  10. Enhanced CD4+ cellular apoptosis by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with progressive HIV-1 infection

    SciTech Connect

    Wade, Jessica; Sterjovski, Jasminka; Gray, Lachlan; Roche, Michael; Chiavaroli, Lisa; Ellett, Anne; Jakobsen, Martin R.; Cowley, Daniel; Fonseca Pereira, Candida da; Saksena, Nitin; Wang, Bin; Purcell, Damian F.J.; Karlsson, Ingrid; Fenyoe, Eva-Maria; Churchill, Melissa; Gorry, Paul R.

    2010-01-20

    CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.

  11. Neisseria gonorrhoeae enhances HIV-1 infection of primary resting CD4+ T cells through TLR2 activation.

    PubMed

    Ding, Jian; Rapista, Aprille; Teleshova, Natalia; Mosoyan, Goar; Jarvis, Gary A; Klotman, Mary E; Chang, Theresa L

    2010-03-15

    Sexually transmitted infections increase the likelihood of HIV-1 transmission. We investigated the effect of Neisseria gonorrheae (gonococcus [GC]) exposure on HIV replication in primary resting CD4(+) T cells, a major HIV target cell during the early stage of sexual transmission of HIV. GC and TLR2 agonists, such as peptidylglycan (PGN), Pam(3)CSK(4), and Pam(3)C-Lip, a GC-derived synthetic lipopeptide, but not TLR4 agonists including LPS or GC lipooligosaccharide enhanced HIV-1 infection of primary resting CD4(+) T cells after viral entry. Pretreatment of CD4(+) cells with PGN also promoted HIV infection. Anti-TLR2 Abs abolished the HIV enhancing effect of GC and Pam(3)C-Lip, indicating that GC-mediated enhancement of HIV infection of resting CD4(+) T cells was through TLR2. IL-2 was required for TLR2-mediated HIV enhancement. PGN and GC induced cell surface expression of T cell activation markers and HIV coreceptors, CCR5 and CXCR4. The maximal postentry HIV enhancing effect was achieved when PGN was added immediately after viral exposure. Kinetic studies and analysis of HIV DNA products indicated that GC exposure and TLR2 activation enhanced HIV infection at the step of nuclear import. We conclude that GC enhanced HIV infection of primary resting CD4(+) T cells through TLR2 activation, which both increased the susceptibility of primary CD4(+) T cells to HIV infection as well as enhanced HIV-infected CD4(+) T cells at the early stage of HIV life cycle after entry. This study provides a molecular mechanism by which nonulcerative sexually transmitted infections mediate enhancement of HIV infection and has implication for HIV prevention and therapeutics. PMID:20147631

  12. Complement opsonization of HIV-1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells

    PubMed Central

    Tjomsland, Veronica; Ellegård, Rada; Burgener, Adam; Mogk, Kenzie; Che, Karlhans F; Westmacott, Garrett; Hinkula, Jorma; Lifson, Jeffrey D; Larsson, Marie

    2013-01-01

    Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery. PMID:23526630

  13. Schistosoma mansoni soluble egg antigens enhance Listeria monocytogenes vector HIV-1 vaccine induction of cytotoxic T cells.

    PubMed

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

    2014-09-01

    Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4(+) T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines. PMID:24990901

  14. Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

    PubMed Central

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J.; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S.; Burton, Dennis R.

    2013-01-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  15. Antibody conjugation approach enhances breadth and potency of neutralization of anti-HIV-1 antibodies and CD4-IgG.

    PubMed

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S; Burton, Dennis R; Barbas, Carlos F

    2013-05-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  16. Enhanced HIV-1 Reverse Transcriptase Inhibitory and Antibacterial Properties in Callus of Catha edulis Forsk.

    PubMed

    Kumari, Aloka; Baskaran, Ponnusamy; Van Staden, Johannes

    2015-06-01

    Developing tissue culture systems for medicinal plants is important in that they may offer an alternative to protect wild populations. However, analysis of bioactivity for tissue culture developed plant tissues is required to offer support and allow acceptance in traditional medicine. The use of propagated callus could provide potential material for therapeutic purposes. This study was aimed at evaluating the anti-HIV and antibacterial properties of a three-month-old tissue culture-derived calli and leaves of cultivated mother plants of Catha edulis Forsk. The calli were derived from leaf explants using different plant growth regulators. The calli obtained from callus cultured on 9.8 μM indole-3-butyric acid plus 2.7 μM naphthalene acetic acid exhibited the highest HIV-1 reverse transcriptase inhibitory effects when compared with other treatments and the mother plants. Different extracts of callus exhibited high antibacterial activity (<1 mg/mL: minimum inhibitory concentration from 0.098 to 0.78 mg/mL) against both gram-positive and gram-negative bacteria. Leaf acetone extracts showed moderate activity (minimum inhibitory concentration of 0.78 mg/mL) against Staphylococcus aureus. The present study indicated that tissue culture-derived calli could be used as therapeutic agents for traditional medicine. The choice of treatment used in the tissue culture system and the age of the callus for production of biomass may significantly influence its therapeutic potential. PMID:25753483

  17. Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

    PubMed Central

    Kanagavelu, Saravana K.; Snarsky, Victoria; Termini, James M.; Gupta, Sachin; Barzee, Suzanne; Wright, Jacqueline A.; Khan, Wasif N.; Kornbluth, Richard S.; Stone, Geoffrey W.

    2011-01-01

    Background DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Methodology and Principal Findings Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8

  18. A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation.

    PubMed Central

    Perkins, N D; Edwards, N L; Duckett, C S; Agranoff, A B; Schmid, R M; Nabel, G J

    1993-01-01

    The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-kappa B to the viral enhancer. In this report, we show that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein-protein interaction must occur between NF-kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross-linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF-kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF-kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF-kappa B and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes. Images PMID:8253080

  19. Immune Focusing and Enhanced Neutralization Induced by HIV-1 gp140 Chemical Cross-Linking

    PubMed Central

    Schiffner, T.; Kong, L.; Duncan, C. J. A.; Back, J. W.; Benschop, J. J.; Shen, X.; Huang, P. S.; Stewart-Jones, G. B.; DeStefano, J.; Seaman, M. S.; Tomaras, G. D.; Montefiori, D. C.; Schief, W. R.

    2013-01-01

    Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design. PMID:23843636

  20. Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

    PubMed Central

    Napuri, Jessica; Pilakka-Kanthikeel, Sudheesh; Raymond, Andrea; Agudelo, Marisela; Yndart-Arias, Adriana; Saxena, Shailendra K.; Nair, Madhavan

    2013-01-01

    Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers. PMID:24391808

  1. HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling

    PubMed Central

    Chen, Duchu; Wang, Huiping; Aweya, Jude Juventus; Chen, Yanheng; Chen, Meihua; Wu, Xiaomeng; Chen, Xiaonan; Lu, Jing

    2016-01-01

    In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway. PMID:27529070

  2. Mycobacterium avium infection in HIV-1-infected subjects increases monokine secretion and is associated with enhanced viral load and diminished immune response to viral antigens.

    PubMed Central

    Denis, M; Ghadirian, E

    1994-01-01

    The complex interaction between HIV-1 infection and Mycobacterium avium was studied. Viral burden was assessed, as well as immune response to HIV-1 in the context of Myco. avium infections. We also examined serum cytokine levels and cytokine release by blood mononuclear cells in HIV-1-infected subjects, infected or not with Myco. avium. Undetectable serum levels of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6 were found in normal controls and in groups I, II and III of HIV-1-infected subjects. Moderate levels of TNF-alpha, IL-1 and IL-6 were found in the sera of group IV patients. When group IV was subdivided into subjects with and without Myco. avium infections, subjects with Myco, avium infections were shown to have higher serum levels of TNF-alpha, IL-1 beta and IL-6 than those with other infections. Blood mononuclear cells from controls and HIV subjects were stimulated with bacterial lipopolysaccharide, and cytokine levels assessed. Cells from group II patients were shown to secrete normal levels of TNF-alpha and IL-6, and lower levels of IL-1 beta; group III subjects released higher levels of IL-6. Patients in group IV had blood cells that released elevated levels of IL-6 and TNF-alpha, and lower levels of IL-1 beta. Group IV subjects with Myco. avium infections had blood cells that released higher levels of TNF-alpha, IL-6 and IL-1 than group IV subjects with other infections. Assessment of viral burden in cells of HIV-1-infected subjects revealed that Myco. avium-infected subjects had a higher level of virus burden and a lower level of lymphoproliferative response to an inactivated gp120-depleted HIV-1 antigen than AIDS subjects with other infections. These data suggest that Myco. avium infections in HIV-1-infected subjects hasten the progression of viral disease, enhance cytokine release and contribute to the anergy to viral antigens. PMID:8033423

  3. Characterization of the Influence of Semen-Derived Enhancer of Virus Infection on the Interaction of HIV-1 with Female Reproductive Tract Tissues

    PubMed Central

    Allen, Shannon A.; Carias, Ann M.; Anderson, Meegan R.; Okocha, Eneniziaogochukwu A.; Benning, Lorie; McRaven, Michael D.; Kelley, Z L.; Lurain, John; Veazey, Ronald S.

    2015-01-01

    ABSTRACT The majority of human immunodeficiency virus type 1 (HIV-1) transmission events occur in women when semen harboring infectious virus is deposited onto the mucosal barriers of the vaginal, ectocervical, and endocervical epithelia. Seminal factors such as semen-derived enhancer of virus infection (SEVI) fibrils were previously shown to greatly enhance the infectivity of HIV-1 in cell culture systems. However, when SEVI is intravaginally applied to living animals, there is no effect on vaginal transmission. To define how SEVI might function in the context of sexual transmission, we applied HIV-1 and SEVI to intact human and rhesus macaque reproductive tract tissues to determine how it influences virus interactions with these barriers. We show that SEVI binds HIV-1 and sequesters most virions to the luminal surface of the stratified squamous epithelium, significantly reducing the number of virions that penetrated the tissue. In the simple columnar epithelium, SEVI was no longer fibrillar in structure and was detached from virions but allowed significantly deeper epithelial virus penetration. These observations reveal that the action of SEVI in intact tissues is very different in the anatomical context of sexual transmission and begin to explain the lack of stimulation of infection observed in the highly relevant mucosal transmission model. IMPORTANCE The most common mode of HIV-1 transmission in women occurs via genital exposure to the semen of HIV-infected men. A productive infection requires the virus to penetrate female reproductive tract epithelial barriers to infect underlying target cells. Certain factors identified within semen, termed semen-derived enhancers of virus infection (SEVI), have been shown to significantly enhance HIV-1 infectivity in cell culture. However, when applied to the genital tracts of living female macaques, SEVI did not enhance virus transmission. Here we show that SEVI functions very differently in the context of intact mucosal

  4. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  5. Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues

    PubMed Central

    Rollenhage, Christiane; Macura, Sherrill L.; Lathrop, Melissa J.; Mackenzie, Todd A.; Doncel, Gustavo F.; Asin, Susana N.

    2015-01-01

    Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic:polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NFκB) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV prophylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be

  6. The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120

    PubMed Central

    Hioe, Catarina E.; Visciano, Maria Luisa; Kumar, Rajnish; Liu, Jianping; Mack, Ethan A.; Simon, Rachel E.; Levy, David N.; Tuen, Michael

    2009-01-01

    The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity, while the ability of Abs to induce antigenic alterations of specific epitopes as a result of immune complex formation have been less well studied. Previously we have shown that the interaction of anti-CD4-binding site (CD4bs) Abs with HIV-1 gp120 induces conformation changes that lead to enhanced antigenicity and immunogenicity of neutralizing epitopes in the V3 loop. The present study shows that significant increases in the antigenicity of the V3 and C1 regions of gp120 were attained for several subtype B gp120s and a subtype C gp120 upon immune complex formation with the anti-CD4bs monoclonal Ab (mAb) 654-D. Such enhancement was observed with immune complexes made with other anti-CD4bs mAbs and anti-V2 mAbs, but not with anti-C2 mAbs, indicating this activity is determined by antigen specificity of the mAb that formed the immune complex. When immune complexes of gp120LAI/654-D and gp120JRFL/654-D were tested as immunogens in mice, serum Abs to gp120 and V3 were generated at significantly higher titers than those induced by the respective uncomplexed gp120s. Notably, the anti-V3 Ab responses had distinct fine specificities; gp120JRFL/654-D stimulated more cross-reactive anti-V3 Abs than gp120LAI/654-D. Neutralizing activities against viruses with heterologous envelope were also detected in sera of mice immunized with gp120JRFL/654-D, although the neutralization breadth was still limited. Overall this study shows the potential use of gp120/Ab complexes to augment the immunogenicity of HIV-1 envelope gp120, but further improvements are needed to elicit virus-neutralizing Ab responses with higher potency and breadth. PMID:19879224

  7. Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6.

    PubMed

    Swaminathan, Gokul; Pascual, Daniel; Rival, Germaine; Perales-Linares, Renzo; Martin-Garcia, Julio; Navas-Martin, Sonia

    2014-09-17

    Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages. PMID:25131930

  8. Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

    PubMed Central

    Trabalza, Antonio; Eleftheriadou, Ioanna; Sgourou, Argyro; Liao, Ting-Yi; Patsali, Petros; Lee, Heyne

    2014-01-01

    ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer

  9. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

    PubMed Central

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  10. Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

    PubMed

    Fan, Peihu; Li, Xiaojun; Su, Weiheng; Kong, Wei; Kong, Xianggui; Wang, Zhenxin; Wang, Youchun; Jiang, Chunlai; Gao, Feng

    2015-01-01

    The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens. PMID:25915630

  11. MicroRNA-mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV-1 infection

    PubMed Central

    Farberov, Luba; Herzig, Eytan; Modai, Shira; Isakov, Ofer; Hizi, Amnon; Shomron, Noam

    2015-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV-1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV-1 inhibition mechanism. As a result, it is expected that during HIV-1 infection miRNAs target genes that hinder or prevent the progression of the HIV-1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV-1 infection are cellular restriction factors. In this study, we identified a direct miRNA target gene that modulates viral spread in T-lymphocytes and HeLa-CCR5 cell lines. Following infection, let-7c, miR-34a or miR-124a were upregulated, and they targeted and downregulated p21 and TASK1 (also known as CDKN1A and KCNK3, respectively) cellular proteins. This eventually led to increased virion release and higher copy number of viral genome transcripts in infected cells. Conversely, by downregulating these miRNAs, we could suppress viral replication and spread. Our data suggest that HIV-1 exploits the host miRNA cellular systems in order to block the innate inhibition mechanism, allowing a more efficient infection process. PMID:25717002

  12. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    SciTech Connect

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  13. Immunogenetic influences on acquisition of HIV-1 infection: consensus findings from two African cohorts point to an enhancer element in IL19 (1q32.2).

    PubMed

    Li, X; Zhang, K; Pajewski, N M; Brill, I; Prentice, H A; Shrestha, S; Kilembe, W; Karita, E; Allen, S; Hunter, E; Kaslow, R A; Tang, J

    2015-01-01

    Numerous reports have suggested that immunogenetic factors may influence human immunodeficiency virus (HIV)-1 acquisition, yet replicated findings that translate between study cohorts remain elusive. Our work aimed to test several hypotheses about genetic variants within the IL10-IL24 gene cluster that encodes interleukin (IL)-10, IL-19, IL-20 and IL-24. In aggregated data from 515 Rwandans and 762 Zambians with up to 12 years of follow-up, 190 single-nucleotide polymorphisms passed quality control procedures. When HIV-1-exposed seronegative subjects (n=486) were compared with newly seroconverted individuals (n=313) and seroprevalent subjects (n=478) who were already infected at enrollment, rs12407485 (G>A) in IL19 showed a robust association signal in adjusted logistic regression models (odds ratio=0.64, P=1.7 × 10(-4) and q=0.033). Sensitivity analyses demonstrated that (i) results from both cohorts and subgroups within each cohort were highly consistent; (ii) verification of HIV-1 infection status after enrollment was critical; and (iii) supporting evidence was readily obtained from Cox proportional hazards models. Data from public databases indicate that rs12407485 is part of an enhancer element for three transcription factors. Overall, these findings suggest that molecular features at the IL19 locus may modestly alter the establishment of HIV-1 infection. PMID:25633979

  14. Second generation bisheteroarylpiperazine (BHAP) HIV-1 reverse transcriptasae inhibitors: Enhancement of antiviral activity and aqueous solubility via 5- and 6-substitution of the indole ring

    SciTech Connect

    Poel, T.; Thomas, R.C.; Romero, D.L.; Hosley, M.J.; Morge, R.A.; Biles, C.; Reusser, F.; Althaus, I.W.; Schinzer, W.C.; Platzer, D.J.

    1993-12-31

    U-87201E, a potent HIV-1 reverse transcriptase inhibitor (RTI) discovered at Upjohn, is currently in Phase II clinical trials. Additional structure-activity studies have identified second-generation BHAPs with enhanced antiviral activity and improved pharmaceutical properties, notably increased aqueous solubility. Capitalizing on initial SAR studies which demonstrated a tolerance for substitution in the indole ring, a series of BHAPs bearing 5- and 6-substituted indoles was evaluated. Substituents such as ethers, sulfonamides, ureas, and sulfamides containing water-solubilizing groups such as polyethers or basic amines provided highly potent BHAPs with greatly enhanced solubility, such as U-93923. The synthesis, antiviral evaluation and solubility properties of these potent HIV-1 RTIs will be detailed.

  15. Chemokine CCL2 enhances NMDA receptor-mediated excitatory postsynaptic current in rat hippocampal slices-a potential mechanism for HIV-1-associated neuropathy?

    PubMed

    Zhou, Yan; Tang, Hongmei; Xiong, Huangui

    2016-06-01

    Human immunodeficiency virus type 1 (HIV-1)-infected mononuclear phagocytes (brain macrophages and microglial cells) release proinflammatory cytokines and chemokines. Elevated levels of chemokine CC motif ligand 2 (CCL2, known previously as monocyte chemoattractant protein-1) have been detected in serum and cerebrospinal fluid (CSF) of HIV-1-infected individuals and the raised CCL2 in the CSF correlates with HIV-1-associated neurocognitive disorders. To understand how elevated CCL2 induces HIV-1-associated neuropathy, we studied effects of CCL2 on excitatory postsynaptic current (EPSCs) in the CA1 region of rat hippocampal brain slices using whole-cell patch recording techniques. The AMPA receptor (AMPAR)-mediated EPSC (EPSCAMPAR) and N-Methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated EPSCs (EPSCNMDAR) were isolated pharmacologically. Bath application of CCL2 produced a significant enhancement of the amplitudes of EPSCs, EPSCAMPAR and EPSCNMDAR. Further studies revealed that CCL2 potentiated NMDAR subtype NR2A-mediated EPSC (EPSCNR2AR) and NR2B-mediated EPSC (EPSCNR2BR). To determine the site of action, we recorded spontaneous mini EPSCs (mEPSC) before and during bath application of CCL2. Our results showed that CCL2 decreased inter event interval (IEI) and increased the frequency of mEPSCs without change on the amplitude, suggesting a presynaptic site of CCL2 action. CCL2 was also found to injure primary rat hippocampal neuronal cultures and neuronal dendrites in the CA1 region of hippocampal slices. The CCL2-associated neuronal and dendritic injuries were blocked by a specific NMDAR antagonist or by a CCR2 receptor antagonist, indicating that CCL2-associated neural injury was mediated via NMDARs and/or CCR2 receptors. Taken together, these results suggest a potential role CCL2 may play in HIV-1-associated neuropathology. PMID:26968849

  16. Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

    PubMed

    Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

    2001-12-13

    We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential

  17. Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

    SciTech Connect

    Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; Perelson, Alan S.; Leitner, Thomas; Kouyos, Roger Dimitri

    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 process 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 fitness

  18. Recombination Enhances HIV-1 Envelope Diversity by Facilitating the Survival of Latent Genomic Fragments in the Plasma Virus Population

    PubMed Central

    Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; Perelson, Alan S.; Leitner, Thomas

    2015-01-01

    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 process 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 fitness

  19. β-chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: Effects on microglial migration

    PubMed Central

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L.; Hauser, Kurt F.; Knapp, Pamela E.

    2010-01-01

    HIV-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or gp120. In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/RANTES, CCL3/MIP-1α, and CCL4/MIP-1β were increased by 12 h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pretreatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous. PMID:20403075

  20. Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

    DOE PAGESBeta

    Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; Perelson, Alan S.; Leitner, Thomas; Kouyos, Roger Dimitri

    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

  1. Unique IL-13Rα2-based HIV-1 vaccine strategy to enhance mucosal immunity, CD8(+) T-cell avidity and protective immunity.

    PubMed

    Ranasinghe, C; Trivedi, S; Stambas, J; Jackson, R J

    2013-11-01

    We have established that mucosal immunization can generate high-avidity human immunodeficiency virus (HIV)-specific CD8(+) T cells compared with systemic immunization, and interleukin (IL)-13 is detrimental to the functional avidity of these T cells. We have now constructed two unique recombinant HIV-1 vaccines that co-express soluble or membrane-bound forms of the IL-13 receptor α2 (IL-13Rα2), which can "transiently" block IL-13 activity at the vaccination site causing wild-type animals to behave similar to an IL-13 KO animal. Following intranasal/intramuscular prime-boost immunization, these IL-13Rα2-adjuvanted vaccines have shown to induce (i) enhanced HIV-specific CD8(+) T cells with higher functional avidity, with broader cytokine/chemokine profiles and greater protective immunity using a surrogate mucosal HIV-1 challenge, and also (ii) excellent multifunctional mucosal CD8(+) T-cell responses, in the lung, genito-rectal nodes (GN), and Peyer's patch (PP). Data revealed that intranasal delivery of these IL-13Rα2-adjuvanted HIV vaccines recruited large numbers of unique antigen-presenting cell subsets to the lung mucosae, ultimately promoting the induction of high-avidity CD8(+) T cells. We believe our novel IL-13R cytokine trap vaccine strategy offers great promise for not only HIV-1, but also as a platform technology against range of chronic infections that require strong sustained high-avidity mucosal/systemic immunity for protection. PMID:23403475

  2. Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides

    PubMed Central

    Rollenhagen, C; Lathrop, M J; Macura, S L; Doncel, G F; Asin, S N

    2014-01-01

    Herpes Simplex virus Type-2 (HSV-2) increases the risk of HIV-1 acquisition, yet the mechanism for this viral pathogen to regulate the susceptibility of the cervicovaginal mucosa to HIV-1 is virtually unknown. Using ex vivo human ectocervical tissue models, we report greater levels of HIV-1 reverse transcription, DNA integration, RNA expression, and virions release in HIV-1/HSV-2 co-infected tissues compared with HIV-1 only infected tissues (P<0.05). Enhanced HIV-1 replication was associated with increased CD4, CCR5, and CD38 transcription (P<0.05) and increased number of CD4+/CCR5+/CD38+ T cells in HIV-1/HSV-2 co-infected tissues compared with tissues infected with HIV-1 alone. Tenofovir (TFV) 1% gel, the leading microbicide candidate, demonstrated only partial protection against HIV-1, when applied vaginally before and after sexual intercourse. It is possible that mucosal inflammation, in particular that induced by HSV-2 infection, may have decreased TFV efficacy. HSV-2 upregulated the number of HIV-1-infected cells and elevated the concentration of TFV needed to decrease HIV-1 infection. Similarly, only high concentrations of TFV inhibited HSV-2 replication in HIV-1/HSV-2-infected tissues. Thus, HSV-2 co-infection and mucosal immune cell activation should be taken into consideration when designing preventative strategies for sexual transmission of HIV-1. PMID:24496317

  3. The Roles of HIV-1 Proteins and Antiretroviral Drug Therapy in HIV-1-Associated Endothelial Dysfunction

    PubMed Central

    Kline, Erik R.; Sutliff, Roy L.

    2008-01-01

    Since the emergence of highly active antiretroviral therapy (HAART), human immunodeficiency virus-1 (HIV-1)-infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite these positive HAART-associated outcomes, it has become increasingly clear that HIV-1 patients have an enhanced risk of developing cardiovascular disease over time. Clinical studies are instrumental in our understanding of vascular dysfunction in the context of HIV-1 infection. However, most clinical studies often do not distinguish whether HIV-1 proteins, HAART, or a combination of these 2 factors cause cardiovascular complications. This review seeks to address the roles of both HIV-1 proteins and antiretroviral drugs in the development of endothelial dysfunction because endothelial dysfunction is the hallmark initial step of many cardiovascular diseases. We analyze recent in vitro and in vivo studies examining endothelial toxicity in response to HIV-1 proteins or in response to the various classes of antiretroviral drugs. Furthermore, we discuss the multiple mechanisms by which HIV-1 proteins and HAART injure the vascular endothelium in HIV-1 patients. By understanding the molecular mechanisms of HIV-1 protein- and antiretroviral-induced cardiovascular disease, we may ultimately improve the quality of life of HIV-1 patients through better drug design and the discovery of new pharmacological targets. PMID:18525451

  4. Psychoneuroimmunology and HIV-1.

    ERIC Educational Resources Information Center

    Antoni, Michael H.; And Others

    1990-01-01

    Presents evidence describing benefits of behavioral interventions such as aerobic exercise training on both psychological and immunological functioning among high risk human immunodeficiency virus-Type 1 (HIV-1) seronegative and very early stage seropositive homosexual men. HIV-1 infection is cast as chronic disease for which early…

  5. An axenic amastigote system for drug screening.

    PubMed Central

    Callahan, H L; Portal, A C; Devereaux, R; Grogl, M

    1997-01-01

    Currently available primary screens for selection of candidate antileishmanial compounds are not ideal. The choices include screens that are designed to closely reflect the situation in vivo but are labor-intensive and expensive (intracellular amastigotes and animal models) and screens that are designed to facilitate rapid testing of a large number of drugs but do not use the clinically relevant parasite stage (promastigote model). The advent of successful in vitro culture of axenic amastigotes permits the development of a primary screen which is quick and easy like the promastigote screen but still representative of the situation in vivo, since it uses the relevant parasite stage. We have established an axenic amastigote drug screening system using a Leishmania mexicana strain (strain M379). A comparison of the 50% inhibitory concentration (IC50) drug sensitivity profiles of M379 promastigotes, intracellular amastigotes, and axenic amastigotes for six clinically relevant antileishmanial drugs (sodium stibogluconate, meglumine antimoniate, pentamidine, paromomycin, amphotericin B, WR6026) showed that M379 axenic amastigotes are a good model for a primary drug screen. Promastigote and intracellular amastigote IC50s differed for four of the six drugs tested by threefold or more; axenic amastigote and intracellular amastigote IC50s differed by twofold for only one drug. This shows that the axenic amastigote susceptibility to clinically used reference drugs is comparable to the susceptibility of amastigotes in macrophages. These data also suggest that for the compounds tested, susceptibility is intrinsic to the parasite stage. This contradicts previous hypotheses that suggested that the activities of antimonial agents against intracellular amastigotes were solely a function of the macrophage. PMID:9087496

  6. Enhanced proliferative cellular responses to HIV-1 V3 peptide and gp120 following immunization with V3:Ty virus-like particles.

    PubMed

    Harris, S J; Gearing, A J; Layton, G T; Adams, S E; Kingsman, A J

    1992-11-01

    The induction of CD4+ T-helper (Th) cell responses is likely to be an important requirement of vaccine candidates designed to prevent or moderate human immunodeficiency virus-1 (HIV-1) infection. We have investigated the ability of hybrid Ty virus-like particles carrying the V3 loop region of the HIV-1 IIIB envelope gp120 (V3:Ty-VLP) to elicit V3-specific proliferative responses. Significant proliferation in response to stimulation in vitro with homologous IIIB V3 peptide was observed following immunization of mice with V3:Ty-VLP either as an aluminium hydroxide precipitate or without adjuvant. Responses to MN V3 peptide were also observed in certain mouse haplotypes. To assess the effect of presenting the V3 loop in this particulate form, we compared the responses induced by V3:Ty-VLP with those obtained with two non-particulate immunogens, recombinant gp120 (rgp120) and V3 peptide conjugated to albumin. V3-specific responses to V3 peptide in vitro were reproducibly higher following immunization with V3:Ty-VLP than with either rgp120 or V3-albumin coagulate (V3-alb). The data indicate that immunization with the V3 loop as a hybrid Ty-VLP results in enhanced proliferative responses to V3 peptide and recognition of rgp120 in vitro. Some cross-reactivity of Th cells for V3 sequences from different isolates was also observed. PMID:1362183

  7. A Chimeric HIV-1 Envelope Glycoprotein Trimer with an Embedded Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Domain Induces Enhanced Antibody and T Cell Responses*

    PubMed Central

    van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Menis, Sergey; Huang, Po-Ssu; Matthews, Katie; Michael, Elizabeth; Berkhout, Ben; Schief, William R.; Moore, John P.; Sanders, Rogier W.

    2011-01-01

    An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric EnvGM-CSF enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines. PMID:21515681

  8. A novel retinoic acid, catechin hydrate and mustard oil-based emulsion for enhanced cytokine and antibody responses against multiple strains of HIV-1 following mucosal and systemic vaccinations

    PubMed Central

    Yu, Mingke; Vajdy, Michael

    2011-01-01

    Non-replicating protein- or DNA-based antigens generally require immune-enhancing adjuvants and delivery systems. It has been particularly difficult to raise antibodies against gp120 of HIV-1, which constitutes an important approach in HIV vaccine design. While almost all effort in adjuvant research has focused on mimicking the pathogens and the danger signals they engender in the host, relatively little effort has been spent on nutritive approaches. In this study, a new nutritive immune-enhancing delivery system (NIDS) composed of vitamin A, a polyphenol-flavonoid catechin hydrate, and mustard oil was tested for its adjuvant effect in immune responses against the gp120 protein of HIV-1CN54. Following a combination of two mucosal and two systemic vaccinations of mice, we found significant enhancement of both local and systemic antibodies as well as cytokine responses. These data have important implications for vaccine and adjuvant design against HIV-1 and other pathogens. PMID:21272602

  9. Uneven Genetic Robustness of HIV-1 Integrase

    PubMed Central

    Rihn, Suzannah J.; Hughes, Joseph; Wilson, Sam J.

    2014-01-01

    ABSTRACT Genetic robustness (tolerance of mutation) may be a naturally selected property in some viruses, because it should enhance adaptability. Robustness should be especially beneficial to viruses like HIV-1 that exhibit high mutation rates and exist in immunologically hostile environments. Surprisingly, however, the HIV-1 capsid protein (CA) exhibits extreme fragility. To determine whether fragility is a general property of HIV-1 proteins, we created a large library of random, single-amino-acid mutants in HIV-1 integrase (IN), covering >40% of amino acid positions. Despite similar degrees of sequence variation in naturally occurring IN and CA sequences, we found that HIV-1 IN was significantly more robust than CA, with random nonsilent IN mutations only half as likely to cause lethal defects. Interestingly, IN and CA were similar in that a subset of mutations with high in vitro fitness were rare in natural populations. IN mutations of this type were more likely to occur in the buried interior of the modeled HIV-1 intasome, suggesting that even very subtle fitness effects suppress variation in natural HIV-1 populations. Lethal mutations, in particular those that perturbed particle production, proteolytic processing, and particle-associated IN levels, were strikingly localized at specific IN subunit interfaces. This observation strongly suggests that binding interactions between particular IN subunits regulate proteolysis during HIV-1 virion morphogenesis. Overall, use of the IN mutant library in conjunction with structural models demonstrates the overall robustness of IN and highlights particular regions of vulnerability that may be targeted in therapeutic interventions. IMPORTANCE The HIV-1 integrase (IN) protein is responsible for the integration of the viral genome into the host cell chromosome. To measure the capacity of IN to maintain function in the face of mutation, and to probe structure/function relationships, we created a library of random single

  10. HIV-1 Adenoviral Vector Vaccines Expressing Multi-Trimeric BAFF and 4-1BBL Enhance T Cell Mediated Anti-Viral Immunity

    PubMed Central

    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

  11. Suppression of HIV-1 Infectivity by Human Glioma Cells.

    PubMed

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2016-05-01

    HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo. PMID:26650729

  12. Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase

    PubMed Central

    Starodubova, E. S.; Isaguliants, M. G.; Kuzmenko, Y. V.; Latanova, A. A.; Krotova, O. A.; Karpov, V. L.

    2014-01-01

    Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-γ and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity. PMID:24772328

  13. HIV-1 Fusion Assay

    PubMed Central

    Cavrois, Marielle; Neidleman, Jason; Greene, Warner C.

    2016-01-01

    The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

  14. Synaptodendritic recovery following HIV-1 Tat exposure: Neurorestoration by phytoestrogens

    PubMed Central

    Bertrand, SJ; Mactutus, CF; Aksenova, MV; Espensen-Sturges, TD; Booze, RM

    2013-01-01

    HIV-1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV-1-associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV-1 infections. The HIV-1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. The present study examined the integrity of the dendritic network after exposure to HIV-1 Tat by labeling filamentous actin (F-actin)-rich structures (puncta) in primary neuronal cultures. After 24 hours of treatment, HIV-1 Tat was associated with the dendritic arbor and produced a significant reduction of F-actin-labeled dendritic puncta as well as loss of dendrites. Pretreatment with either of two plant-derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV-1 Tat treatment. Additionally, 6 days after HIV-1 Tat treatment, treatment with either daidzein or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV-1 Tat-induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV-1, but also promote recovery from synaptodendritic damage. PMID:23875777

  15. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  16. Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

    PubMed

    Bunders, Madeleine J; van Hamme, John L; Jansen, Machiel H; Boer, Kees; Kootstra, Neeltje A; Kuijpers, Taco W

    2014-01-01

    Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4(+) T cells from HIV-1-exposed children and healthy controls. At baseline CD4(+) T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4(+) T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4(+) T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7(+) CD4(+) T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4(+) T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant. PMID:25341640

  17. Antibody-Mediated Enhancement of HIV-1 and HIV-2 Production from BST-2/Tetherin-Positive Cells▿

    PubMed Central

    Miyagi, Eri; Andrew, Amy; Kao, Sandra; Yoshida, Takeshi; Strebel, Klaus

    2011-01-01

    BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2+ cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding. PMID:21917971

  18. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    SciTech Connect

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  19. HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1.

    PubMed

    Schoofs, Till; Klein, Florian; Braunschweig, Malte; Kreider, Edward F; Feldmann, Anna; Nogueira, Lilian; Oliveira, Thiago; Lorenzi, Julio C C; Parrish, Erica H; Learn, Gerald H; West, Anthony P; Bjorkman, Pamela J; Schlesinger, Sarah J; Seaman, Michael S; Czartoski, Julie; McElrath, M Juliana; Pfeifer, Nico; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-05-20

    3BNC117 is a broad and potent neutralizing antibody to HIV-1 that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 affects host antibody responses in viremic individuals. In comparison to untreated controls that showed little change in their neutralizing activity over a 6-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1. PMID:27199429

  20. K65R and K65A substitutions in HIV-1 reverse transcriptase enhance polymerase fidelity by decreasing both dNTP misinsertion and mispaired primer extension efficiencies

    PubMed Central

    Garforth, Scott J.; Domaoal, Robert A.; Lwatula, Chisanga; Landau, Mark J.; Meyer, Amanda J.; Anderson, Karen S.; Prasad, Vinayaka R.

    2010-01-01

    The Lys65 residue, in the fingers domain of HIV reverse transcriptase (RT), interacts in a sequence independent fashion with the incoming dNTP. Previously, we showed that a 5 amino acid deletion spanning Lys65 and a K65A substitution both enhanced the fidelity of dNTP insertion. We hypothesized that the Lys65 residue enhances dNTP misinsertion via interactions with the γ-phosphate of the incoming dNTP. We now examine this hypothesis in pre-steady state kinetic studies using wild type HIV-1 RT and two substitution mutants: K65A and K65R. The K65R mutation did not greatly increase misinsertion fidelity, but the K65A mutation led to higher incorporation fidelity. For a misinsertion to become a permanent error, it needs to be accompanied by the extension of the mispaired terminus thus formed. Both mutants, and the wild-type enzyme, discriminated against the mismatched primer at the catalytic step (kpol). Additionally, K65A and K65R mutants displayed a further decrease in mismatch extension efficiency, primarily at the level of dNTP binding. We employed hydroxyl radical footprinting to determine the position of the RT on the primer-template. The wild-type and Lys65 substituted enzymes occupied the same position at the primer terminus; the presence of a mismatched primer terminus caused all three enzymes to be displaced to a −2 position relative to the primer 3’ end. In the context of an efficiently extended mismatched terminus, the presence of the next complementary nucleotide overcame the displacement, resulting in a complex resembling the matched terminus. The results are consistent with the observed reduction in kpol in mispaired primer extension being due to the position of the enzyme at a mismatched terminus. Our work shows the influence of stabilizing interactions of Lys65 with the incoming dNTP in affecting two different aspects of polymerase fidelity. PMID:20538005

  1. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

    PubMed Central

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-01-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection

  2. TopoisomeraseIIβ in HIV-1 transactivation.

    PubMed

    Chekuri, Anil; Bhaskar, C; Bollimpelli, V Satish; Kondapi, Anand K

    2016-03-01

    TopoisomeraseIIβ, an isoform of type II topoisomerase, was found to be functional in various viral infections. Its plausible role in HIV life cycle was also suggested earlier, but not clearly established. In the present study, we have investigated the role of TopoIIβ in HIV-1 infection by its gain and loss of function. Overexpression of TopoIIβ lead to an increase in viral replication, resulting in enhanced virion production. HIV-1 replication was impaired when TopoIIβ was down regulated by siRNA and inhibited by ICRF-193 and merbarone. The role of TopoIIβ in HIV-1 transcription was shown through its interaction with Tat and recruitement to long terminal repeat (LTR) region by co-immunoprecipitation and ChIP assays. Involvement of TopoIIβ in transactivation of HIV-1 LTR was confirmed by luciferase assay in reporter cell line, TZM bl and also by transfection of reporter exogenously. It was also observed that LTR transactivation commensurated with the expression of TopoIIβ in the presence of Tat. In addition, a decreased viral gene expression on treatment with merbarone exemplifies the importance of catalytic activity of TopoIIβ in viral replication. These observations indicate that TopoIIβ is involved in the cascade of coactivator complexes that are recruited to LTR for regulation of HIV-1 transcription. PMID:26876283

  3. 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

  4. The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model.

    PubMed

    San Román, Beatriz; De Andrés, Ximena; Muñoz, Pilar-María; Obregón, Patricia; Asensio, Aaron-C; Garrido, Victoria; Mansilla, Cristina; Arribillaga, Laura; Lasarte, Juan-José; De Andrés, Damián; Amorena, Beatriz; Grilló, María-Jesús

    2012-03-28

    The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus. PMID:22326778

  5. Revisiting HIV-1 uncoating.

    PubMed

    Arhel, Nathalie

    2010-01-01

    HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus. It is an obligatory step of HIV-1 early infection and accompanies the transition between reverse transcription complexes (RTCs), in which reverse transcription occurs, and pre-integration complexes (PICs), which are competent to integrate into the host genome. The study of the nature and timing of HIV-1 uncoating has been paved with difficulties, particularly as a result of the vulnerability of the capsid assembly to experimental manipulation. Nevertheless, recent studies of capsid structure, retroviral restriction and mechanisms of nuclear import, as well as the recent expansion of technical advances in genome-wide studies and cell imagery approaches, have substantially changed our understanding of HIV uncoating. Although early work suggested that uncoating occurs immediately following viral entry in the cell, thus attributing a trivial role for the capsid in infected cells, recent data suggest that uncoating occurs several hours later and that capsid has an all-important role in the cell that it infects: for transport towards the nucleus, reverse transcription and nuclear import. Knowing that uncoating occurs at a later stage suggests that the viral capsid interacts extensively with the cytoskeleton and other cytoplasmic components during its transport to the nucleus, which leads to a considerable reassessment of our efforts to identify potential therapeutic targets for HIV therapy. This review discusses our current understanding of HIV uncoating, the functional interplay between infectivity and timely uncoating, as well as exposing the appropriate methods to study uncoating and addressing the many questions that remain unanswered. PMID:21083892

  6. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  7. Enhanced Immune Responses to HIV-1 Envelope Elicited by a Vaccine Regimen Consisting of Priming with Newcastle Disease Virus Expressing HIV gp160 and Boosting with gp120 and SOSIP gp140 Proteins.

    PubMed

    Khattar, Sunil K; DeVico, Anthony L; LaBranche, Celia C; Panda, Aruna; Montefiori, David C; Samal, Siba K

    2016-02-01

    Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost. PMID:26581986

  8. The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor.

    PubMed

    Tomasicchio, Michele; Avenant, Chanel; Du Toit, Andrea; Ray, Roslyn M; Hapgood, Janet P

    2013-01-01

    The glucocorticoid receptor (GR) regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr), can modulate the transcriptional response of the GR. Glucocorticoids (GCs) and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA) is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A). We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4(+) T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex), cortisol and MPA induce apoptosis in primary CD4(+) T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4(+) T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4(+) T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to susceptibility and

  9. Transplanting Supersites of HIV-1 Vulnerability

    PubMed Central

    Yang, Yongping; Gorman, Jason; Ofek, Gilad; Srivatsan, Sanjay; Druz, Aliaksandr; Lees, Christopher R.; Lu, Gabriel; Soto, Cinque; Stuckey, Jonathan; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Kwon, Peter D.

    2014-01-01

    One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose9-N-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 N-linked glycans and ∼25 Env residues, can be segregated

  10. Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch, Enhances Membrane Association, and Polyubiquitination of Gag

    PubMed Central

    Hahn, Friedrich; Setz, Christian; Friedrich, Melanie; Rauch, Pia; Solbak, Sara Marie; Frøystein, Nils Åge; Henklein, Petra; Votteler, Jörg; Fossen, Torgils; Schubert, Ulrich

    2014-01-01

    The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag. PMID:25279819

  11. Antiviral activity of CYC202 in HIV-1-infected cells.

    PubMed

    Agbottah, Emmanuel; de La Fuente, Cynthia; Nekhai, Sergie; Barnett, Anna; Gianella-Borradori, Athos; Pumfery, Anne; Kashanchi, Fatah

    2005-01-28

    There are currently 40 million individuals in the world infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality. Unfortunately, up to 25% of patients discontinue their initial HAART regimen. Current HIV-1 inhibitors target the fusion of the virus to the cell and two viral proteins, reverse transcriptase and protease. Here, we examined whether other targets, such as an activated transcription factor, could be targeted to block HIV-1 replication. We specifically asked whether we could target a cellular kinase needed for HIV-1 transcription using CYC202 (R-roscovitine), a pharmacological cyclin-dependent kinase inhibitor. We targeted the cdk2-cyclin E complex in HIV-1-infected cells because both cdk2 and cyclin E are nonessential during mammalian development and are likely replaced by other kinases. We found that CYC202 effectively inhibits wild type and resistant HIV-1 mutants in T-cells, monocytes, and peripheral blood mononuclear cells at a low IC(50) and sensitizes these cells to enhanced apoptosis resulting in a dramatic drop in viral titers. Interestingly, the effect of CYC202 is independent of cell cycle stage and more specific for the cdk2-cyclin E complex. Finally, we show that cdk2-cyclin E is loaded onto the HIV-1 genome in vivo and that CYC202 is able to inhibit the uploading of this cdk-cyclin complex onto HIV-1 DNA. Therefore, targeting cellular enzymes necessary for HIV-1 transcription, which are not needed for cell survival, is a compelling strategy to inhibit wild type and mutant HIV-1 strains. PMID:15531588

  12. The structure of Leishmania major amastigote lipophosphoglycan.

    PubMed

    Moody, S F; Handman, E; McConville, M J; Bacic, A

    1993-09-01

    Intracellular amastigotes of Leishmania major produce 6 x 10(4) copies/cell of a lipophosphoglycan (LPG) that is structurally distinct from the LPG produced by the extracellular promastigote form of L. major, Leishmania donovani, and Leishmania mexicana (reviewed by McConville, M. J. (1991) Cell Biol. Int. Rep. 15, 779-798). L. major amastigote LPG is composed of a lysoalkyl phosphatidylinositol lipid anchor that links via a diphosphorylated hexasaccharide core to a phosphoglycan (6-100 kDa). The structures of the anchor, the core, and the phosphoglycan were determined by monosaccharide and linkage analysis, fast atom bombardment-mass spectrometry, one-dimensional 1H NMR spectroscopy, and exoglycosidase microsequencing. The lipid anchor contains predominantly 1-O-alkylglycerols with 24:0 and 22:0 alkyl chains. The lipids are linked via a glycerol-myo-inositol-PO4 to a core glycan with the structure -PO4-6)Gal(alpha 1-)Gal(alpha 1-) Galf(beta 1-)[Glc(alpha 1-PO4-)]Man(alpha 1-)Man(alpha 1-)GlcN(alpha 1-). The chromatographic characteristics of the core glycan suggest that the saccharide components are linked similarly in amastigote and promastigote LPG. The phosphoglycan attached to the core consists of -PO4-6)Gal(beta 1-4)Man(alpha 1- repeats units which are either unsubstituted (70%) or substituted (30%) at the 3-position of the Gal residues with oligosaccharide side chains containing primarily Gal and some Glc. Thirteen different types of side chains were identified with the structures [Gal(beta 1-3)]x, where x = 1-11, or Glc(1-3)Glc(1-3), or Glc(1-3)Gal(beta 1-3), where glucose is probably in the beta-configuration. All monosaccharides in the phosphoglycan domain are in the pyranose configuration. The average number of repeat units per molecule is 36. The nonreducing terminus of the phosphoglycan chains probably terminates predominantly in the neutral disaccharide Gal(beta 1-4)Man(alpha 1-. Comparison of the structure of L. major amastigote LPG to L. major

  13. Medroxyprogesterone Acetate Regulates HIV-1 Uptake and Transcytosis but Not Replication in Primary Genital Epithelial Cells, Resulting in Enhanced T-Cell Infection.

    PubMed

    Ferreira, Victor H; Dizzell, Sara; Nazli, Aisha; Kafka, Jessica K; Mueller, Kristen; Nguyen, Philip V; Tremblay, Michel J; Cochrane, Alan; Kaushic, Charu

    2015-06-01

    Although clinical and experimental evidence indicates that female sex hormones and hormonal contraceptives regulate susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, the underlying mechanism remains unknown. Genital epithelial cells (GECs) are the first cells to encounter HIV during sexual transmission and their interaction with HIV may determine the outcome of exposure. This is the first report that HIV uptake by GECs increased significantly in the presence of the hormonal contraceptive medroxyprogesterone acetate (MPA) and progesterone and that uptake occurred primarily via endocytosis. No productive infection was detected, but endocytosed virus was released into apical and basolateral compartments. Significantly higher viral transcytosis was observed in the presence of MPA. In GEC and T-cell cocultures, maximum viral replication in T cells was observed in the presence of MPA, which also broadly upregulated chemokine production by GECs. These results suggest that MPA may play a significant role in regulating susceptibility to HIV. PMID:25538276

  14. Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1

    PubMed Central

    Pollara, Justin; McGuire, Erin; Fouda, Genevieve G.; Rountree, Wes; Eudailey, Josh; Overman, R. Glenn; Seaton, Kelly E.; Deal, Aaron; Edwards, R. Whitney; Tegha, Gerald; Kamwendo, Deborah; Kumwenda, Jacob; Nelson, Julie A. E.; Liao, Hua-Xin; Brinkley, Christie; Denny, Thomas N.; Ochsenbauer, Christina; Ellington, Sascha; King, Caroline C.; Jamieson, Denise J.; van der Horst, Charles; Kourtis, Athena P.; Tomaras, Georgia D.; Ferrari, Guido

    2015-01-01

    ABSTRACT Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody

  15. Broadly Neutralizing Antibody VRC01 Prevents HIV-1 Transmission from Plasmacytoid Dendritic Cells to CD4 T Lymphocytes

    PubMed Central

    Lederle, Alexandre; Laumond, Géraldine; Ducloy, Camille; Schmidt, Sylvie; Decoville, Thomas

    2014-01-01

    Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-α) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-α secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition. PMID:24965460

  16. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

    PubMed

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells. PMID:26184775

  17. A single amino acid substitution in the C4 region in gp120 confers enhanced neutralization of HIV-1 by modulating CD4 binding sites and V3 loop.

    PubMed

    Ringe, Rajesh; Sharma, Deepak; Zolla-Pazner, Susan; Phogat, Sanjay; Risbud, Arun; Thakar, Madhuri; Paranjape, Ramesh; Bhattacharya, Jayanta

    2011-09-30

    Identification of vulnerability in the HIV-1 envelope (Env) will aid in Env-based vaccine design. We recently found an HIV-1 clade C Env clone (4-2.J45) amplified from a recently infected Indian patient showing exceptional neutralization sensitivity to autologous plasma in contrast to other autologous Envs obtained at the same time point. By constructing chimeric Envs and fine mapping between sensitive and resistant Env clones, we found that substitution of highly conserved isoleucine (I) with methionine (M) (ATA to ATG) at position 424 in the C4 domain conferred enhanced neutralization sensitivity of Env-pseudotyped viruses to autologous and heterologous plasma antibodies. When tested against monoclonal antibodies targeting different sites in gp120 and gp41, Envs expressing M424 showed significant sensitivity to anti-V3 monoclonal antibodies and modestly to sCD4 and b12. Substitution of I424M in unrelated Envs also showed similar neutralization phenotype, indicating that M424 in C4 region induces exposure of neutralizing epitopes particularly in CD4 binding sites and V3 loop. PMID:21851958

  18. Genital HIV-1 RNA Quantity Predicts Risk of Heterosexual HIV-1 Transmission

    PubMed Central

    Baeten, Jared M.; Kahle, Erin; Lingappa, Jairam R.; Coombs, Robert W.; Delany-Moretlwe, Sinead; Nakku-Joloba, Edith; Mugo, Nelly R.; Wald, Anna; Corey, Lawrence; Donnell, Deborah; Campbell, Mary S.; Mullins, James I.; Celum, Connie

    2011-01-01

    High plasma HIV-1 RNA concentrations are associated with an increased risk of HIV-1 transmission. Although plasma and genital HIV-1 RNA concentrations are correlated, no study has evaluated the relationship between genital HIV-1 RNA and the risk of heterosexual HIV-1 transmission. In a prospective study of 2521 African HIV-1 serodiscordant couples, we assessed genital HIV-1 RNA quantity and HIV-1 transmission risk. HIV-1 transmission linkage was established within the partnership by viral sequence analysis. We tested endocervical samples from 1805 women, including 46 who transmitted HIV-1 to their partner, and semen samples from 716 men, including 32 who transmitted HIV-1 to their partner. Genital and plasma HIV-1 concentrations were correlated: For endocervical swabs, Spearman’s rank correlation coefficient rho was 0.56 (p<0.001), and for semen rho was 0.55 (p<0.001). Each 1 log10 increase in genital HIV-1 RNA was associated with a 2.20-fold (for endocervical swabs, 95% confidence interval 1.60–3.04, p<0.001) and a 1.79-fold (for semen, 95% confidence interval 1.30–2.47, p<0.001) increased risk of HIV-1 transmission. Genital HIV-1 RNA independently predicted HIV-1 transmission risk after adjusting for plasma HIV-1 quantity (hazard ratio 1.67 for endocervical swabs and 1.68 for semen). Seven female-to-male and four male-to-female HIV-1 transmissions (incidence <1% per year) occurred from persons with undetectable genital HIV-1 RNA, but in all eleven plasma HIV-1 RNA was detected. Thus, higher genital HIV-1 RNA concentrations are associated with greater risk of heterosexual HIV-1 transmission, and this effect was independent of plasma HIV-1 concentrations. These data suggest that HIV-1 RNA in genital secretions could be used as a marker of HIV-1 sexual transmission risk. PMID:21471433

  19. Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages.

    PubMed

    Kelleher, M; Moody, S F; Mirabile, P; Osborn, A H; Bacic, A; Handman, E

    1995-01-01

    Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors. PMID:7806383

  20. Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

    PubMed Central

    Paskaleva, Elena E; Lin, Xudong; Duus, Karen; McSharry, James J; Veille, Jean-Claude L; Thornber, Carol; Liu, Yanze; Lee, David Yu-Wei; Canki, Mario

    2008-01-01

    Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 μg/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 μg. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 μg/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development. PMID:18197976

  1. A Case of Seronegative HIV-1 Infection

    PubMed Central

    Spivak, Adam M.; Brennan, Tim; O'Connell, Karen; Sydnor, Emily; Williams, Thomas M.; Siliciano, Robert F.; Gallant, Joel E.; Blankson, Joel N.

    2009-01-01

    Patients infected with HIV-1 typically seroconvert within weeks of primary infection. In rare cases, patients do not develop antibodies against HIV-1 despite demonstrable infection. We describe an HLA-B*5802 positive individual who presented with AIDS despite repeatedly negative HIV-1 antibody screening tests. Phylogenetic analysis of env clones revealed little sequence diversity, and weak HIV-1 specific CD8+ T cell responses were present to Gag epitopes. The patient seroconverted after immune reconstitution on HAART. Lack of an antibody response to HIV-1 is rare and appears to be due to a defect in HIV-1-specific immunity rather than infection with attenuated virus. PMID:20039801

  2. CRISPR-mediated Activation of Latent HIV-1 Expression.

    PubMed

    Limsirichai, Prajit; Gaj, Thomas; Schaffer, David V

    2016-03-01

    Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here, we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate viral gene expression in cell line models of HIV-1 latency. We further demonstrate that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases can also promote viral gene activation. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection. PMID:26607397

  3. HIV-1 assembly in macrophages

    PubMed Central

    2010-01-01

    The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines. Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells

  4. Structural basis for enhanced neutralization of HIV-1 by a dimeric IgG form of the glycan-recognizing antibody 2G12

    PubMed Central

    Wu, Yunji; West, Anthony P.; Kim, Helen J.; Thornton, Matthew E.; Ward, Andrew B.; Bjorkman, Pamela J.

    2013-01-01

    SUMMARY The human IgG 2G12 recognizes high-mannose carbohydrates on the HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally-distinct dimers, demonstrating flexibility of the (Fab)2–Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12. PMID:24316082

  5. Phenotypic Correlates of HIV-1 Macrophage Tropism

    PubMed Central

    Arrildt, Kathryn T.; LaBranche, Celia C.; Joseph, Sarah B.; Dukhovlinova, Elena N.; Graham, William D.; Ping, Li-Hua; Schnell, Gretja; Sturdevant, Christa B.; Kincer, Laura P.; Mallewa, Macpherson; Heyderman, Robert S.; Van Rie, Annelies; Cohen, Myron S.; Spudich, Serena; Price, Richard W.; Montefiori, David C.

    2015-01-01

    ABSTRACT HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4+ T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE HIV-1 typically replicates in CD4+ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses

  6. HIV-1 Tat elongates the G1 phase and indirectly promotes HIV-1 gene expression in cells of glial origin.

    PubMed

    Kundu, M; Sharma, S; De Luca, A; Giordano, A; Rappaport, J; Khalili, K; Amini, S

    1998-04-01

    Human immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-kappaB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex. PMID:9525916

  7. Neutralizing antibodies decrease the envelope fluidity of HIV-1

    SciTech Connect

    Harada, Shinji Monde, Kazuaki; Tanaka, Yuetsu; Kimura, Tetsuya; Maeda, Yosuke; Yusa, Keisuke

    2008-01-05

    For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46 antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.

  8. Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization

    PubMed Central

    2010-01-01

    Background HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. Results We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). Conclusion These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa. PMID:20546571

  9. Bioinformatic Analysis of HIV-1 Entry and Pathogenesis

    PubMed Central

    Aiamkitsumrit, Benjamas; Dampier, Will; Antell, Gregory; Rivera, Nina; Martin-Garcia, Julio; Pirrone, Vanessa; Nonnemacher, Michael R.; Wigdahl, Brian

    2015-01-01

    The evolution of human immunodeficiency virus type 1 (HIV-1) with respect to co-receptor utilization has been shown to be relevant to HIV-1 pathogenesis and disease. The CCR5-utilizing (R5) virus has been shown to be important in the very early stages of transmission and highly prevalent during asymptomatic infection and chronic disease. In addition, the R5 virus has been proposed to be involved in neuroinvasion and central nervous system (CNS) disease. In contrast, the CXCR4-utilizing (X4) virus is more prevalent during the course of disease progression and concurrent with the loss of CD4+ T cells. The dual-tropic virus is able to utilize both co-receptors (CXCR4 and CCR5) and has been thought to represent an intermediate transitional virus that possesses properties of both X4 and R5 viruses that can be encountered at many stages of disease. The use of computational tools and bioinformatic approaches in the prediction of HIV-1 co-receptor usage has been growing in importance with respect to understanding HIV-1 pathogenesis and disease, developing diagnostic tools, and improving the efficacy of therapeutic strategies focused on blocking viral entry. Current strategies have enhanced the sensitivity, specificity, and reproducibility relative to the prediction of co-receptor use; however, these technologies need to be improved with respect to their efficient and accurate use across the HIV-1 subtypes. The most effective approach may center on the combined use of different algorithms involving sequences within and outside of the env-V3 loop. This review focuses on the HIV-1 entry process and on co-receptor utilization, including bioinformatic tools utilized in the prediction of co-receptor usage. It also provides novel preliminary analyses for enabling identification of linkages between amino acids in V3 with other components of the HIV-1 genome and demonstrates that these linkages are different between X4 and R5 viruses. PMID:24862329

  10. Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

    PubMed Central

    Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

    2015-01-01

    Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

  11. HIV-1, human interaction database: current status and new features

    PubMed Central

    Ako-Adjei, Danso; Fu, William; Wallin, Craig; Katz, Kenneth S.; Song, Guangfeng; Darji, Dakshesh; Brister, J. Rodney; Ptak, Roger G.; Pruitt, Kim D.

    2015-01-01

    The ‘Human Immunodeficiency Virus Type 1 (HIV-1), Human Interaction Database’, available through the National Library of Medicine at http://www.ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/interactions, serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. Each HIV-1 human protein interaction can be retrieved without restriction by web-based downloads and ftp protocols and includes: Reference Sequence (RefSeq) protein accession numbers, National Center for Biotechnology Information Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. In addition to specific HIV-1 protein–human protein interactions, included are interaction effects upon HIV-1 replication resulting when individual human gene expression is blocked using siRNA. A total of 3142 human genes are described participating in 12 786 protein–protein interactions, along with 1316 replication interactions described for each of 1250 human genes identified using small interfering RNA (siRNA). Together the data identifies 4006 human genes involved in 14 102 interactions. With the inclusion of siRNA interactions we introduce a redesigned web interface to enhance viewing, filtering and downloading of the combined data set. PMID:25378338

  12. Combinatorial Latency Reactivation for HIV-1 Subtypes and Variants▿ †

    PubMed Central

    Burnett, John C.; Lim, Kwang-il; Calafi, Arash; Rossi, John J.; Schaffer, David V.; Arkin, Adam P.

    2010-01-01

    The eradication of HIV-1 will likely require novel clinical approaches to purge the reservoir of latently infected cells from a patient. We hypothesize that this therapy should target a wide range of latent integration sites, act effectively against viral variants that have acquired mutations in their promoter regions, and function across multiple HIV-1 subtypes. By using primary CD4+ and Jurkat cell-based in vitro HIV-1 latency models, we observe that single-agent latency reactivation therapy is ineffective against most HIV-1 subtypes. However, we demonstrate that the combination of two clinically promising drugs—namely, prostratin and suberoylanilide hydroxamic acid (SAHA)—overcomes the limitations of single-agent approaches and can act synergistically for many HIV-1 subtypes, including A, B, C, D, and F. Finally, by identifying the proviral integration position of latent Jurkat cell clones, we demonstrate that this drug combination does not significantly enhance the expression of endogenous genes nearest to the proviral integration site, indicating that its effects may be selective. PMID:20357084

  13. Strategies for Eliciting HIV-1 Inhibitory Antibodies

    PubMed Central

    Tomaras, Georgia D.; Haynes, Barton F.

    2012-01-01

    Purpose of review Major roadblocks persist in the development of vaccines that elicit potent neutralizing antibodies targeting diverse HIV-1 strains, similar to known broadly neutralizing HIV-1 human monoclonal antibodies. Alternatively, other types of anti-HIV-1 envelope antibodies that may not neutralize HIV-1 in traditional neutralization assays but have other anti-HIV-1 activities (hereafter termed HIV-1 inhibitory antibodies) can be elicited by current vaccine strategies, and numerous studies are exploring their roles in preventing HIV-1 acquisition. We review examples of strategies for eliciting potentially protective HIV-1 inhibitory antibodies. Recent Findings Heterologous prime-boost strategies can yield anti-HIV immune responses; although only one (canarypox prime, Env protein boost) has been tested and shown positive results in an efficacy trial (RV144). Although the immune correlates of protection are as yet undefined, the reduced rate of acquisition without a significant effect on initial viral loads or CD4+ T cell counts, have raised the hypothesis of an RV144 vaccine-elicited transient protective B cell response. Summary In light of the RV144 trial, there is a critical need to define the entire functional spectrum of anti-HIV-1 antibodies, how easily each can be elicited, and how effective different types of antibody effector mechanisms can be in prevention of HIV-1 transmission. PMID:20978384

  14. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells

    PubMed Central

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-01-01

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways. PMID:26848681

  15. Tetraspanins regulate cell-to-cell transmission of HIV-1

    PubMed Central

    Krementsov, Dimitry N; Weng, Jia; Lambelé, Marie; Roy, Nathan H; Thali, Markus

    2009-01-01

    Background The presence of the tetraspanins CD9, CD63, CD81 and CD82 at HIV-1 budding sites, at the virological synapse (VS), and their enrichment in HIV-1 virions has been well-documented, but it remained unclear if these proteins play a role in the late phase of the viral replication cycle. Here we used overexpression and knockdown approaches to address this question. Results Neither ablation of CD9, CD63 and/or CD81, nor overexpression of these tetraspanins was found to affect the efficiency of virus release. However, confirming recently reported data, tetraspanin overexpression in virus-producing cells resulted in the release of virions with substantially reduced infectivity. We also investigated the roles of these tetraspanins in cell-to-cell transmission of HIV-1. Overexpression of CD9 and CD63 led to reduced cell-to-cell transmission of this virus. Interestingly, in knockdown experiments we found that ablation of CD63, CD9 and/or CD81 had no effect on cell-free infectivity. However, knockdown of CD81, but not CD9 and CD63, enhanced productive particle transmission to target cells, suggesting additional roles for tetraspanins in the transmission process. Finally, tetraspanins were found to be downregulated in HIV-1-infected T lymphocytes, suggesting that HIV-1 modulates the levels of these proteins in order to maximize the efficiency of its transmission within the host. Conclusion Altogether, these results establish an active role of tetraspanins in HIV-1 producer cells. PMID:19602278

  16. Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation.

    PubMed

    Paopang, Porntip; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Seesuriyachan, Phisit; Butr-Indr, Bordin

    2016-04-01

    The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments. PMID:25831436

  17. A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells from elite controllers.

    PubMed

    Leng, Jin; Ho, Hsin-Pin; Buzon, Maria J; Pereyra, Florencia; Walker, Bruce D; Yu, Xu G; Chang, Emmanuel J; Lichterfeld, Mathias

    2014-06-11

    HIV-1 reverse transcription represents the predominant target for pharmacological inhibition of viral replication, but cell-intrinsic mechanisms that can block HIV-1 reverse transcription in a clinically significant way are poorly defined. We find that effective HIV-1 reverse transcription depends on the phosphorylation of viral reverse transcriptase by host cyclin-dependent kinase (CDK) 2 at a highly conserved Threonine residue. CDK2-dependent phosphorylation increased the efficacy and stability of viral reverse transcriptase and enhanced viral fitness. Interestingly, p21, a cell-intrinsic CDK inhibitor that is upregulated in CD4(+) T cells from "elite controllers," potently inhibited CDK2-dependent phosphorylation of HIV-1 reverse transcriptase and significantly reduced the efficacy of viral reverse transcription. These data suggest that p21 can indirectly block HIV-1 reverse transcription by inhibiting host cofactors supporting HIV-1 replication and identify sites of viral vulnerability that are effectively targeted in persons with natural control of HIV-1 replication. PMID:24922574

  18. Elevated levels of Macrophage Migration Inhibitory Factor (MIF) in the plasma of HIV-1-infected patients and in HIV-1-infected cell cultures: a relevant role on viral replication

    PubMed Central

    Regis, Eduardo G.; Barreto-de-Souza, Victor; Morgado, Mariza M.; Bozza, Marcelo T.; Leng, Lin; Bucala, Richard; Bou-Habib, Dumith C.

    2011-01-01

    The cytokine macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of inflammatory and infectious diseases, however its role in HIV-1 infection is unknown. Here we show that HIV-1-infected patients present elevated plasma levels of MIF, that HIV-1-infected peripheral blood mononuclear cells (PBMCs) release a greater amount of MIF, and that the HIV-1 envelope glycoprotein gp120 induces MIF secretion from uninfected PBMCs. The HIV-1 replication in PBMCs declines when these cells were treated with anti-MIF antibodies, or when treated with the ABC-transporter inhibitor probenecid, which also inhibited MIF secretion. The addition of recombinant MIF (rhMIF) to HIV-1-infected PBMCs enhances viral replication of CCR5- or CXCR4-tropic HIV-1 isolates. Using a T CD4+ cell lineage containing an HIV long terminal repeats (LTR)-Luciferase construct, we detected that rhMIF promotes transcription from HIV-1 LTR. Our results show that HIV-1 induces MIF secretion and suggest that MIF influences the HIV-1 biology through activation of HIV-1 LTR. PMID:20085845

  19. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  20. Stimulation of HIV-1 Replication in Immature Dendritic Cells in Contact with Primary CD4 T or B Lymphocytes ▿

    PubMed Central

    Holl, Vincent; Xu, Ke; Peressin, Maryse; Lederle, Alexandre; Biedma, Marina Elizabeth; Delaporte, Maryse; Decoville, Thomas; Schmidt, Sylvie; Laumond, Géraldine; Aubertin, Anne-Marie; Moog, Christiane

    2010-01-01

    Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence. PMID:20147388

  1. A Prime-Boost Strategy Combining Intravaginal and Intramuscular Administration of Homologous Adenovirus to Enhance Immune Response Against HIV-1 in Mice.

    PubMed

    Ji, Zhonghua; Xie, Zhaolu; Wang, Qin; Zhang, Zhirong; Gong, Tao; Sun, Xun

    2016-03-01

    Immune responses to HIV in the vaginal tract effectively trigger both systemic and mucosal protection, providing a double layer of defense. However, recombinant adenoviral (rAd) vectors delivered intravaginally do not effectively penetrate the mucus layer and vaginal epithelium, and instead are rapidly cleared. To overcome these barriers, we previously synthesized a novel cationic polyethylene glycol derivative that can self-assemble into nanocomplexes with rAd. These nanocomplexes can help rAd bypass the mucus layer and enhance mucosal immune response to the encoded antigen. However, the resulting cellular and humoral responses were still lower than those elicited by single intramuscular injection of rAd. Therefore, in the present study we investigated a new vaccination strategy involving intravaginal priming with our nanocomplexes, followed by an intramuscular boost with rAd-gag. Mice immunized in this way showed more potent humoral and cellular responses, as well as higher IgA levels, than animals primed and boosted intravaginally with nanocomplexes. These results show the promise of a prime-boost strategy for developing vaccine candidates against HIV. PMID:26715124

  2. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

    PubMed Central

    Aiken, C

    1997-01-01

    Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA. PMID:9223476

  3. Human cervicovaginal mucus contains an activity that hinders HIV-1 movement.

    PubMed

    Shukair, S A; Allen, S A; Cianci, G C; Stieh, D J; Anderson, M R; Baig, S M; Gioia, C J; Spongberg, E J; Kauffman, S M; McRaven, M D; Lakougna, H Y; Hammond, C; Kiser, P F; Hope, T J

    2013-03-01

    Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function. PMID:22990624

  4. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal.

    PubMed

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C; Mahmoudi, Tokameh

    2016-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal. PMID:26870822

  5. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal

    PubMed Central

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C.; Mahmoudi, Tokameh

    2015-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal. PMID:26870822

  6. HIV-1 Infection and the PPARγ-Dependent Control of Adipose Tissue Physiology

    PubMed Central

    Giralt, Marta; Domingo, Pere; Villarroya, Francesc

    2009-01-01

    PPARγ is a ligand-dependent master transcription factor controlling adipocyte differentiation as well as multiple biological processes taking place in other cells present in adipose tissue depots such as macrophages. Recent research indicates that HIV-1 infection-related events may alter adipose tissue biology through several mechanisms involving PPARγ, ranging from direct effects of HIV-1-encoded proteins on adipocytes to the promotion of a proinflammatory environment that interferes with PPARγ actions. This effect of HIV-1 on adipose tissue cells can occur even in the absence of direct infection of adipocytes, as soluble HIV-1-encoded proteins such as Vpr may enter cells and inhibit PPARγ action. Moreover, repression of PPARγ actions may relieve inhibitory pathways of HIV-1 gene transcription, thus enhancing HIV-1 effects in infected cells. HIV-1 infection-mediated interference of PPARγ-dependent pathways in adipocytes and other cells inside adipose depots such as macrophages is likely to create an altered local environment that, after antiretroviral treatment, leads to lipodystrophy in HIV-1-infected and HAART-treated patients. PMID:19081837

  7. GADD45 proteins inhibit HIV-1 replication through specific suppression of HIV-1 transcription.

    PubMed

    Liang, Zhibin; Liu, Ruikang; Zhang, Hui; Zhang, Suzhen; Hu, Xiaomei; Tan, Juan; Liang, Chen; Qiao, Wentao

    2016-06-01

    GADD45 proteins are a group of stress-induced proteins and participate in various cellular pathways including cell cycle regulation, cell survival and death, DNA repair and demethylation. It was recently shown that HIV-1 infection induces the expression of GADD45 proteins. However, the effect of GADD45 on HIV-1 replication has not been studied. Here, we report that overexpression of GADD45 proteins reduces HIV-1 production through suppressing transcription from the HIV-1 LTR promoter. This inhibitory effect is specific to HIV-1, since GADD45 proteins neither inhibit the LTR promoters from other retroviruses nor reduce the production of these viruses. Knockdown of endogenous GADD45 modestly activates HIV-1 in the J-Lat A72 latency cell line, which suggests GADD45 proteins might play a role in maintaining HIV-1 latency. PMID:26994425

  8. Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance

    PubMed Central

    Kmiec, Dorota; Iyer, Shilpa S.; Stürzel, Christina M.; Sauter, Daniel; Hahn, Beatrice H.

    2016-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) groups M, N, O, and P are the result of independent zoonotic transmissions of simian immunodeficiency viruses (SIVs) infecting great apes in Africa. Among these, only Vpu proteins of pandemic HIV-1 group M strains evolved potent activity against the restriction factor tetherin, which inhibits virus release from infected cells. Thus, effective Vpu-mediated tetherin antagonism may have been a prerequisite for the global spread of HIV-1. To determine whether this particular function enhances primary HIV-1 replication and interferon resistance, we introduced mutations into the vpu genes of HIV-1 group M and N strains to specifically disrupt their ability to antagonize tetherin, but not other Vpu functions, such as degradation of CD4, down-modulation of CD1d and NTB-A, and suppression of NF-κB activity. Lack of particular human-specific adaptations reduced the ability of HIV-1 group M Vpu proteins to enhance virus production and release from primary CD4+ T cells at high levels of type I interferon (IFN) from about 5-fold to 2-fold. Interestingly, transmitted founder HIV-1 strains exhibited higher virion release capacity than chronic control HIV-1 strains irrespective of Vpu function, and group M viruses produced higher levels of cell-free virions than an N group HIV-1 strain. Thus, efficient virus release from infected cells seems to play an important role in the spread of HIV-1 in the human population and requires a fully functional Vpu protein that counteracts human tetherin. PMID:27531907

  9. Cytoplasmic dynein promotes HIV-1 uncoating.

    PubMed

    Pawlica, Paulina; Berthoux, Lionel

    2014-11-01

    Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating. PMID:25375884

  10. Elimination of Leishmania donovani amastigotes by activated macrophages.

    PubMed Central

    Haidaris, C G; Bonventre, P F

    1981-01-01

    Tissue macrophages are the obligatory host cells for Leishmania donovani, the causative agent of visceral leishmaniasis. In this study we sought to determine whether activated macrophages, as defined by the functional criterion of tumor cell cytotoxicity, were also able to exert a microbicidal effect on ingested L. donovani amastigotes. We found that mouse peritoneal macrophages activated by a variety of means exerted a cytotoxic effect on tumor cell targets but were not able to kill L. donovani amastigotes unless the infected macrophages were exposed continually to an activating stimulus. Corynebacterium parvum, Mycobacterium tuberculosis H37Ra, and lymphokine-activated peritoneal macrophages from C57BL/6J mice were cytotoxic for EMT6 tumor cell targets. However, L. donovani Sudan strain 1S amastigotes were not killed by these macrophages unless the activated state was maintained by daily addition of lymphokine to the infected monolayers for several days postinfection. The killing of amastigotes was dependent on the time of exposure to lymphokine, as well as on the concentration of lymphokine added to the culture. Images PMID:7287190

  11. In vitro Uncoating of HIV-1 Cores

    PubMed Central

    Shah, Vaibhav B.; Aiken, Christopher

    2011-01-01

    The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating. PMID:22105356

  12. In vitro uncoating of HIV-1 cores.

    PubMed

    Shah, Vaibhav B; Aiken, Christopher

    2011-01-01

    The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone(1, 2). Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form(3) in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells(4). This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability(4, 5, 6). It should also be useful for studying the role of cellular factors in HIV-1 uncoating. PMID:22105356

  13. Macrophages and HIV-1: An Unhealthy Constellation.

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-01

    Lentiviruses have a long-documented association with macrophages. Abundant evidence exists for in vitro and, in a tissue-specific manner, in vivo infection of macrophages by the primate lentiviruses HIV-1 and SIV. However, macrophage contribution to aspects of HIV-1 and SIV pathogenesis, and their role in viral persistence in individuals on suppressive antiretroviral therapy, remains unclear. Here we discuss recent evidence implicating macrophages in HIV-1-mediated disease and highlight directions for further investigation. PMID:26962941

  14. HIV-1 RNA quantification in CRF02_AG HIV-1 infection: too easy to make mistakes.

    PubMed

    Tatarelli, Paola; Taramasso, Lucia; Di Biagio, Antonio; Sticchi, Laura; Nigro, Nicola; Barresi, Renata; Viscoli, Claudio; Bruzzone, Bianca

    2016-04-01

    The number of patients newly infected by HIV-1 non-B subtypes and circulating recombinant forms (CRFs) is increasing worldwide, including in the western countries. We report on a primary HIV-1 infection in a Caucasian patient. A routine quantitative assay (Nuclisens EasyQ HIV-1 2.0, BioMérieux SA) showed 6,700 HIV-1 RNA copies/ml. A combined antiretroviral therapy (cART) consistent with low baseline HIV-1 RNA was started. Few days later, the analysis performed with REGA HIV-1 Subtyping Tool - Version 3.0 attributed the HIV-1 sequence to the CRF02_AG recombinant form. Therefore, a second real-time PCR assay was performed, using the Versant HIV-1 RNA 1.0 Assay (kPCR) (Siemens HealthCare Diagnostics) which revealed a HIV-1 RNA of 230,000 copies/ml. Consequently, the ongoing cART was potentiated. This case suggests that the wide genetic variability of HIV-1 subtypes may affect the capability of the commonly used assays to detect and accurately quantify HIV-1 RNA in non-B subtypes and CRFs. In presence of CRFs different commercial HIV-1 RNA tests should be performed to find the most reliable for viral load quantification at the diagnosis, because it influences the choice of cART, and during the follow-up. Indeed, international guidelines for HIV-1 infection management suggest to monitor patient' HIV-RNA with the same assay over the course of treatment. As different commercial tests can be performed in the same laboratory with considerable difficulty, the laboratory should select an assay that is suitable not only for the more prevalent strain, but also for less frequent ones that, nevertheless, can occur. Then, knowing and investigating the spread of non-B strains has essential clinical and laboratory implications. PMID:27196556

  15. Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

    SciTech Connect

    Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.; Sheri, Venkat Reddy; Kassekert, Luke A.; Chen, Shujing; Agniswamy, Johnson; Wang, Yuan-Fang; Hayashi, Hironori; Aoki, Manabu; Weber, Irene T.; Mitsuya, Hiroaki

    2015-10-30

    Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.

  16. HIV-1 Eradication: Early Trials (and Tribulations).

    PubMed

    Spivak, Adam M; Planelles, Vicente

    2016-01-01

    Antiretroviral therapy (ART) has rendered HIV-1 infection a manageable illness for those with access to treatment. However, ART does not lead to viral eradication owing to the persistence of replication-competent, unexpressed proviruses in long-lived cellular reservoirs. The potential for long-term drug toxicities and the lack of access to ART for most people living with HIV-1 infection have fueled scientific interest in understanding the nature of this latent reservoir. Exploration of HIV-1 persistence at the cellular and molecular level in resting memory CD4(+) T cells, the predominant viral reservoir in patients on ART, has uncovered potential strategies to reverse latency. We review recent advances in pharmacologically based 'shock and kill' HIV-1 eradication strategies, including comparative analysis of early clinical trials. PMID:26691297

  17. Development of prophylactic vaccines against HIV-1

    PubMed Central

    2013-01-01

    The focus of most current HIV-1 vaccine development is on antibody-based approaches. This is because certain antibody responses correlated with protection from HIV-1 acquisition in the RV144 phase III trial, and because a series of potent and broad spectrum neutralizing antibodies have been isolated from infected individuals. Taken together, these two findings suggest ways forward to develop a neutralizing antibody-based vaccine. However, understanding of the correlates of protection from disease in HIV-1 and other infections strongly suggests that we should not ignore CTL-based research. Here we review recent progress in the field and highlight the challenges implicit in HIV-1 vaccine design and some potential solutions. PMID:23866844

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

    SciTech Connect

    Leitner, Thomas; Campbell, Mary S; Mullins, James I; Hughes, James P; Wong, Kim G; Raugi, Dana N; Scrensen, Stefanie

    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 determination 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

  19. Exosomes: Implications in HIV-1 Pathogenesis

    PubMed Central

    Madison, Marisa N.; Okeoma, Chioma M.

    2015-01-01

    Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission. PMID:26205405

  20. Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel.

    PubMed

    Hsu, Kate; Seharaseyon, Jegatheesan; Dong, Peihong; Bour, Stephan; Marbán, Eduardo

    2004-04-23

    Sequence analysis predicted significant structural homology between the HIV-1 accessory protein Vpu and the N-terminal region of TASK-1, a mammalian background K(+) channel. If the homology resulted from molecular piracy during HIV-1 evolution, these two proteins may have important functional interactions. Here we demonstrate that TASK and Vpu physically interact in cultured cells and in AIDS lymphoid tissues. The functional consequences were potentially destructive for both components: Vpu abolished TASK-1 current, while overexpressing TASK led to a marked impairment of Vpu's ability to enhance viral particle release. Further, the first 40 amino acids of TASK-1 (part of the homology to Vpu) were capable of enhancing HIV-1 particle release. This virus-host interaction may influence HIV-1/AIDS progression, as well as electrical signaling in infected host tissues. PMID:15099524

  1. HIV-1 TAT Inhibits Microglial Phagocytosis of Aβ Peptide

    PubMed Central

    Giunta, Brian; Zhou, Yuyan; Hou, Huayan; Rrapo, Elona; Fernandez, Francisco; Tan, Jun

    2008-01-01

    Human immunodeficiency virus (HIV)-associated dementia (HAD) is a subcortical neuropsychiatric syndrome that has increased in prevalence in the era of highly active antiretroviral therapy (HAART). Several studies demonstrated increased amyloidosis in brains of HIV patients and suggested that there may be a significant number of long-term HIV survivors with co-morbid Alzheimer's disease (AD) in the future. We show HIV-1 Tat protein inhibits microglial uptake of Aβ1-42 peptide, a process that is enhanced by interferon-gamma (IFN-γ) and rescued by the STAT1 inhibitor (-)-epigallocatechin-3-gallate (EGCG). It is hypothesized that reduced Aβ uptake occurs through IFN-γ mediated STAT1 activation. This process promotes a switch from a phagocytic to an antigen presenting phenotype in microglia through activation of class II transactivator (CIITA). Additionally, we show that HIV-1 Tat significantly disrupts apolipoprotein-3 (Apo-E3) promoted microglial Aβ uptake. As Tat has been shown to directly interact with the low density lipoprotein (LRP) receptor and thus inhibit the uptake of its ligands including apolipoprotein E4 (Apo-E4) and Aβ peptide in neurons, we further hypothesize that a similar inhibition of LRP may occur in microglia. Future studies will be required to fully characterize the mechanisms underlying IFN-γ enhancement of HIV-1 Tats disruption of microglial phagocytosis of Aβ and Apo-E3. PMID:18784813

  2. Dendritic Cells from HIV Controllers Have Low Susceptibility to HIV-1 Infection In Vitro but High Capacity to Capture HIV-1 Particles

    PubMed Central

    Hamimi, Chiraz; David, Annie; Versmisse, Pierre; Weiss, Laurence; Bruel, Timothée; Zucman, David; Appay, Victor; Moris, Arnaud; Ungeheuer, Marie-Noëlle; Lascoux-Combe, Caroline; Barré-Sinoussi, Françoise; Muller-Trutwin, Michaela; Boufassa, Faroudy; Lambotte, Olivier; Pancino, Gianfranco; Sáez-Cirión, Asier

    2016-01-01

    HIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia. PMID:27505169

  3. Dendritic Cells from HIV Controllers Have Low Susceptibility to HIV-1 Infection In Vitro but High Capacity to Capture HIV-1 Particles.

    PubMed

    Hamimi, Chiraz; David, Annie; Versmisse, Pierre; Weiss, Laurence; Bruel, Timothée; Zucman, David; Appay, Victor; Moris, Arnaud; Ungeheuer, Marie-Noëlle; Lascoux-Combe, Caroline; Barré-Sinoussi, Françoise; Muller-Trutwin, Michaela; Boufassa, Faroudy; Lambotte, Olivier; Pancino, Gianfranco; Sáez-Cirión, Asier

    2016-01-01

    HIV controllers (HICs), rare HIV-1 infected individuals able to control viral replication without antiretroviral therapy, are characterized by an efficient polyfunctional and cytolytic HIV-specific CD8+ T cell response. The mechanisms underlying the induction and maintenance of such response in many HICs despite controlled viremia are not clear. Dendritic cells play a crucial role in the generation and reactivation of T cell responses but scarce information is available on those cells in HICs. We found that monocyte derived dendritic cells (MDDCs) from HICs are less permissive to HIV-1 infection than cells from healthy donors. In contrast MDDCs from HICs are particularly efficient at capturing HIV-1 particles when compared to cells from healthy donors or HIV-1 patients with suppressed viral load on antiretroviral treatment. MDDCs from HICs expressed on their surface high levels of syndecan-3, DC-SIGN and MMR, which could cooperate to facilitate HIV-1 capture. The combination of low susceptibility to HIV-1 infection but enhanced capacity to capture particles might allow MDDCs from HICs to preserve their function from the deleterious effect of infection while facilitating induction of HIV-specific CD8+ T cells by cross-presentation in a context of low viremia. PMID:27505169

  4. High-Throughput Humanized Mouse Models for Evaluation of HIV-1 Therapeutics and Pathogenesis.

    PubMed

    Thomas, Tynisha; Seay, Kieran; Zheng, Jian Hua; Zhang, Cong; Ochsenbauer, Christina; Kappes, John C; Goldstein, Harris

    2016-01-01

    enhance mucosal transmission of HIV-1. PMID:26714715

  5. HIV-1 Tat and Cocaine Impair Survival of Cultured Primary Neuronal Cells via a Mitochondrial Pathway.

    PubMed

    De Simone, Francesca Isabella; Darbinian, Nune; Amini, Shohreh; Muniswamy, Madesh; White, Martyn K; Elrod, John W; Datta, Prasun K; Langford, Dianne; Khalili, Kamel

    2016-06-01

    Addictive stimulant drugs, such as cocaine, are known to increase the risk of exposure to HIV-1 infection and hence predispose towards the development of AIDS. Previous findings suggested that the combined effect of chronic cocaine administration and HIV-1 infection enhances cell death. Neuronal survival is highly dependent on the health of mitochondria providing a rationale for assessing mitochondrial integrity and functionality following cocaine treatment, either alone or in combination with the HIV-1 viral protein Tat, by monitoring ATP release and mitochondrial membrane potential (ΔΨm). Our results indicate that exposing human and rat primary hippocampal neurons to cocaine and HIV-1 Tat synergistically decreased both mitochondrial membrane potential and ATP production. Additionally, since previous studies suggested HIV-1 infection alters autophagy in the CNS, we investigated how HIV-1 Tat and cocaine affect autophagy in neurons. The results indicated that Tat induces an increase in LC3-II levels and the formation of Parkin-ring-like structures surrounding damaged mitochondria, indicating the possible involvement of the Parkin/PINK1/DJ-1 (PPD) complex in neuronal degeneration. The importance of mitochondrial damage is also indicated by reductions in mitochondrial membrane potential and ATP content induced by HIV-1 Tat and cocaine. PMID:27032771

  6. Maturation-Induced Cloaking of Neutralization Epitopes on HIV-1 Particles

    PubMed Central

    Joyner, Amanda S.; Willis, Jordan R.; Crowe, James E.; Aiken, Christopher

    2011-01-01

    To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER. PMID:21931551

  7. Exercise and Human Immunodeficiency Virus (HIV-1) Infection

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales; Jackson, Catherine G. R.; Greenleaf, John E.

    1995-01-01

    The human immune system is highly efficient and remarkably protective when functioning properly. Similar to other physiological systems, it functions best when the body is maintained with a balanced diet, sufficient rest and a moderately stress-free lifestyle. It can be disrupted by inappropriate drug use and extreme emotion or exertion. The functioning of normal or compromised immune systems can be enhanced by properly prescribed moderate exercise conditioning regimens in healthy people, and in some human immunodeficiency virus (HIV-1)-infected patients but not in others who unable to complete an interval training program. Regular exercise conditioning in healthy people reduces cardiovascular risk factors, increases stamina, facilitates bodyweight control, and reduces stress by engendering positive feelings of well-being. Certain types of cancer may also be suppressed by appropriate exercise conditioning. Various exercise regimens are being evaluated as adjunct treatments for medicated patients with the HIV-1 syndrome. Limited anecdotal evidence from patients suggests that moderate exercise conditioning is per se responsible for their survival well beyond expectancy. HIV-1-infected patients respond positively, both physiologically and psychologically, to moderate exercise conditioning. However, the effectiveness of any exercise treatment programme depends on its mode, frequency, intensity and duration when prescribed o complement the pathological condition of the patient. The effectiveness of exercise conditioning regimens in patients with HIV-1 infection is reviewed in this article. In addition, we discuss mechanisms and pathways, involving the interplay of psychological and physiological factors, through which the suppressed immune system can be enhanced. The immune modulators discussed are endogenous opioids, cytokines, neurotransmitters and other hormones. Exercise conditioning treatment appears to be more effective when combined with other stress management

  8. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    PubMed Central

    Xiao, Xiaodong; Chen, Weizao; Feng, Yang; Dimitrov, Dimiter S.

    2009-01-01

    Several human monoclonal antibodies (hmAbs) and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG) lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM) affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i) antibodies in HIV-1-infected patients (X5 is a CD4i antibody) as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and intermediate antibodies

  9. Envelope gene evolution and HIV-1 neuropathogenesis

    PubMed Central

    Vázquez-Santiago, Fabián J.; Rivera-Amill, Vanessa

    2016-01-01

    In the era of combined antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) account for 40 to 56% of all HIV+ cases. During the acute stage of HIV-1 infection (<6 months), the virus invades and replicates within the central nervous system (CNS). Compared to peripheral tissues, the local CNS cell population expresses distinct levels of chemokine receptors, which levels exert selective pressure on the invading virus. HIV-1 envelope (env) sequences recovered from the brains and cerebrospinal fluid (CSF) of neurocognitively impaired HIV+ subjects often display higher nucleotide variability as compared to non-impaired HIV+ subjects. Specifically, env evolution provides HIV-1 with the strategies to evade host immune response, to reduce chemokine receptor dependence, to increase co-receptor binding efficiency, and to potentiate neurotoxicity. The evolution of env within the CNS leads to changes that may result in the emergence of novel isolates with neurotoxic and neurovirulent features. However, whether specific factors of HIV-1 evolution lead to the emergence of neurovirulent and neurotropic isolates remains ill-defined. HIV-1 env evolution is an ongoing phenomenon that occurs independently of neurological and neurocognitive disease severity; thus HIV env evolution may play a pivotal and reciprocal role in the etiology of HAND. Despite the use of cART, the reactivation of latent viral reservoirs represents a clinical challenge because of the replenishment of the viral pool that may subsequently lead to persistent infection. Therefore, gaining a more complete understanding of how HIV-1 env evolves over the course of the disease should be considered for the development of future therapies aimed at controlling CNS burden, diminishing persistent viremia, and eradicating viral reservoirs. Here we review the current literature on the role of HIV-1 env evolution in the setting of HAND disease progression and on the impact of cART on the dynamics of

  10. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.

    PubMed

    Usami, Yoshiko; Wu, Yuanfei; Göttlinger, Heinrich G

    2015-10-01

    HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS. PMID:26416733

  11. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

    PubMed Central

    Usami, Yoshiko; Wu, Yuanfei; Göttlinger, Heinrich G.

    2015-01-01

    HIV-1 Nef and the unrelated murine leukemia virus glycoGag strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins SERINC3 and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of SERINC3 together with SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivities. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS. PMID:26416733

  12. Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells.

    PubMed

    Sundstrom, J Bruce; Little, Dawn M; Villinger, Francois; Ellis, Jane E; Ansari, Aftab A

    2004-04-01

    Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection. PMID:15034054

  13. Determinants of Cyclophilin A-dependent TRIM5α restriction against HIV-1

    PubMed Central

    Lin, Tsai-Yu; Emerman, Michael

    2008-01-01

    TRIM5α is a host protein that can bind to incoming retroviral capsid (CA) and inhibit retroviruses in a species-specific manner. The CA protein of HIV-1 also interacts with high affinity to the host protein cyclophilin A (CypA). This binding has been shown to positively affect some early stage of the viral life-cycle in human cells. However, the CypA/CA interaction also renders HIV-1 more susceptible to rhesus TRIM5α (rhTRIM5α) restriction. We find that the ability of old world monkey TRIM5α genes to restrict HIV-1 in a CypA-dependent manner is widespread. On the other hand, we find that simian immunodeficiency viruses from tantalus monkeys (SIVagmTAN), is unlike HIV-1 in that CypA does not enhance the rhTRIM5α restriction against the virus even though the CA of this virus, like HIV-1, does bind CypA. Mapping of the determinants for this phenotype by swapping regions on CA between SIVagmTAN and HIV-1 showed that when SIVagmTAN contains loops between helices 4/5 (4–5 loop) and 6/7 (6–7 loop) from HIV-1 CA, it becomes susceptible to the CypA-enhanced rhTRIM5α restriction. Surprisingly, when SIVagmTAN contains either loop from HIV-1 CA, it gains sensitivity to TRIM5α from species which originally have no effect on the wild-type virus. Moreover, we find that CypA/CA interaction occurs early after viral entry but the CypA-enhanced restriction mostly acts on the stage after reverse transcription. PMID:18678385

  14. Interleukin-1- and Type I Interferon-Dependent Enhanced Immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef Vaccine Vector with Dual Deletions of Type I and Type II Interferon-Binding Proteins

    PubMed Central

    Delaloye, Julie; Filali-Mouhim, Abdelali; Cameron, Mark J.; Haddad, Elias K.; Harari, Alexandre; Goulet, Jean-Pierre; Gomez, Carmen E.; Perdiguero, Beatriz; Esteban, Mariano; Pantaleo, Giuseppe; Roger, Thierry; Sékaly, Rafick-Pierre

    2015-01-01

    ABSTRACT NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4+ T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV

  15. The Host Proteins Transportin SR2/TNPO3 and Cyclophilin A Exert Opposing Effects on HIV-1 Uncoating

    PubMed Central

    Shah, Vaibhav B.; Shi, Jiong; Hout, David R.; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S.; Engelman, Alan

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  16. The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating.

    PubMed

    Shah, Vaibhav B; Shi, Jiong; Hout, David R; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S; Engelman, Alan; Aiken, Christopher

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  17. 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

  18. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

    PubMed Central

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S. Y.

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This “shock” approach is then followed by “kill” of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  19. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules.

    PubMed

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  20. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression.

    PubMed

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

    2016-01-01

    The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-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 m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. PMID:27371828

  1. Neuropathology of early HIV-1 infection.

    PubMed

    Gray, F; Scaravilli, F; Everall, I; Chretien, F; An, S; Boche, D; Adle-Biassette, H; Wingertsmann, L; Durigon, M; Hurtrel, B; Chiodi, F; Bell, J; Lantos, P

    1996-01-01

    Early HIV-1 invasion of the central nervous system has been demonstrated by many cerebrospinal fluid studies; however, most HIV-1 carriers remain neurologically unimpaired during the so called "asymptomatic" period lasting from seroconversion to symptomatic AIDS. Therefore, neuropathological studies in the early pre-AIDS stages are very few, and the natural history of central nervous system changes in HIV-1 infection remains poorly understood. Examination of brains of asymptomatic HIV-1 positive individuals who died accidentally and of rare cases with acute fatal encephalopathy revealing HIV infection, and comparison with experimental simian immunodeficiency virus and feline immunodeficiency virus infections suggest that, invasion of the CNS by HIV-1 occurs at the time of primary infection and induces an immunological process in the central nervous system. This includes an inflammatory T-cell reaction with vasculitis and leptomeningitis, and immune activation of brain parenchyma with increased number of microglial cells, upregulation of major histocompatibility complex class II antigens and local production of cytokines. Myelin pallor and gliosis of the white matter are usually found and are likely to be the consequence of opening of the blood brain barrier due to vasculitis; direct damage to oligodendrocytes by cytokines may also interfere. These white matter changes may explain, at least partly, the early cerebral atrophy observed, by magnetic resonance imaging, in asymptomatic HIV-1 carriers. In contrast, cortical damage seems to be a late event in the course of HIV-1 infection. There is no significant neuronal loss at the early stages of the disease, no accompanying increase in glial fibrillary acid protein staining in the cortex, and only exceptional neuronal apoptosis. Although HIV-1 proviral DNA may be demonstrated in a number of brains, viral replication remains very low during the asymptomatic stage of HIV-1 infection. This makes it likely that, although

  2. STAT1 signaling modulates HIV-1-induced inflammatory responses and leukocyte transmigration across the blood-brain barrier.

    PubMed

    Chaudhuri, Anathbandhu; Yang, Bo; Gendelman, Howard E; Persidsky, Yuri; Kanmogne, Georgette D

    2008-02-15

    The relationship among neuroinflammation, blood-brain barrier (BBB) dysfunction, and progressive HIV-1 infection as they affect the onset and development of neuroAIDS is incompletely understood. One possible link is signal transducers and activators of transcription (STATs) pathways. These respond to proinflammatory and regulatory factors and could affect neuroinflammatory responses induced from infected cells and disease-affected brain tissue. Our previous works demonstrated that HIV-1 activates pro-inflammatory and interferon-alpha-inducible genes in human brain microvascular endothelial cells (HBMECs) and that these genes are linked to the Janus kinase (JAK)/STAT pathway. We now demonstrate that HIV-1 activates STAT1, induces IL-6 expression, and diminishes expression of claudin-5, ZO-1, and ZO-2 in HBMECs. The STAT1 inhibitor, fludarabine, blocked HIV-1-induced IL-6, diminished HIV-1-induced claudin-5 and ZO-1 down-regulation, and blocked HIV-1- and IL-6-induced monocyte migration across a BBB model. Enhanced expression and activation of STAT1 and decreased claudin-5 were observed in microvessels from autopsied brains of patients with HIV-1-associated dementia. These data support the notion that STAT1 plays an integral role in HIV-1-induced BBB damage and is relevant to viral neuropathogenesis. Inhibition of STAT1 activation could provide a unique therapeutic strategy to attenuate HIV-1-induced BBB compromise and as such improve clinical outcomes. PMID:18003888

  3. HIV-1 Genetic Variability and Clinical Implications

    PubMed Central

    Santoro, Maria Mercedes; Perno, Carlo Federico

    2013-01-01

    Despite advances in antiretroviral therapy that have revolutionized HIV disease management, effective control of the HIV infection pandemic remains elusive. Beyond the classic non-B endemic areas, HIV-1 non-B subtype infections are sharply increasing in previous subtype B homogeneous areas such as Europe and North America. As already known, several studies have shown that, among non-B subtypes, subtypes C and D were found to be more aggressive in terms of disease progression. Luckily, the response to antiretrovirals against HIV-1 seems to be similar among different subtypes, but these results are mainly based on small or poorly designed studies. On the other hand, differences in rates of acquisition of resistance among non-B subtypes are already being observed. This different propensity, beyond the type of treatment regimens used, as well as access to viral load testing in non-B endemic areas seems to be due to HIV-1 clade specific peculiarities. Indeed, some non-B subtypes are proved to be more prone to develop resistance compared to B subtype. This phenomenon can be related to the presence of subtype-specific polymorphisms, different codon usage, and/or subtype-specific RNA templates. This review aims to provide a complete picture of HIV-1 genetic diversity and its implications for HIV-1 disease spread, effectiveness of therapies, and drug resistance development. PMID:23844315

  4. The hunt for HIV-1 integrase inhibitors.

    PubMed

    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results. PMID:16839248

  5. Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

    PubMed Central

    Hanauske-Abel, Hartmut M.; Saxena, Deepti; Palumbo, Paul E.; Hanauske, Axel-Rainer; Luchessi, Augusto D.; Cambiaghi, Tavane D.; Hoque, Mainul; Spino, Michael; Gandolfi, Darlene D'Alliessi; Heller, Debra S.; Singh, Sukhwinder; Park, Myung Hee; Cracchiolo, Bernadette M.; Tricta, Fernando; Connelly, John; Popowicz, Anthony M.; Cone, Richard A.; Holland, Bart; Pe’ery, Tsafi; Mathews, Michael B.

    2013-01-01

    HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal

  6. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle. PMID:20016921

  7. HIV-1 Reservoirs During Suppressive Therapy.

    PubMed

    Barton, Kirston; Winckelmann, Anni; Palmer, Sarah

    2016-05-01

    The introduction of antiretroviral therapy (ART) 20 years ago has dramatically reduced morbidity and mortality associated with HIV-1. Initially there was hope that ART would be curative, but it quickly became clear that even though ART was able to restore CD4(+) T cell counts and suppress viral loads below levels of detection, discontinuation of treatment resulted in a rapid rebound of infection. This is due to persistence of a small reservoir of latently infected cells with a long half-life, which necessitates life-long ART. Over the past few years, significant progress has been made in defining and characterizing the latent reservoir of HIV-1, and here we review how understanding the latent reservoir during suppressive therapy will lead to significant advances in curative approaches for HIV-1. PMID:26875617

  8. HIV-1 immunopathogenesis in humanized mouse models

    PubMed Central

    Zhang, Liguo; Su, Lishan

    2012-01-01

    In recent years, the technology of constructing chimeric mice with humanized immune systems has markedly improved. Multiple lineages of human immune cells develop in immunodeficient mice that have been transplanted with human hematopoietic stem cells. More importantly, these mice mount functional humoral and cellular immune responses upon immunization or microbial infection. Human immunodeficiency virus type I (HIV-1) can establish an infection in humanized mice, resulting in CD4+ T-cell depletion and an accompanying nonspecific immune activation, which mimics the immunopathology in HIV-1-infected human patients. This makes humanized mice an optimal model for studying the mechanisms of HIV-1 immunopathogenesis and for developing novel immune-based therapies. PMID:22504952

  9. Population genomics of intrapatient HIV-1 evolution.

    PubMed

    Zanini, Fabio; Brodin, Johanna; Thebo, Lina; Lanz, Christa; Bratt, Göran; Albert, Jan; Neher, Richard A

    2015-01-01

    Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100 bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. PMID:26652000

  10. 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.

  11. MAS NMR of HIV-1 protein assemblies.

    PubMed

    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. PMID:25797001

  12. Novel vaccine vectors for HIV-1

    PubMed Central

    Picker, Louis J.

    2014-01-01

    The ultimate solution to the global HIV-1 epidemic will probably require the development of a safe and effective vaccine. Multiple vaccine platforms have been evaluated in both preclinical and clinical trials, but, given the disappointing results of the clinical efficacy studies so far, novel vaccine approaches are needed. In this Opinion article, we discuss the scientific basis and clinical potential of novel adenovirus and cytomegalovirus vaccine vectors for HIV-1 as two contrasting, but potentially complementary, vector approaches. Both of these vector platforms have demonstrated partial protection against stringent simian immunodeficiency virus challenges in rhesus monkeys using different immunological mechanisms. PMID:25296195

  13. Laparoscopic sterilization in HIV-1-positive women.

    PubMed

    Intaraprasert, S; Taneepanichskul, S; Chaturachinda, K

    1996-11-01

    Laparoscopic sterilizations in HIV-1-positive women were performed. Patients, who were HIV-1-positive, underwent voluntary laparoscopic sterilization. The mean age of patients was 27.5 +/- 3.8 years. Most were of low socioeconomic status. The mean duration of the operation was 14.4 +/- 5.4 min. No accidental injury to the surgical team was recorded, and no complications occurred among the patients. It was concluded that laparoscopic sterilization in HIV-positive patients was safe with low risk of HIV transmission to the surgical team. PMID:8934065

  14. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  15. Induction of Strong HIV-1-specific CD4+ T Cell Responses using an HIV-1 gp120/NefTat Vaccine adjuvanted with AS02A in ARV Treated HIV-1-Infected Individuals

    PubMed Central

    Lichterfeld, Mathias; Gandhi, Rajesh T.; Simmons, Rachel P.; Flynn, Teresa; Sbrolla, Amy; Yu, Xu G.; Basgoz, Nesli; Mui, Stanley; Williams, Katie; Streeck, Hendrik; Burgett-Yandow, Nicole; Roy, Gilbert; Janssens, Michel; Pedneault, Louise; Vandepapelière, Pierre; Koutsoukos, Marguerite; Demoitié, Marie-Ange; Bourguignon, Patricia; McNally, Lisa; Voss, Gerald; Altfeld, Marcus

    2011-01-01

    Background Induction of HIV-1-specific CD4+ T cell responses by therapeutic vaccination represents an attractive intervention to potentially increase immune control of HIV-1. Methods We performed a double-blinded, randomized, placebo-controlled clinical trial to determine the safety and immunogenicity of GSK Biologicals' HIV-1 gp120/NefTat subunit protein vaccine formulated with the AS02A adjuvant in subjects with well controlled chronic HIV-1 infection on HAART. Ten individuals received the vaccine; while adjuvant alone or placebo was given to five subjects each. Immunogenicity was monitored by intracellular cytokine flow cytometry and CFSE-based proliferation assays. Results The vaccine was well tolerated with no related SAEs. Vaccine recipients had significantly stronger gp120-specific CD4+ T cell responses which persisted until week 48 and greater gp120-specific CD4+ T cell proliferation activity as compared to controls. In the vaccine group, the number of participants that demonstrated positive responses for both gp120-specific CD4+ T cell IL-2 production and gp120-specific CD8+ T cell proliferation was significantly higher at week 6. Conclusions The gp120/NefTat/AS02A vaccine induced strong gp120-specific CD4+ T cell responses, and a higher number of vaccinees developed both HIV-1-specific CD4+ T cell responses and CD8+ T cell proliferation. The induction of these responses may be important in enhancing immune-mediated viral control. PMID:21963936

  16. Altered Dendritic Cell Phenotype in Response to Leishmania amazonensis Amastigote Infection Is Mediated by MAP Kinase, ERK

    PubMed Central

    Boggiatto, Paola Mercedes; Jie, Fei; Ghosh, Mousumi; Gibson-Corley, Katherine Nicole; Ramer-Tait, Amanda Ellen; Jones, Douglas Elliot; Petersen, Christine Anne

    2009-01-01

    Initiation of productive immune responses against Leishmania depends on the successful transition of dendritic cells (DC) from an immature to a mature phenotype. This process is characterized by high CD40 surface expression as well as interleukin-12 production, which are frequently seen in response to L. major infection. In vivo footpad infection of C3HeB/FeJ mice for 7 days with L. amazonensis promoted an immature CD11c+ DC phenotype characterized by both significantly low CD40 surface expression and significantly decreased interleukin-12p40 production compared with L. major infection of these same mice. In vitro infection of bone marrow-derived dendritic cells with L. amazonensis amastigotes resulted in rapid and significant phosphorylation of the mitogen activated protein kinase, extracellular signal-regulated kinase 1/2, observed within minutes of exposure to the parasite. Infection with L. amazonensis promastigotes led to increased 1/2 phosphorylation after 4 hours of infection compared with L. major infection, which correlated with promastigote transformation into amastigotes. Treatment of bone marrow-derived dendritic cells with a mitogen activated protein kinase kinase-specific inhibitor, PD98059, led to regained surface CD40 expression and interleukin-12p40 production following L. amazonensis amastigote infection compared with non-treated, infected DC. Treatment of L. amazonensis-infected mice with the highly-specific mitogen activated protein kinase kinase inhibitor, CI-1040, enhanced surface CD40 expression on CD11c+ DC obtained from the draining lymph node. L. amazonensis amastigotes, through activation of extracellular signal-regulated kinase 1/2, inhibit the ability of DC to undergo proper maturation both in vitro and in vivo. PMID:19349356

  17. Interaction of the phospholipid scramblase 1 with HIV-1 Tat results in the repression of Tat-dependent transcription

    SciTech Connect

    Kusano, Shuichi Eizuru, Yoshito

    2013-04-19

    Highlights: •PLSCR1 specifically interacted with HIV-1 Tat in vitro and in vivo. •PLSCR1 repressed Tat-dependent transactivation of the HIV-1 LTR. •Suppression of PLSCR1 expression enhanced the levels of HIV-1 transcripts. •PLSCR1 reduced the nuclear localization of Tat. -- Abstract: Human phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)-stimulated gene and possesses an IFN-mediated antiviral function. We show here that PLSCR1 directly interacts with human immunodeficiency virus type-1 (HIV-1) Tat. This interaction occurs both in vitro and in vivo through amino acids 160–250 of PLSCR1. Overexpression of PLSCR1 efficiently represses the Tat-dependent transactivation of the HIV-1 long terminal repeat (LTR) and reduces the nuclear translocation of Tat. In addition, shRNA-mediated suppression of endogenous PLSCR1 expression enhances the levels of gag mRNA in an HIV-1-infected T-cell line. These findings indicate that PLSCR1 negatively regulates the Tat-dependent transactivation of the HIV-1 LTR during HIV-1 infection.

  18. Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte-derived dendritic cells.

    PubMed

    Argueta-Donohué, Jesús; Wilkins-Rodríguez, Arturo A; Aguirre-García, Magdalena; Gutiérrez-Kobeh, Laila

    2016-06-01

    Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C-type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte-derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC-SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC-SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes. PMID:26399218

  19. The HPA axis in HIV-1 infection.

    PubMed

    Kumar, Mahendra; Kumar, Adarsh M; Waldrop, Drenna; Antoni, Michael H; Schneiderman, Neil; Eisdorfer, Carl

    2002-10-01

    Several lines of evidence suggest that neuroendocrine abnormalities in general and HPA axis activity in particular occur in both HIV-1 infection and individuals engaging in chronic drug use. For instance, our studies showing attenuated norepinephrine as well as ACTH and cortisol responses to a cold pressor challenge in asymptomatic HIV-1 persons support such a concept. Furthermore, our data on investigations on mirror-star tracing and speech challenges also support the finding that neuroendocrine responses are compromised in HIV-1 infection. Although the mechanisms leading to adverse effects on HPA axis activity in HIV infection are not fully understood, several lines of evidence suggest that a number of mechanisms may be involved, including homologies in molecular structures of various mediators of neuroendocrine activity and HIV-related structures, HIV as a chronic stress model, and virus-induced toxic factors. This article reviews our recent findings in this area and also presents research hypotheses needed for testing and understanding the mechanisms involved in the development of neuroendocrine abnormalities in HIV-1-infected injection drug users. PMID:12394788

  20. HIV-1 remodels the nuclear pore complex

    PubMed Central

    Monette, Anne; Panté, Nelly

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) commandeers host cell proteins and machineries for its replication. Our earlier work showed that HIV-1 induced the cytoplasmic retention of nucleocytoplasmic shuttling and ribonucleic acid (RNA)–binding proteins. This retention is dependent on nuclear export of the viral genomic RNA and on changes in the localization and expression level of the nucleoporin (Nup) p62 (Nup62). To further characterize the extent of perturbation induced by HIV-1, we performed proteomics analyses of nuclear envelopes (NEs) isolated from infected T cells. Infection induced extensive changes in the composition of the NE and its associated proteins, including a remarkable decrease in the abundance of Nups. Immunogold electron microscopy revealed the translocation of Nups into the cytoplasm. Nup62 was identified as a component of purified virus, and small interfering RNA depletion studies revealed an important role for this Nup in virus gene expression and infectivity. This detailed analysis highlights the profound effects on NE composition induced by HIV-1 infection, providing further evidence of the magnitude of viral control over the cell biology of its host. PMID:21576391

  1. HIV-1 integrase: from biology to chemotherapeutics.

    PubMed

    Zeinalipour-Loizidou, Eriketi; Nicolaou, Christos; Nicolaides, Athanasios; Kostrikis, Leondios G

    2007-07-01

    AIDS has claimed the lives of 25 million people worldwide, an additional 40 million people are HIV-infected and new cases are being diagnosed every year. Despite the fact that HAART has moved AIDS from the category of terminal diseases to that of treatable chronic illnesses, its long-term therapeutic success may be compromised by the development of resistance to the currently used drugs. Despite the availability of RT, PR and fusion inhibitors, the development of further drugs such as inhibitors that target the third enzyme IN is essential for the clinical management of HIV-infected patients. The absence of cellular homolgues to IN and the unique nature of the reactions catalyzed by IN, make it an ideal target for drug design. Considerable progress towards designing HIV-1 IN inhibitors has been made over the last years and several lead compounds have been identified, synthesized and clinically studied. This review focuses on the existing knowledge of the biology of HIV-1 IN with emphasis on the mechanism of integration, structure and function and the technologies for measuring IN activity. This is followed by the current trends on designing HIV-1 IN inhibitors with the aid of molecular informatics and a review on the main classes of HIV-1 IN inhibitors reported this far with special emphasis on the clinical candidates. PMID:17627500

  2. HIV-1 transcription and latency: an update

    PubMed Central

    2013-01-01

    Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs. PMID:23803414

  3. Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

    PubMed

    Shrivastava, Surya; Trivedi, Jay; Mitra, Debashis

    2016-03-25

    Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis. PMID:26915805

  4. The Dual Role of Dendritic cells in the Immune Response to HIV-1 Infection

    PubMed Central

    Hogue, Ian B.; Bajaria, Seema H.; Fallert, Beth A.; Qin, Shulin; Reinhart, Todd A.; Kirschner, Denise E.

    2009-01-01

    Many aspects of the complex interaction between HIV-1 and the human immune system remain elusive. Our objective is to study these interactions, focusing on the specific roles of dendritic cells (DCs). DCs enhance HIV-1 infection processes as well as promote an anti-viral immune response. We explore the implications of these dual roles. We present and analyse a mathematical model describing the dynamics of HIV-1, CD4+ and CD8+ T-cells, and DCs interacting in a human lymph node. We validate the behaviour of our model against non-human primate SIV experimental data and published human HIV-1 data. Our model qualitatively and quantitatively recapitulates clinical HIV-1 infection dynamics. We perform sensitivity analyses on the model to determine which mechanisms strongly affect infection dynamics. Sensitivity analysis identifies system interactions that contribute to infection progression, including DC-related mechanisms. We compare DC-dependent and DC-independent routes of CD4+ T-cell infection. The model predicts that simultaneous priming and infection of T cells by DCs drives early infection dynamics when activated T-helper cell numbers are low. Further, our model predicts that, while direct failure of DC function and an indirect failure due to loss of CD4+ T-cell help are both significant contributors to infection dynamics, our results support the hypothesis that the former has a more significant impact on HIV-1 immunopathogenesis. PMID:18753232

  5. Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors

    PubMed Central

    Perdigão, Pedro; Gaj, Thomas; Santa-Marta, Mariana; Goncalves, Joao

    2016-01-01

    The presence of replication-competent HIV-1 –which resides mainly in resting CD4+ T cells–is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection. PMID:26933881

  6. Evaluation of Health Disparity in Bacterial Vaginosis and the Implications for HIV-1 Acquisition in African American Women.

    PubMed

    Alcendor, Donald J

    2016-08-01

    There is a health disparity for both bacterial vaginosis (BV) and human immunodeficiency virus type 1 (HIV-1) infection in African American women that may be linked. The evidence that BV predisposes women to higher risk for HIV infection is well documented. The underlying mechanisms to support the epidemiological connections will require further investigations. This review explores the risk factors for BV disease with implications for HIV-1 acquisition in the context of race as a potential driver of the 20-fold increase in HIV-1 acquisition for African American women compared to white women. Specifically, it explores (i) disparities for BV in African American women, (ii) racial disparity for HIV-1 acquisition in African American women, (iii) common factors associated with BV and HIV acquisition in African American women, and (iv) potential mechanisms of the enhancement of HIV-1 transmission by BV. PMID:26847837

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

    PubMed Central

    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.

    2014-01-01

    Background 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. Methods We conducted a nested case-control analysis using data from two prospective cohort studies of heterosexual HIV-1 serodiscordant couples from 6 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 non-transmitting 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 infected 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. Results HIV-1 subtype C was not associated with increased risk of HIV-1 transmission compared to 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. Conclusion 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 to non-C subtypes, arguing against the hypothesis that subtype C is more transmissible compared to other common subtypes. PMID:24413311

  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. PMID:27245560

  9. Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies

    PubMed Central

    Klein, Florian; Horwitz, Joshua A.; Halper-Stromberg, Ariel; Sather, D. Noah; Marcovecchio, Paola M.; Lee, Terri; West, Anthony P.; Gao, Han; Seaman, Michael S.; Stamatatos, Leonidas; Nussenzweig, Michel C.; Bjorkman, Pamela J.

    2013-01-01

    Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46G54W variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46G54W–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination. PMID:23712429

  10. THE MAJOR SURFACE PROTEASE (MSP OR GP63) IN THE INTRACELLULAR AMASTIGOTE STAGE OF LEISHMANIA CHAGASI

    PubMed Central

    Christine Hsiao, Chia-Hung; Yao, Chaoqun; Storlie, Patricia; Donelson, John E; Wilson, Mary E

    2009-01-01

    The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes. Promastigotes express MSPS and MSPL RNAs, and two forms of MSPC RNA, whereas amastigotes express only MSPL RNA and one MSPC transcript. We confirmed the presence of MSPC protein in both promastigotes and amastigotes by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). More than 10 MSP isoforms were visualized in both amastigotes and promastigotes using two-dimensional immunoblots, but amastigote MSPs migrated at a more acidic pI. Promastigote MSPs were N-glycosylated, whereas most amastigote MSPs were not. Immuno-electron microscopy showed that two-thirds of the promastigote MSP is distributed along the cell surface. In contrast, most amastigote MSP localized at the flagellar pocket, the major site of leishmania endocytosis/exocytosis. Biochemical analyses indicated that most amastigote MSP is soluble in the cytosol, vesicles or organelles, whereas most promastigote MSP is membrane-associated and GPI anchored. Activity gels and immunoblots confirmed the presence of a novel proteolytically active amastigote MSP of higher Mr than the promastigote MSPs. Furthermore, promastigote MSP is shed extracellularly whereas MSP is not shed from axenic amastigotes. We conclude that amastigote and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently between the parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp. PMID:18067978

  11. Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors

    PubMed Central

    Gupta, Sachin; Termini, James M.; Issac, Biju; Guirado, Elizabeth; Stone, Geoffrey W.

    2016-01-01

    Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG), including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1). Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5) expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration. PMID:26849062

  12. Interactions between neutrophils and Leishmania braziliensis amastigotes facilitate cell activation and parasite clearance

    PubMed Central

    Carlsen, Eric D.; Jie, Zuliang; Liang, Yuejin; Henard, Calvin A.; Hay, Christie; Sun, Jiaren; de Matos Guedes, Herbert; Soong, Lynn

    2015-01-01

    Leishmania braziliensis and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in South America. However, patient prognosis and the host immune response differ considerably depending on the infecting parasite species. The mechanisms underlying these differences appear to be multifactorial, with both host and parasite components contributing to disease outcome. Because neutrophils are a prominent component of the inflammatory infiltrate in chronic cutaneous, diffuse cutaneous, and mucocutaneous lesions, we examined neutrophil activation and microbicidal activity against amastigotes of L. amazonensis and L. braziliensis. We found that murine neutrophils internalized L. braziliensis amastigotes with greater efficiency than did L. amazonensis amastigotes. Additionally, L. braziliensis infection was a potent trigger for neutrophil activation, oxidative burst, degranulation, and production of IL-22 and IL-10, while L. amazonensis amastigotes poorly induced these responses. Finally, neutrophils were able to kill L. braziliensis amastigotes, especially when cells were activated with phorbol myristate acetate (PMA). L. amazonensis amastigotes, however, were highly resistant to neutrophil microbicidal mechanisms. This study reveals, for the first time, differential neutrophil responsiveness to distinct species of Leishmania amastigotes and highlights the complexity of neutrophil-amastigote interactions during chronic leishmaniasis. PMID:25766649

  13. Multiscale Computer Simulation of the Immature HIV-1 Virion

    PubMed Central

    Ayton, Gary S.; Voth, Gregory A.

    2010-01-01

    Multiscale computer simulations, employing a combination of experimental data and coarse-graining methods, are used to explore the structure of the immature HIV-1 virion. A coarse-grained (CG) representation is developed for the virion membrane shell and Gag polypeptides using molecular level information. Building on the results from electron cryotomography experiments, the simulations under certain conditions reveal the existence of an incomplete p6 hexameric lattice formed from hexameric bundles of the Gag CA domains. In particular, the formation and stability of the immature Gag lattice at the CG level requires enhanced interfacial interactions of the CA protein C-terminal domains (CTDs). An exact mapping of the CG representation back to the molecular level then allows for detailed atomistic molecular dynamics studies to confirm the existence of these enhanced CACTD interactions and to probe their possible origin. The multiscale simulations further provide insight into potential CACTD mutations that may disrupt or modify the Gag immature lattice assembly process in the immature HIV-1 virion. PMID:21044572

  14. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    SciTech Connect

    Di Nunzio, Francesca; Fricke, Thomas; Miccio, Annarita; Valle-Casuso, Jose Carlos; Perez, Patricio; Souque, Philippe; Rizzi, Ermanno; Severgnini, Marco; Mavilio, Fulvio; Charneau, Pierre; Diaz-Griffero, Felipe

    2013-05-25

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.

  15. Proline transport in Leishmania donovani amastigotes: dependence on pH gradients and membrane potential.

    PubMed

    Glaser, T A; Mukkada, A J

    1992-03-01

    Amastigotes of Leishmania donovani develop and multiply within the acidic phagolysosomes of mammalian macrophages. Isolated amastigotes are acidophilic; they catabolize substrates and synthesize macromolecules optimally at pH 5.5. Substrate transport in amastigotes has not been characterized. Here we show that amastigotes exhibit an uphill transport of proline (active transport) with an acid pH optimum (pH 5.5). It is dependent upon metabolic energy and is driven by proton motive force. Agents which selectively disturb the component forces of proton motive force, such as carbonyl cyanide chlorophenylhydrazone, nigericin and valinomycin, inhibit proline transport. Transport is sensitive to dicyclohexylcarbodiimide and insensitive to ouabain, demonstrating the involvement of a proton ATPase in the maintenance of proton motive force. It is suggested that the plasma membrane pH gradient probably makes the greatest contribution to proton motive force that drives substrate transport in the amastigote stage. PMID:1533014

  16. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  17. Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo

    PubMed Central

    2014-01-01

    Background Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively. Results CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition. Conclusions In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in

  18. Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

    PubMed Central

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-01-01

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6,564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research. PMID:25445329

  19. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  20. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGESBeta

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  1. Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation

    PubMed Central

    Duverger, Alexandra; Wolschendorf, Frank; Anderson, Joshua C.; Wagner, Frederic; Bosque, Alberto; Shishido, Takao; Jones, Jennifer; Planelles, Vicente; Willey, Christopher; Cron, Randall Q.

    2014-01-01

    Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation. PMID:24155393

  2. Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

    PubMed

    Duverger, Alexandra; Wolschendorf, Frank; Anderson, Joshua C; Wagner, Frederic; Bosque, Alberto; Shishido, Takao; Jones, Jennifer; Planelles, Vicente; Willey, Christopher; Cron, Randall Q; Kutsch, Olaf

    2014-01-01

    Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation. PMID:24155393

  3. Reprogramming Neutral Lipid Metabolism in Mouse Dendritic Leucocytes Hosting Live Leishmania amazonensis Amastigotes

    PubMed Central

    Lecoeur, Hervé; Giraud, Emilie; Prévost, Marie-Christine; Milon, Geneviève; Lang, Thierry

    2013-01-01

    Background After loading with live Leishmania (L) amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. Methodology/Principal Findings Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i) long-chain fatty acids (LCFA) and cholesterol uptake/transport, (ii) LCFA and cholesterol (re)-esterification to triacyl-sn-glycerol (TAG) and cholesteryl esters (CE), respectively. As these neutral lipids are known to make up the lipid body (LB) core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. Conclusions/Significance As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin? PMID:23785538

  4. High sensitivity detection of HIV-1 using two genomic targets compared with single target PCR.

    PubMed

    Shah, Krishna; Ragupathy, Viswanath; Saga, Anusha; Hewlett, Indira

    2016-06-01

    The genetic diversity of Human Immunodeficiency Virus type-1(HIV-1) has been shown to affect the performance of Nucleic Acid Testing (NAT) of Human Immunodeficiency Virus type-1. Although, majority NAT assays were designed to detect the conserved regions of HIV-1 mutations at the primer or probe binding regions may lead to false negatives. In this study, we evaluated the feasibility of detecting two genomic targets for enhanced sensitivity. A total of 180 tests using HIV-1 VQA RNA quantitation standard, 240 tests using EQAPOL HIV-1 viral diversity subtype panel, and 30 clinical plasma samples from Cameroon were evaluated. The analysis was based on probit and hit rate. The genomic targets LOD estimated by PROBIT for the gag target was 118 cps/ml (95%CI 64 cps/ml lower bound), Pol or POL/LTR was at 40 cps/ml (95%CI 17, 16 cps/ml), LTR 45 cps/ml (95%CI 20 cps/ml lower bound), and Gag/LTR at 67.8 cps/ml (95%CI 32 cps/ml lower bound). For HIV-1 subtypes the overall reactivity was 55-100% when tested at 100 and 1000 cps/ml and combination of genomic targets detection increased the reactivity to 100%. The plasma samples evaluation showed LTR or pol/LTR combination yielded higher sensitivity for patients with lower viral load (<40 cps/ml). We conclude that detection of two HIV-1 genomic targets improved sensitivity for detection of genetically diverse HIV-1 strains. J. Med. Virol. 88:1092-1097, 2016. © 2015 Wiley Periodicals, Inc. PMID:26575693

  5. Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood.

    PubMed

    Chargin, Amanda; Yin, Fangfang; Song, Min; Subramaniam, Srividyabhuvaneswari; Knutson, Grace; Patterson, Bruce K

    2015-01-01

    Plasma viral load and CD4 counts are effective for clinical monitoring, but they do not give a full representation of HIV-1 quasispecies in cellular reservoirs, the major repository of replication-competent HIV-1 in infected individuals. We sought to develop a diagnostic system that might stimulate the replication-competent HIV-1 reservoirs for enhanced clinical monitoring, including selection of antiretroviral regimens. Whole-blood samples from 45 HIV-infected individuals were collected into 1 ViraStim HIV-1 activation tube and 1 EDTA tube. Samples were tested for viral load and cell type-specific HIV-1 replication. Further, 7 matched activated/nonactivated samples were sequenced using the Trugene HIV-1 genotyping kit. The percentage of patients with replication-competent virus in peripheral blood mononuclear cells (PBMCs) varied, depending on the baseline plasma viral load in the EDTA tubes. Six out of 24 patients with a starting plasma viral load of <20 copies/ml (cp/ml), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml, and 8 out of 13 patients with starting viral loads of >1,000 all showed increases in viral replication of >5-fold. These increases came from cellular reservoirs in blood as determined by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). When resistance genotypes in plasma from activation tubes were compared to those from EDTA tubes for 7 patients, all patients showed additional mutations in the activation tube, while 3 patients demonstrated additional genotypic resistance determinants. We show that HIV-1 viral replication can be stimulated directly from infected whole blood. The sequencing results showed that 3 of 7 cases demonstrated additional drug resistance following stimulation. PMID:25339403

  6. Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

    PubMed Central

    Michienzi, Alessandro; Cagnon, Laurence; Bahner, Ingrid; Rossi, John J.

    2000-01-01

    The HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4+ and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing. PMID:10922055

  7. Increased Risk of HIV-1 Transmission in Pregnancy: A Prospective Study among African HIV-1 Serodiscordant Couples

    PubMed Central

    MUGO, Nelly R.; HEFFRON, Renee; DONNELL, Deborah; WALD, Anna; WERE, Edwin O.; REES, Helen; CELUM, Connie; KIARIE, James N.; COHEN, Craig R.; KAYINTEKORE, Kayitesi; BAETEN, Jared M.

    2011-01-01

    Background Physiologic and behavioral changes during pregnancy may alter HIV-1 susceptibility and infectiousness. Prospective studies exploring pregnancy and HIV-1 acquisition risk in women have found inconsistent results. No study has explored the effect of pregnancy on HIV-1 transmission risk from HIV-1 infected women to male partners. Methods In a prospective study of African HIV-1 serodiscordant couples, we evaluated the relationship between pregnancy and the risk of 1) HIV-1 acquisition among women and 2) HIV-1 transmission from women to men. Results 3321 HIV-1 serodiscordant couples were enrolled, 1085 (32.7%) with HIV-1 susceptible female partners and 2236 (67.3%) with susceptible male partners. HIV-1 incidence in women was 7.35 versus 3.01 per 100 person-years during pregnant and non-pregnant periods (hazard ratio [HR] 2.34, 95% confidence interval [CI] 1.33–4.09). This effect was attenuated and not statistically significant after adjusting for sexual behavior and other confounding factors (adjusted HR 1.71, 95% CI 0.93–3.12). HIV-1 incidence in male partners of infected women was 3.46 versus 1.58 per 100 person-years when their partners were pregnant versus not pregnant (HR 2.31, 95% CI 1.22–4.39). This effect was not attenuated in adjusted analysis (adjusted HR 2.47, 95% CI 1.26–4.85). Conclusions HIV-1 risk increased two-fold during pregnancy. Elevated risk of HIV-1 acquisition in pregnant women appeared in part to be explained by behavioral and other factors. This is the first study to show pregnancy increased the risk of female-to-male HIV-1 transmission, which may reflect biological changes of pregnancy that could increase HIV-1 infectiousness. PMID:21785321

  8. SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells

    PubMed Central

    Bruel, Timothée; Cardinaud, Sylvain; Porrot, Françoise; Prado, Julia G.; Moris, Arnaud

    2015-01-01

    ABSTRACT Monocyte-derived dendritic cells (MDDC) stimulate CD8+ cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. IMPORTANCE Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early

  9. Productive Replication and Evolution of HIV-1 in Ferret Cells

    PubMed Central

    Fadel, Hind J.; Saenz, Dyana T.; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary

    2012-01-01

    A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1

  10. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  11. An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

    PubMed Central

    Bonsignori, Mattia; Wiehe, Kevin; Grimm, Sebastian K.; Lynch, Rebecca; Yang, Guang; Kozink, Daniel M.; Perrin, Florence; Cooper, Abby J.; Hwang, Kwan-Ki; Chen, Xi; Liu, Mengfei; McKee, Krisha; Parks, Robert J.; Eudailey, Joshua; Wang, Minyue; Clowse, Megan; Criscione-Schreiber, Lisa G.; Moody, M. Anthony; Ackerman, Margaret E.; Boyd, Scott D.; Gao, Feng; Kelsoe, Garnett; Verkoczy, Laurent; Tomaras, Georgia D.; Liao, Hua-Xin; Kepler, Thomas B.; Montefiori, David C.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells. PMID:24614107

  12. Immunodominant HIV-1 Cd4+ T Cell Epitopes in Chronic Untreated Clade C HIV-1 Infection

    PubMed Central

    Ramduth, Danni; Day, Cheryl L.; Thobakgale, Christina F.; Mkhwanazi, Nompumelelo P.; de Pierres, Chantal; Reddy, Sharon; van der Stok, Mary; Mncube, Zenele; Nair, Kriebashne; Moodley, Eshia S.; Kaufmann, Daniel E.; Streeck, Hendrik; Coovadia, Hoosen M.; Kiepiela, Photini; Goulder, Philip J. R.; Walker, Bruce D.

    2009-01-01

    Background A dominance of Gag-specific CD8+ T cell responses is significantly associated with a lower viral load in individuals with chronic, untreated clade C human immunodeficiency virus type 1 (HIV-1) infection. This association has not been investigated in terms of Gag-specific CD4+ T cell responses, nor have clade C HIV-1–specific CD4+ T cell epitopes, likely a vital component of an effective global HIV-1 vaccine, been identified. Methodology/Principal Findings Intracellular cytokine staining was conducted on 373 subjects with chronic, untreated clade C infection to assess interferon-gamma (IFN-γ) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-γ–producing CD4+ T cell responses were detected in 261/373 (70%) subjects, with the Gag responders having a significantly lower viral load and higher CD4 count than those with no detectable Gag response (p<0.0001 for both parameters). To identify individual peptides targeted by HIV-1–specific CD4+ T cells, separate ELISPOT screening was conducted on CD8-depleted PBMCs from 32 chronically infected untreated subjects, using pools of overlapping peptides that spanned the entire HIV-1 clade C consensus sequence, and reconfirmed by flow cytometry to be CD4+ mediated. The ELISPOT screening identified 33 CD4+ peptides targeted by 18/32 patients (56%), with 27 of the 33 peptides located in the Gag region. Although the breadth of the CD4+ responses correlated inversely with viral load (p = 0.015), the magnitude of the response was not significantly associated with viral load. Conclusions/Significance These data indicate that in chronic untreated clade C HIV-1 infection, IFN-γ–secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control. PMID:19352428

  13. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells

    PubMed Central

    2012-01-01

    Background Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. Results Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. Conclusion Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks. PMID:23092122

  14. Inhibition of PP2A by LIS1 increases HIV-1 gene expression

    PubMed Central

    Epie, Nicolas; Ammosova, Tatyana; Turner, Willie; Nekhai, Sergei

    2006-01-01

    Background Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. Results Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. Conclusion Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A. PMID:17018134

  15. Active replication of HIV-1 at the lymphoepithelial surface of the tonsil.

    PubMed Central

    Frankel, S. S.; Tenner-Racz, K.; Racz, P.; Wenig, B. M.; Hansen, C. H.; Heffner, D.; Nelson, A. M.; Pope, M.; Steinman, R. M.

    1997-01-01

    Cells that are infected with HIV-1 were visualized at the mucosal surface of the nasopharyngeal and palatine tonsils in 14 specimens from patients with CD4+ T-cell counts of 200 to 900/microliter and 2- to 10-year histories of HIV-1 infection. Most of the cells with intracellular HIV-1 protein were small but multinucleated. The majority of these syncytia could be double labeled for HIV-1 RNA and a dendritic cell marker S100. In the palatine tonsil, the infected cells were not found in the stratified squamous epithelium that is adjacent to the pharynx. Instead, the S100+ infected syncytia were localized to the surface of tonsil invaginations or crypts. This mucosa, termed lymphoepithelium, contains antigen-transporting M cells that lie above regions where S100+ dendritic cells are juxtaposed with CD4+ lymphocytes. Likewise, infected cells were found in lymphoepithelium and not respiratory epithelium of nasopharyngeal tonsils or adenoids. We propose that lymphoepithelia, the histological term that describes the specialized regions where antigens access mucosa-associated lymphoid tissue, are sites where HIV-1 replication can be enhanced in syncytia derived from dendritic cells. Images Figure 1 Figure 2 Figure 3 PMID:9212735

  16. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1{sub IIIB} infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  17. The Nucleoside Analog D-carba T Blocks HIV-1 Reverse Transcription

    PubMed Central

    Boyer, Paul L.; Vu, B. Christie; Ambrose, Zandrea; Julias, John G.; Warnecke, Svenja; Liao, Chenzhong; Meier, Chris; Marquez, Victor E.; Hughes, Stephen H.

    2009-01-01

    A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3′ hydroxyl on the pseudosugar. The 3′ hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections. PMID:19678643

  18. A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter.

    PubMed

    Perrone, Rosalba; Nadai, Matteo; Frasson, Ilaria; Poe, Jerrod A; Butovskaya, Elena; Smithgall, Thomas E; Palumbo, Manlio; Palù, Giorgio; Richter, Sara N

    2013-08-22

    G-Quadruplexes, noncanonical nucleic acid structures, act as silencers in the promoter regions of human genes; putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes. Here we show that also the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity. Computational analysis on 953 HIV-1 strains substantiated a highly conserved G-rich sequence corresponding to Sp1 and NF-κB binding sites. Biophysical/biochemical analysis proved that two mutually exclusive parallel-like intramolecular G-quadruplexes, stabilized by small molecule ligands, primarily fold in this region. Mutations disrupting G-quadruplex formation enhanced HIV promoter activity in cells, whereas treatment with a G-quadruplex ligand impaired promoter activity and displayed antiviral effects. These findings disclose the possibility of inhibiting the HIV-1 LTR promoter by G-quadruplex-interacting small molecules, providing a new pathway to development of anti-HIV-1 drugs with unprecedented mechanism of action. PMID:23865750

  19. Galectin-1 promotes HIV-1 infectivity in macrophages through stabilization of viral adsorption

    SciTech Connect

    Mercier, Simon; St-Pierre, Christian; Pelletier, Isabelle; Ouellet, Michel; Tremblay, Michel J. Sato, Sachiko

    2008-02-05

    Following primary infection with human immunodeficiency virus type-1 (HIV-1), macrophages are thought to play an important role, as they are one of the first target cells the virus encounters and can also sustain a significant production of viruses over extended periods of time. While the interaction between the primary cellular receptor CD4 and the virus-encoded external envelope glycoprotein gp120 initiates the infection process, it has been suggested that various host factors are exploited by HIV-1 to facilitate adsorption onto the cell surface. Macrophages and other cells found at the infection site can secrete a soluble mammalian lectin, galectin-1, which binds to {beta}-galactoside residues through its carbohydrate recognition domain. Being a dimer, galectin-1 can cross-link ligands expressed on different constituents to mediate adhesion between cells or between cells and pathogens. We report here that galectin-1, but not galectin-3, increased HIV-1 infectivity in monocyte-derived macrophages (MDMs). This phenomenon was likely due to an enhancement of virus adsorption kinetics, which facilitates HIV-1 entry. The fusion inhibitors T-20 and TAK779 remained effective at reducing infection even in the presence of galectin-1, indicating that the galectin-1-mediated effect is occurring at a step prior to fusion. Together, our data suggest that galectin-1 can facilitate HIV-1 infection in MDMs by promoting early events of the virus replicative cycle (i.e. adsorption)

  20. Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

    PubMed

    Chen, Adrienne; Boulton, Ian C; Pongoski, Jodi; Cochrane, Alan; Gray-Owen, Scott D

    2003-03-01

    Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression. PMID:12598784

  1. Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase.

    PubMed

    Lee, Won-Gil; Frey, Kathleen M; Gallardo-Macias, Ricardo; Spasov, Krasimir A; Chan, Albert H; Anderson, Karen S; Jorgensen, William L

    2015-11-01

    Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data. PMID:26166629

  2. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.

    PubMed

    Li, C J; Friedman, D J; Wang, C; Metelev, V; Pardee, A B

    1995-04-21

    Infection by human immunodeficiency virus-type 1 (HIV-1) is typified by the progressive depletion of CD4 T lymphocytes and deterioration of immune function in most patients. A central unresolved issue in acquired immunodeficiency syndrome (AIDS) pathogenesis is the mechanism underlying this T cell depletion. HIV-1 Tat protein was shown to induce cell death by apoptosis in a T cell line and in cultured peripheral blood mononuclear cells from uninfected donors. This Tat-induced apoptosis was inhibitable by growth factors and was associated with enhanced activation of cyclin-dependent kinases. PMID:7716549

  3. Immune-Based Approaches to the Prevention of Mother-to-child-Transmission of HIV-1: Active and Passive Immunization

    PubMed Central

    Lohman-Payne, Barb; Slyker, Jennifer; Rowland-Jones, Sarah L.

    2010-01-01

    Synopsis Despite more than two decades of research, an effective vaccine that can prevent HIV-1 infection in populations exposed to the virus remains elusive. In the pursuit of an HIV-1 vaccine, does prevention of exposure to maternal HIV-1 in utero, at birth or in early life through breast-milk require special consideration? In this article we will review what is known about the immune mechanisms of susceptibility and resistance to mother-to-child transmission (MTCT) of HIV-1 and will summarise studies that have used passive or active immunisation strategies to interrupt -MTCT of HIV-1. We will also describe potentially modifiable infectious co-factors that may enhance transmission and/or disease progression (especially in the developing world). Ultimately an effective prophylactic vaccine against HIV-1 infection will need to be deployed as part of the Extended Programme of Immunisation (EPI) recommended by the World Health Organisation (WHO) for use in developing countries, so it is important to understand how the infant immune system responds to HIV-1 antigens, both in natural infection and presented by candidate vaccines. PMID:21078451

  4. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells.

    PubMed

    Lichinchi, Gianluigi; Gao, Shang; Saletore, Yogesh; Gonzalez, Gwendolyn Michelle; Bansal, Vikas; Wang, Yinsheng; Mason, Christopher E; Rana, Tariq M

    2016-01-01

    N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m(6)A in the immune system or its role in host-pathogen interactions. Here, we investigate the topology, dynamics and bidirectional influences of the viral-host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m(6)A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m(6)A for viral replication was demonstrated by silencing of the m(6)A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev response element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system. PMID:27572442

  5. Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components

    SciTech Connect

    Iida, K.; Whitlow, M.B.; Nussenzweig, V.

    1989-03-01

    We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated /sup 86/Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

  6. Suppression of HIV-1 replication by propolis and its immunoregulatory effect.

    PubMed

    Harish, Z; Rubinstein, A; Golodner, M; Elmaliah, M; Mizrachi, Y

    1997-01-01

    In the current study we show that propolis, a non-toxic natural bee-hive product, suppresses HIV-1 replication and modulates in vitro immune responses. CEM cells were treated with propolis at nontoxic concentrations prior to or following infection with HIV-1. Propolis abolished syncytium formation at 4.5 micrograms/ml and inhibited it at lower doses in a concentration-dependent manner. Propolis decreased p24 antigen production by as much as 90-100% in a concentration-dependent manner. Furthermore, modulation of peripheral blood mononuclear cells (PBMCs) mitogenic responses upon the addition of propolis was noted, reducing the elevated responses to Concanavalin A (Con A) and enhancing suppressed mitogenic responses to pokeweed mitogen (PWM). In summary, propolis may constitute a non-toxic natural product with both anti HIV-1 and immunoregulatory effects. PMID:9309384

  7. An empiric risk scoring tool for identifying high-risk heterosexual HIV-1 serodiscordant couples for targeted HIV-1 prevention

    PubMed Central

    KAHLE, Erin M.; HUGHES, James P.; LINGAPPA, Jairam R.; JOHN-STEWART, Grace; CELUM, Connie; NAKKU-JOLOBA, Edith; NJUGUNA, Stella; MUGO, Nelly; BUKUSI, Elizabeth; MANONGI, Rachel; BAETEN, Jared M.

    2012-01-01

    Background and objectives Heterosexual HIV-1 serodiscordant couples are increasingly recognized as an important source of new HIV-1 infections in sub-Saharan Africa. A simple risk assessment tool could be useful for identifying couples at highest risk for HIV-1 transmission. Methods Using data from three prospective studies of HIV-1 serodiscordant couples from seven African countries and standard methods for development of clinical prediction rules, we derived and validated a risk scoring tool developed from multivariate modeling and composed of key predictors for HIV-1 risk that could be measured in standard research and clinical settings. Results The final risk score included age of the HIV-1 uninfected partner, married and/or cohabiting partnership, number of children, unprotected sex, uncircumcised male HIV-1 uninfected partner, and plasma HIV-1 RNA in the HIV-1 infected partner. The maximum risk score was 12, scores ≥5 were associated with an annual HIV-1 incidence of >3%, and couples with a score ≥6 accounted for only 28% of the population but 67% of HIV-1 transmissions. The area under the curve for predictive ability of the score was 0.74 (95% CI 0.70–0.78). Internal and external validation showed similar predictive ability of the risk score, even when plasma viral load was excluded from the risk score. Conclusions A discrete combination of clinical and behavioral characteristics defines highest-risk HIV-1 serodiscordant couples. Discriminating highest-risk couples for HIV-1 prevention programs and clinical trials using a validated risk score could improve research efficiency and maximize the impact of prevention strategies for reducing HIV-1 transmission. PMID:23187945

  8. Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

    PubMed Central

    Jehuda-Cohen, T; Slade, B A; Powell, J D; Villinger, F; De, B; Folks, T M; McClure, H M; Sell, K W; Ahmed-Ansari, A

    1990-01-01

    Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals. Images PMID:2111024

  9. Contribution of the 80s loop of HIV-1 protease to the multidrug-resistance mechanism: crystallographic study of MDR769 HIV-1 protease variants

    SciTech Connect

    Yedidi, Ravikiran S.; Proteasa, Georghe; Martinez, Jorge L.; Vickrey, John F.; Martin, Philip D.; Wawrzak, Zdzislaw; Liu, Zhigang; Kovari, Iulia A.; Kovari, Ladislau C.

    2011-09-06

    The flexible flaps and the 80s loops (Pro79-Ile84) of HIV-1 protease are crucial in inhibitor binding. Previously, it was reported that the crystal structure of multidrug-resistant 769 (MDR769) HIV-1 protease shows a wide-open conformation of the flaps owing to conformational rigidity acquired by the accumulation of mutations. In the current study, the effect of mutations on the conformation of the 80s loop of MDR769 HIV-1 protease variants is reported. Alternate conformations of Pro81 (proline switch) with a root-mean-square deviation of 3-4.8 {angstrom} in the C{alpha} atoms of the I10V mutant and a side chain with a 'flipped-out' conformation in the A82F mutant cause distortion in the S1/S1' binding pockets that affects inhibitor binding. The A82S and A82T mutants show local changes in the electrostatics of inhibitor binding owing to the mutation from nonpolar to polar residues. In summary, the crystallographic studies of four variants of MDR769 HIV-1 protease presented in this article provide new insights towards understanding the drug-resistance mechanism as well as a basis for design of future protease inhibitors with enhanced potency.

  10. High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution.

    PubMed

    Tongo, Marcel; Dorfman, Jeffrey R; Martin, Darren P

    2016-03-01

    The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56-61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine

  11. Rational development of radiopharmaceuticals for HIV-1.

    PubMed

    Lau, Chuen-Yen; Maldarelli, Frank; Eckelman, William C; Neumann, Ronald D

    2014-04-01

    The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A "rational" development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings. PMID:24607432

  12. Novel Approaches to Inhibiting HIV-1 Replication

    PubMed Central

    Adamson, Catherine S.; Freed, Eric O.

    2009-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. PMID:19782103

  13. Novel approaches to inhibiting HIV-1 replication.

    PubMed

    Adamson, Catherine S; Freed, Eric O

    2010-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010. PMID:19782103

  14. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  15. Potentiation of the immune response in HIV-1+ individuals.

    PubMed

    Schmitz, T; Underwood, R; Khiroya, R; Bachovchin, W W; Huber, B T

    1996-03-15

    T cells from HIV-1+ individuals have a defect in mounting an antigen specific response. HIV-1 Tat has been implicated as the causative agent of this immunosuppression. We have previously shown that HIV-1 Tat inhibits antigen specific proliferation of normal T cells in vitro by binding to the accessory molecule CD26, a dipeptidase expressed on the surface of activated T cells. We now demonstrate that the defective in vitro recall antigen response in HIV-1 infected individuals can be restored by the addition of soluble CD26, probably by serving as a decoy receptor for HIV-1 Tat. The restored response is comparable to that of an HIV-1- individual, suggesting that early in HIV infection there is a block in the memory cell response, rather than deletion of these cells. PMID:8617888

  16. Purinergic Receptors: Key Mediators of HIV-1 Infection and Inflammation.

    PubMed

    Swartz, Talia H; Dubyak, George R; Chen, Benjamin K

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) causes a chronic infection that afflicts more than 30 million individuals worldwide. While the infection can be suppressed with potent antiretroviral therapies, individuals infected with HIV-1 have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV-1 pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here, we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets. PMID:26635799

  17. Purinergic Receptors: Key Mediators of HIV-1 Infection and Inflammation

    PubMed Central

    Swartz, Talia H.; Dubyak, George R.; Chen, Benjamin K.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) causes a chronic infection that afflicts more than 30 million individuals worldwide. While the infection can be suppressed with potent antiretroviral therapies, individuals infected with HIV-1 have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV-1 pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here, we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets. PMID:26635799

  18. HIV-1 Nef: Taking Control of Protein Trafficking.

    PubMed

    Pereira, Estela A; daSilva, Luis L P

    2016-09-01

    The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions. PMID:27161574

  19. Biochemical and Biologic Characterization of Exosomes and Microvesicles as Facilitators of HIV-1 Infection in Macrophages1

    PubMed Central

    Kadiu, Irena; Narayanasamy, Prabagaran; Dash, Prasanta K.; Zhang, Wei; Gendelman, Howard E.

    2013-01-01

    Exosomes and microvesicles are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Lipidomic, proteomic and cell biologic approaches uncovered processes by which the human immunodeficiency virus type-1 (HIV-1) can use exosomes and MV to facilitate its dissemination. Macrophage MV and exosomes were isolated by immunoaffinity and sucrose cushion centrifugation and characterized by morphologic, biochemical and molecular assays. HIV-1 was “entrapped” in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV and exosomes facilitated viral infection that was affected by a range of cell surface receptors and adhesion proteins. HIV-1 readily completed its life cycle in human monocyte-derived macrophages but not in CD4 negative cells. The data support a significant role for exosomes as facilitators of viral infection. PMID:22711894

  20. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  1. Purification, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexicana amastigotes.

    PubMed

    Ilg, T; Stierhof, Y D; McConville, M J; Overath, P

    1995-02-01

    The intracellular amastigote form of the parasitic protozoon Leishmania mexicana expresses a high-molecular weight phosphoglycan, which is antigenically related to the surface glycolipid lipophosphoglycan and the secreted enzyme acid phosphatase of Leishmania promastigotes. This antigen was purified from a cell-free homogenate of infected mouse tissue and from amastigotes. Compositional and immunological analysis of the purified components indicate a proteophosphoglycan structure consisting of serine-rich polypeptide chains and mild acid-labile phosphooligosaccharides capped by mannooligosaccharides. Immunofluorescence and immunoelectron microscopy of parasitized mouse peritoneal macrophages and infected mouse tissue suggest that the proteophosphoglycan is secreted in large amounts by amastigotes via their flagellar pockets into the parasitophorous vacuoles of host cells. In some infected macrophages proteophosphoglycan is also located in vesicles apparently originating from the parasitophorous vacuole, which demonstrates redistribution of a secreted amastigote antigen in parasitized host cells. PMID:7774606

  2. Genome editing strategies: potential tools for eradicating HIV-1/AIDS

    PubMed Central

    Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-01-01

    Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

  3. Broad activation of latent HIV-1 in vivo.

    PubMed

    Barton, Kirston; Hiener, Bonnie; Winckelmann, Anni; Rasmussen, Thomas Aagaard; Shao, Wei; Byth, Karen; Lanfear, Robert; Solomon, Ajantha; McMahon, James; Harrington, Sean; Buzon, Maria; Lichterfeld, Mathias; Denton, Paul W; Olesen, Rikke; Østergaard, Lars; Tolstrup, Martin; Lewin, Sharon R; Søgaard, Ole Schmeltz; Palmer, Sarah

    2016-01-01

    The 'shock and kill' approach to cure human immunodeficiency virus (HIV) includes transcriptional induction of latent HIV-1 proviruses using latency-reversing agents (LRAs) with targeted immunotherapy to purge infected cells. The administration of LRAs (panobinostat or vorinostat) to HIV-1-infected individuals on antiretroviral therapy induces a significant increase in cell-associated unspliced (CA-US) HIV-1 RNA from CD4(+) T cells. However, it is important to discern whether the increases in CA-US HIV-1 RNA are due to limited or broad activation of HIV-1 proviruses. Here we use single-genome sequencing to find that the RNA transcripts observed following LRA administration are genetically diverse, indicating activation of transcription from an extensive range of proviruses. Defective sequences are more frequently found in CA HIV-1 RNA than in HIV-1 DNA, which has implications for developing an accurate measure of HIV-1 reservoir size. Our findings provide insights into the effects of panobinostat and vorinostat as LRAs for latent HIV-1. PMID:27605062

  4. Targeting TNF-Alpha in HIV-1 Infection.

    PubMed

    Kumar, Amit; Coquard, Laurie; Herbein, Georges

    2016-01-01

    Highly active antiretroviral therapy (HAART) has dramatically extended the lifespan and quality of life of individuals infected with human immunodeficiency virus type 1 (HIV-1). HAART comprises of a cocktail of various pharmacological inhibitors which interfere with almost every stages of HIV-1 life cycle. However, constant application of drugs often results in the evolution of hostpathogen relationship resulting in the emergence of drug resistant viral strains. Drug resistant HIV-1 is a potent threat for the humankind. Therefore, there is a constant need to search for novel therapeutic molecules. HIV-1 infection results in the depletion of CD4+/CD8+T cells and alters the cytokine network in the infected individuals. Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, plays a critical role in HIV-1 pathogenesis. HIV-1 utilizes the TNF-alpha signaling pathway for expanding its reservoir. Several HIV-1 proteins mimic and regulate the TNF-alpha signaling pathway. TNF-alpha inhibitors have been used in several inflammatory pathologies with success to some extent. In the present mini review we will discuss the role of TNF-alpha in HIV-1 pathogenesis. Furthermore we will evaluate the TNF-alpha inhibitors as an additional therapeutic option for HIV-1 infection. PMID:26073859

  5. Modeling Bone Marrow Progenitor Cell Differentiation and Susceptibility to HIV-1 Infection

    PubMed Central

    Alexaki, Aikaterini; Quiterio, Shane J; Nonnemacher, Michael R; Shah, Sonia; Liu, Yujie; Banerjee, Anupam; Li, Luna; Passic, Shendra; Pirrone, Vanessa; Kilareski, Evelyn; Petrovas, Constantinos; Wigdahl, Brian

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection of the monocytic lineage is involved in the pathologic events associated with AIDS and HIV-1-associated dementia (HAD). Hematopoietic progenitor cells (HPCs) within the bone marrow are refractile to HIV-1 infection, while their progeny of the monocyte-macrophage lineage are susceptible. Previous studies, using phorbol-myristate-acetate (PMA) as a differentiating agent, have suggested that the CD34+/CD38+ TF-1 cell line may be used as one model to study the differentiation processes of HPCs. In the present study, medium that has been conditioned by PMA-treated TF-1 cells but is devoid of any traces of PMA, was utilized to induce differentiation of TF-1 cells. The conditioned medium (CM) from this bone marrow-derived cell population is enriched with respect to numerous cytokines and induces differentiation and activation of TF-1 cells, as indicated by changes in the expression of CD34, CD38, and CD69 cell surface molecules. Furthermore, treatment with CM was also shown to induce the expression of CCR5 and CXCR4, while maintaining the expression of CD4, which was ultimately correlated with increased susceptibility to HIV-1. Additionally, the activation of the TF-1 cells was shown to lead to increased LTR activity, with specificity protein (Sp) and nuclear factor kappa-light-chain-enhancer of activated B cells) NF-κB factors playing a crucial role in HIV-1 long terminal repeat (LTR)-mediated transcription and possibly overall TF-1 permissivity. Interleukin (IL)-1β, which is elevated in the CM, recapitulates some of the CM effects. In summary, these studies suggest that the TF-1 cell line could serve as a model to study the susceptibility of bone marrow progenitor cells to HIV-1 infection. PMID:26229980

  6. HIV-1 and Human PEG10 Frameshift Elements Are Functionally Distinct and Distinguished by Novel Small Molecule Modulators

    PubMed Central

    Sleebs, Brad E.; Lackovic, Kurt; Parisot, John P.; Moss, Rebecca M.; Crowe-McAuliffe, Caillan; Mathew, Suneeth F.; Edgar, Christina D.; Kleffmann, Torsten; Tate, Warren P.

    2015-01-01

    Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using −1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 μM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same

  7. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM

    PubMed Central

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-01-01

    Abstract A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1–infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1–infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART. PMID:26579828

  8. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM.

    PubMed

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-11-01

    A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1-infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1-infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART. PMID:26579828

  9. Transcriptional Bursting from the HIV-1 Promoter is a Significant Source of Stochastic Noise in HIV-1 Gene Expression

    SciTech Connect

    Singh, A; Razooky, B; Cox, Chris D.; Simpson, Michael L; Weinberger, Leor S.

    2010-01-01

    Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1 s long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2 10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

  10. HIV-1 Treatment-as-Prevention

    PubMed Central

    Tang, Zhenzhu; Lan, Guanghua; Chen, Ying Qing; Zhu, Qiuying; Yang, Xiaoyi; Shen, Zhiyong; Chen, Yi; Zhang, Heng; Kan, Wei; Xing, Hui; Ruan, Yuhua; Shao, Yiming

    2015-01-01

    Abstract The Chinese national observational cohort study suggests that the treatment-as-prevention (TasP) approach can be an effective public health HIV-1 prevention strategy. However, results from that study may have been biased because the follow-up time of index patients prior to their initiation of antiretroviral therapy (ART) was excluded. In this study, we correct for such bias by using an extended time-dependent Cox regression model to conduct a cohort study analysis of serodiscordant couples in Guangxi of China, inclusive of all follow-up time. During the follow-up of this observational cohort study of HIV-1 sero-discordant couples, the positive index partners may have never be treated with ART, or enter untreated but subsequently began treatment, or may have been treated immediately upon entry into the public health system. The treatment effectiveness of ART in HIV-1 acquisition among HIV-negative partners is assessed by the extended Cox regression model with treatment status as a time-varying covariate. A total of 6548 sero-discordant couples were included in the cohort study analysis. Among them, 348 negative partners sero-converted. HIV seroincidence was significantly higher among the nontreated (4.3 per 100 person-years, 3.7–4.9) compared with those receiving ART (1.8 per 100 person-years, 1.5–2.0). An overall 35% reduction in risk of HIV transmission was associated with receiving ART (adjusted hazard ratio [AHR] 0.65, 95% confidence interval [CI] 0.51–0.83), and the yearly risk reduction was also significant in the first 3 consecutive years of follow-up. Moreover, ART was found to be significantly inversely associated with multiple baseline characteristics of index partners. TasP may be feasible on a national or regional scale. In addition to other proven preventive strategies such as the use of condoms, ART adherence to maintain viral suppression would then be the key challenge for successful TasP implementation.

  11. Bioactivity of flavonoids isolated from Lychnophora markgravii against Leishmania amazonensis amastigotes.

    PubMed

    Salvador, Marcos José; Sartori, Fabiana Terezinha; Sacilotto, Ana Claudia B C; Pral, Elizabeth M F; Alfieri, Silvia Celina; Vichnewski, Walter

    2009-01-01

    The bioactivity of the flavonoids pinostrobin (1), pinocembrin (2), tectochrysin (3), galangin 3-methyl ether (4), and tiliroside (5) isolated from Lychnophora markgravii aerial parts was investigated in vitro against amastigote stages of Leishmania amazonensis. The compounds were isolated by several chromatographic techniques and their chemical structures were established by ESI-MS and NMR spectroscopic data. The flavonoids 1 and 3 were the most active compounds; they markedly reduced the viability of Leishmania amastigotes. PMID:19791501

  12. Leishmania amazonensis Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence

    PubMed Central

    Henard, Calvin A.; Carlsen, Eric D.; Hay, Christie; Kima, Peter E.; Soong, Lynn

    2014-01-01

    Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO−), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO− cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania's ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis. PMID:25033301

  13. A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

    PubMed Central

    Du, Sean X.; Xu, Li; Zhang, Wenge; Tang, Susan; Boenig, Rebecca I.; Chen, Helen; Mariano, Ellaine B.; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Wrin, Terri; Petropoulos, Christos J.; Ballantyne, John A.; Chambers, Michael; Whalen, Robert G.

    2011-01-01

    A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences. PMID:21738594

  14. Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents.

    PubMed Central

    Sereno, D; Lemesre, J L

    1997-01-01

    Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microassay, previously described as a means of quantifying Leishmania amazonensis in vitro at the amastigote stage (D. Sereno and J. L. Lemesre, Parisitol. Res., in press), we have compared the activities of seven drugs, including those currently used to treat leishmaniasis, against axenically grown amastigote and promastigote forms of three Leishmania species (L. amazonensis, L. mexicana, and L. infantum, responsible for diffuse cutaneous, cutaneous, and visceral leishmaniasis, respectively). The ability of axenically cultured amastigote organisms to be used in an investigation of antileishmanial agents was first evaluated. We have confirmed the toxicities of sodium stibogluconate (Pentostam), pentamidine, and amphotericin B to active and dividing populations of axenically cultured amastigotes. The toxicity of potassium antimonyl tartrate trihydrate, which is generally higher than that of Pentostam, seemed to indicate that pentavalent antimony can be metabolized in vivo to compounds, possibly trivalent in nature, which are more active against the amastigote organisms. When the drug susceptibilities of parasites at both stages were compared, great variations were found for all the drugs studied. These major differences, which show the specific chemosusceptibility of the parasite at the mammalian stage, demonstrate the potential of using cultured amastigotes instead of promastigotes in a drug-screening procedure for early detection. This in vitro model may help in the isolation of active compounds, particularly those with low-grade activities, against the mammalian stage of the parasite. PMID:9145854

  15. HIV-1 Assembly, Budding, and Maturation

    PubMed Central

    Sundquist, Wesley I.; Kräusslich, Hans-Georg

    2012-01-01

    A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis. PMID:22762019

  16. Evaluation of Immune Survival Factors in Pediatric HIV-1 Infection

    PubMed Central

    SHEARER, WILLIAM T.; EASLEY, KIRK A.; GOLDFARB, JOHANNA; JENSON, HAL B.; ROSENBLATT, HOWARD M.; KOVACS, ANDREA; MCINTOSH, KENNETH

    2015-01-01

    Peripheral blood CD4+ and CD8+ T cells, CD19+/20+ B cells, and serum immunoglobulins (Igs) have been implicated as survival factors for pediatric HIV-1 infection. To determine which of these immune factors might be important in predicting survival, we studied HIV-1 vertically infected (HIV-1+) children over a 5-year period. Peripheral blood lymphocytes and Igs were measured in 298 HIV-1+ children, who were classified as survivors or nonsurvivors, and in 463 HIV-1 vertically exposed and noninfected (HIV-1–) children. Measurements of other possible survival factors were included in this study: albumin, hemoglobin, lactic dehydrogenase (LDH), and HIV-1 RNA levels. Survivors had significantly higher CD4+ T-cell, CD8+ T-cell, and CD19+/CD20+ B-cell counts and serum IgG levels, but lower serum IgA and IgM levels than nonsurvivors. Serum albumin and blood hemoglobin levels were higher, but serum LDH and HIV-1 RNA levels were lower in the survivors compared to non-survivors. In univariable analysis, factors affecting survival were baseline CD4+ T-cell and CD8+ T-cell counts, IgG, albumin, hemoglobin, LDH, and HIV-1 RNA (all p < 0.001). In multivariable analysis, high baseline CD4+ T-cell count, IgG and albumin levels, and low baseline HIV-1 RNA load remained important factors for survival. Serum IgG level has been identified as an immune factor that independently predicts survival, in addition to the already established CD4+ T-cell count. The HIV-1 RNA and serum albumin levels also predicted survival. PMID:11144332

  17. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication. PMID:12482665

  18. Macrophage Inflammatory Protein-3 Alpha (MIP-3α)/CCL20 in HIV-1-Infected Individuals

    PubMed Central

    Aziz, Najib; Detels, Roger; Chang, L Cindy; Butch, Anthony W

    2016-01-01

    Objective Uncontrolled HIV infection progresses to the depletion of systemic and mucosal CD4 and AIDS. Early HIV infection may be associated with increases in the concentration of MIP-3α in the blood and gut fluids. MIP-3α/CCL20 is the only chemokine known to interact with CCR6 receptors which are expressed on immature dendritic cells and both effector and memory CD8+ and CD4+ T cells. The role and prognostic value of blood levels of MIP-3α in HIV-infected individuals has yet to be described. Methods We determined the serum levels of MIP-3α, and IFN-γ, in 167 HIV-1-infected and 27 HIV-1-uninfected men participating in the Multicenter AIDS Cohort Study (MACS). The blood biomarkers were measured using enzyme-linked immunosorbent assays (ELISA) and the cell phenotypes using flow cytometry. Results Median serum levels of MIP-3α in HIV-1-infected and uninfected men was significantly different (p<0.0001) and were 21.3 pg/mL and 6.4 pg/mL respectively. The HIV-1-infected men with CD4+ T cell count <200 cells/μL showed the highest median serum MIP-3α (23.1 pg/mL). Serum levels of MIP-3α in HIV-1 infected (n=167) were negatively correlated with absolute number of CD4+ T cell (p=0.01) and were positively correlated with CD38 molecules on CD8+ T cells (p=0.0002) and with serum levels of IFN-γ (0.006). Conclusion Serum levels of MIP-3α concomitantly increase with plasma levels of IFN-γ, CD38 expression on CD8+ T cells, and decreased of absolute CD4+ T cells in HIV-1-infected men. A higher blood level of MIP-3α may be representation of locally high level of MIP-3α and more recruitment of immature dendritic cell at site of infection. Involvement of CCR6/CCL20 axis and epithelial cells at the recto-colonel level may enhance sexual transmission of HIV-1 in MSM and may be useful as a prognostic marker in HIV-1-infection and AIDS.

  19. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed Central

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R. B.; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules. PMID:26785380

  20. The fate of HIV-1 capsid: a biochemical assay for HIV-1 uncoating.

    PubMed

    Yang, Yang; Luban, Jeremy; Diaz-Griffero, Felipe

    2014-01-01

    The uncoating process of HIV-1 is a poorly understood process, so the development of a reliable assay to study uncoating is critical for moving the field forward. Here we describe an uncoating assay that currently represents the state-of-the-art biochemical procedure for monitoring uncoating and core stability during infection. This assay is based on the biochemical separation of soluble capsid protein from particulate capsid cores and provides information about the fate of the capsid during infection. PMID:24158811

  1. Breast Milk from Tanzanian Women Has Divergent Effects on Cell-Free and Cell-Associated HIV-1 Infection In Vitro

    PubMed Central

    Lyimo, Magdalena A.; Mosi, Matilda Ngarina; Housman, Molly L.; Zain-Ul-Abideen, Muhammad; Lee, Frederick V.; Howell, Alexandra L.; Connor, Ruth I.

    2012-01-01

    Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-LewisX from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding. PMID:22952771

  2. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    SciTech Connect

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

    2007-10-25

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

  3. Tetraspanin CD63 is a regulator of HIV-1 replication

    PubMed Central

    Fu, Enqing; Pan, Lei; Xie, Yonghong; Mu, Deguang; Liu, Wei; Jin, Faguang; Bai, Xuefan

    2015-01-01

    Macrophages and CD4+ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4+ T-cells, dendritic cells, and a CD4+ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4+ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4+ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2). PMID:25973004

  4. [A new unique HIV-1 recombinant form detected in Belarus].

    PubMed

    Eremin, V F; Gasich, E L; Sosinovich, S V

    2012-01-01

    Republican Research-and-Practical Center for Epidemiology and Microbiology, Ministry of Health of Belarus, Minsk The paper presents data on the molecular genetic characteristics of a new HIV-1 recombinant form. The study has shown that the virus is referred to as HIV-1 subtype B in terms of the gag gene and HIV-1 subtype A in terms of the pol and env genes. At the same time the new isolate is closer, in terms of the gag gene, to the HIV-1 DQ207943 strain isolated in Georgia, in terms of the pol gene, to the HIV-1 AF413987.1 strain isolated in Ukraine and, in terms of the env gene to the HIV-1 AY500393 strain isolated in Russia. Thus, the described new HIV-1 recombinant form has the following structure: BgagApolAenv. The gag, pol, and env gene sequences from the new unique HIV-1 recombinant form have been registered in the international database EMBL/Genbank/DDBJ under accession numbers FR775442.1, FN995656.1, and FR775443.1. PMID:22905420

  5. Reliable Genotypic Tropism Tests for the Major HIV-1 Subtypes

    PubMed Central

    Cashin, Kieran; Gray, Lachlan R.; Harvey, Katherine L.; Perez-Bercoff, Danielle; Lee, Guinevere Q.; Sterjovski, Jasminka; Roche, Michael; Demarest, James F.; Drummond, Fraser; Harrigan, P. Richard; Churchill, Melissa J.; Gorry, Paul R.

    2015-01-01

    Over the past decade antiretroviral drugs have dramatically improved the prognosis for HIV-1 infected individuals, yet achieving better access to vulnerable populations remains a challenge. The principal obstacle to the CCR5-antagonist, maraviroc, from being more widely used in anti-HIV-1 therapy regimens is that the pre-treatment genotypic “tropism tests” to determine virus susceptibility to maraviroc have been developed primarily for HIV-1 subtype B strains, which account for only 10% of infections worldwide. We therefore developed PhenoSeq, a suite of HIV-1 genotypic tropism assays that are highly sensitive and specific for establishing the tropism of HIV-1 subtypes A, B, C, D and circulating recombinant forms of subtypes AE and AG, which together account for 95% of HIV-1 infections worldwide. The PhenoSeq platform will inform the appropriate use of maraviroc and future CCR5 blocking drugs in regions of the world where non-B HIV-1 predominates, which are burdened the most by the HIV-1 pandemic. PMID:25712827

  6. A novel small-molecule inhibitor of HIV-1 entry

    PubMed Central

    Heredia, Alonso; Latinovic, Olga S; Barbault, Florent; de Leeuw, Erik PH

    2015-01-01

    Background Antiretroviral therapy has transformed HIV-1 infection into a managed condition with near-normal life expectancy. However, a significant number of patients remain with limited therapeutic options due to HIV-1 resistance, side effects, or drug costs. Further, it is likely that current drugs will not retain efficacy, due to risks of side effects and transmitted resistance. Results We describe compound 5660386 (3-ethyl-2-[3-(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)-1-propen-1-yl]-1,3-benzothiazol-3-ium) as a novel inhibitor of HIV-1 entry. Compound 5660386 inhibits HIV-1 entry in cell lines and primary cells, binds to HIV-1 envelope protein, and inhibits the interaction of GP120 to CD4. Further, compound 5660386 showed a unique and broad-range activity against primary HIV-1 isolates from different subtypes and geographical areas. Conclusion Development of small-molecule entry inhibitors of HIV-1 such as 5660386 may lead to novel classes of anti-HIV-1 therapeutics. These inhibitors may be particularly effective against viruses resistant to current antiretroviral drugs and could have potential applications in both treatment and prevention. PMID:26491257

  7. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells

    PubMed Central

    Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M.; Ballana, Ester

    2016-01-01

    Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

  8. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

    PubMed

    Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M; Vives-Pi, Marta; Esté, José A; Ballana, Ester

    2016-08-01

    Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

  9. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    PubMed Central

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  10. Low-Level Detection and Quantitation of Cellular HIV-1 DNA and 2-LTR Circles Using Droplet Digital PCR

    PubMed Central

    Henrich, Timothy J.; Gallien, Sebastien; Li, Jonathan Z.; Pereyra, Florencia; Kuritzkes, Daniel R.

    2012-01-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  11. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection

    PubMed Central

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-01-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca2+ influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4+ T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  12. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  13. Effective Cytotoxic T Lymphocyte Targeting of Persistent HIV-1 during Antiretroviral Therapy Requires Priming of Naive CD8+ T Cells

    PubMed Central

    Smith, Kellie N.; Mailliard, Robbie B.; Piazza, Paolo A.; Fischer, Will; Korber, Bette T.; Fecek, Ronald J.; Ratner, Deena; Gupta, Phalguni; Mullins, James I.

    2016-01-01

    ABSTRACT Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8+ T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8+ T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4+ T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8+ T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. PMID:27247230

  14. GSK3β-Activation is a Point of Convergence for HIV-1 and Opiate-Mediated Interactive Neurotoxicity

    PubMed Central

    Masvekar, Ruturaj R.; El-Hage, Nazira; Hauser, Kurt F.; Knapp, Pamela E.

    2015-01-01

    Infection of the CNS with HIV-1 occurs rapidly after primary peripheral infection. HIV-1 can induce a wide range of neurological deficits, collectively known as HIV-1-associated neurocognitive disorders. Our previous work has shown that the selected neurotoxic effects induced by individual viral proteins, Tat and gp120, and by HIV+ supernatant are enhanced by co-exposure to morphine. This mimics co-morbid neurological effects observed in opiate-abusing HIV+ patients. Although there is a correlation between opiate drug abuse and progression of HIV-1-associated neurocognitive disorders, the mechanisms underlie interactions between HIV-1 and opiates remain obscure. Previous studies have shown that HIV-1 induces neurotoxic effects through abnormal activation of GSK3β. Interestingly, expression of GSK3β has shown to be elevated in brains of young opiate abusers indicating that GSK3β is also linked to neuropathology seen with opiate abusing patients. Thus, we hypothesize that GSK3β activation is a point of convergence for HIV- and opiate-mediated interactive neurotoxic effects. Neuronal cultures were treated with supernatant from HIV-1SF162-infected THP-1 cells, in the presence or absence of morphine and GSK3β inhibitors. Our results show that GSK3β inhibitors, including valproate and small molecule inhibitors, significantly reduce HIV-1-mediated neurotoxic outcomes, and also negate interactions with morphine that result in cell death, suggesting that GSK3β-activation is an important point of convergence and a potential therapeutic target for HIV- and opiate-mediated neurocognitive deficits. PMID:25616162

  15. Mucosal Correlates of Protection in HIV-1-Exposed Seronegative Persons

    PubMed Central

    Shen, Ruizhong; Smith, Phillip D.

    2014-01-01

    Resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) persons offers a promising opportunity to identify mechanisms of “natural” protection. Unique features of the mucosa in particular may contribute to this protection. Here we highlight several key issues pertaining to the mucosal correlates of protection in HESN persons, including humoral immune responses, mechanisms of mucosal HIV-1-neutralization, immune cell activation, and role of the microbiota in mucosal responses. We also discuss mucosal model systems that can be used to investigate the mechanisms of resistance in HESN subjects. A clear understanding of the mucosal correlates of protection against HIV-1 in HESN persons will provide critical new insights for the development of effective vaccine and microbicide strategies for the prevention of HIV-1 transmission. PMID:24428610

  16. Multifarious immunotherapeutic approaches to cure HIV-1 infection

    PubMed Central

    Imami, Nesrina; Herasimtschuk, Anna A

    2015-01-01

    Immunotherapy in the context of treated HIV-1 infection aims to improve immune responses to achieve better control of the virus. To date, multifaceted immunotherapeutic approaches have been shown to reduce immune activation and increase CD4 T-lymphocyte counts, further to the effects of antiretroviral therapy alone, in addition to improving HIV-1-specific T-cell responses. While sterilizing cure of HIV-1 would involve elimination of all replication-competent virus, a functional cure in which the host has long-lasting control of viral replication may be more feasible. In this commentary, we discuss novel strategies aimed at targeting the latent viral reservoir with cure of HIV-1 infection being the ultimate goal, an achievement that would have considerable impact on worldwide HIV-1 infection. PMID:26048144

  17. HIV-1 infection, microenvironment and endothelial cell dysfunction.

    PubMed

    Mazzuca, Pietro; Caruso, Arnaldo; Caccuri, Francesca

    2016-09-01

    HIV-1 promotes a generalized immune activation that involves the main targets of HIV-1 infection but also cells that are not sensitive to viral infection. ECs display major dysfunctions in HIV+ patients during long-standing viral infection that persist even in the current cART era, in which new-generation drugs have reduced dysmetabolic side effects and successfully impeded viral replication. In vivo studies have failed to demonstrate the presence of replicating virus in ECs suggesting that a direct role of the virus is unlikely, and implying that the mechanism accounting for vascular dysfunction may rely on the indirect action of molecules released in the microenvironment by HIV-1-infected cells. This article reviews the current understanding of how HIV-1 infection can contribute to vascular dysfunction. In particular, we discuss the emerging role played by different HIV-1 proteins in driving inflammation and EC dysregulation, and highlight the need to target them for therapeutic benefit. PMID:27602413

  18. The role of human dendritic cells in HIV-1 infection.

    PubMed

    Ahmed, Zahra; Kawamura, Tatsuyoshi; Shimada, Shinji; Piguet, Vincent

    2015-05-01

    Dendritic cells (DCs) and their subsets have multifaceted roles in the early stages of HIV-1 transmission and infection. DC studies have led to remarkable discoveries, including identification of restriction factors, cellular structures promoting viral transmission including the infectious synapse or the interplay of the C-type lectins, Langerin on Langerhans cells (LCs), and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin on other DC subsets, limiting or facilitating HIV transmission to CD4(+) T cells, respectively. LCs/DCs are also exposed to encountering HIV-1 and other sexually transmitted infections (herpes simplex virus-2, bacteria, fungi), which reprogram HIV-1 interaction with these cells. This review will summarize advances in the role of DCs during HIV-1 infection and discuss their potential involvement in the development of preventive strategies against HIV-1 and other sexually transmitted infections. PMID:25407434

  19. The use of Nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells.

    PubMed

    Jaworski, Elizabeth; Saifuddin, Mohammed; Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

    2014-01-01

    HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

  20. The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

    PubMed Central

    Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

    2014-01-01

    HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

  1. Distinct Characteristics of Endometrial and Decidual Macrophages and Regulation of Their Permissivity to HIV-1 Infection by SAMHD1

    PubMed Central

    Quillay, Héloïse; El Costa, Hicham; Marlin, Romain; Duriez, Marion; Cannou, Claude; Chrétien, Fabrice; Fernandez, Hervé; Lebreton, Anne; Ighil, Julien; Schwartz, Olivier; Barré-Sinoussi, Françoise; Nugeyre, Marie-Thérèse

    2014-01-01

    ABSTRACT In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages are the main HIV-1 target cells in the decidua. Here we report that in vitro, macrophages and T cells are the main HIV-1 targets in the endometrium in nonpregnant women. As reported for decidual macrophages (dM), endometrial macrophages (eM) were found to have an M2-like phenotype (CD68+ CD163+ CD206+ IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of proinflammatory and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role for SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with FRT mucosae in nonpregnant women should help to identify mechanisms of mucosal protection against HIV-1 infection. IMPORTANCE The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the endometrium in nonpregnant women could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium and that infection of macrophages from both the endometrium and the decidua is restricted by

  2. Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

    PubMed Central

    Spotin, Adel; Rouhani, Soheila; Ghaemmaghami, Parnazsadat; Haghighi, Ali; Zolfaghari, Mohammad Reza; Amirkhani, Aref; Farahmand, Mahin; Bordbar, Ali; Parvizi, Parviz

    2015-01-01

    Background: Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Methods: Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Results: Only L. major was identified in patients containing regular amastigotes' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075). Conclusion: Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. PMID:26081070

  3. The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo

    PubMed Central

    Søgaard, Ole S.; Graversen, Mette E.; Leth, Steffen; Olesen, Rikke; Brinkmann, Christel R.; Nissen, Sara K.; Kjaer, Anne Sofie; Schleimann, Mariane H.; Denton, Paul W.; Hey-Cunningham, William J.; Koelsch, Kersten K.; Pantaleo, Giuseppe; Krogsgaard, Kim; Sommerfelt, Maja; Fromentin, Remi; Chomont, Nicolas; Rasmussen, Thomas A.; Østergaard, Lars; Tolstrup, Martin

    2015-01-01

    Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-1 infection. However, until now, approaches to reverse HIV-1 latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-1 infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7–7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-1 transcription quantified as copies of cell-associated un-spliced HIV-1 RNA increased significantly from baseline during treatment (range of fold-increase: 2.4–5.0; p = 0.03). Plasma HIV-1 RNA increased from <20 copies/mL at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46–103 copies/mL following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade 1–2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-1 transcription resulting in plasma HIV-1 RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-1 latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-1 reservoir. Trial Registration clinicaltrials.gov NTC02092116 PMID:26379282

  4. HIV-1-infected macrophages induce astrogliosis by SDF-1{alpha} and matrix metalloproteinases

    SciTech Connect

    Okamoto, Mika; Wang, Xin; Baba, Masanori . E-mail: baba@m.kufm.kagoshima-u.ac.jp

    2005-11-04

    Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1{alpha} or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1{alpha} production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1{alpha} was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1{alpha} and MMP production, which implies a mechanism of astrogliosis in HAD.

  5. The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

    SciTech Connect

    Wang, Yong; Liu, Zhigang; Brunzelle, Joseph S.; Kovari, Iulia A.; Dewdney, Tamaria G.; Reiter, Samuel J.; Kovari, Ladislau C.

    2011-11-17

    Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.

  6. HIV1-viral protein R (Vpr) mutations: associated phenotypes and relevance for clinical pathologies.

    PubMed

    Soares, Rui; Rocha, Graça; Meliço-Silvestre, António; Gonçalves, Teresa

    2016-09-01

    Over the last 30 years, research into HIV has advanced the knowledge of virus genetics and the development of efficient therapeutic strategies. HIV-1 viral protein R (Vpr) is a specialized and multifunctional protein that plays important roles at multiple stages of the HIV-1 viral life cycle. This protein interacts with a number of cellular and viral proteins and with multiple activities including nuclear transport of the pre-integration complex (PIC) to the nucleus, transcriptional activation, cell cycle arrest at G2/M transition phase and induction of cell death via apoptosis. Specifically, Vpr has been shown to control many host cell functions through a variety of biological processes and by interaction with several cellular pathways. The different functions of Vpr may enhance viral replication and impair the immune system in HIV-1 infected patients. Importantly, functional defects induced by mutations in the Vpr protein correlate with slow disease progression of HIV-infected patients. Vpr is also associated with other concomitant pathologies developed by these patients, which may lead it to be considered as a potential novel therapeutic target. This review will focus on HIV-1 Vpr, mainly on the importance of its structural mutations on the progression of HIV infection, associated phenotypes and relevance for clinical pathologies. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27264019

  7. Interfacial Cavity Filling To Optimize CD4-Mimetic Miniprotein Interactions with HIV-1 Surface Glycoprotein

    SciTech Connect

    Morellato-Castillo, Laurence; Acharya, Priyamvada; Combes, Olivier; Michiels, Johan; Descours, Anne; Ramos, Oscar H.P.; Yang, Yongping; Vanham, Guido; Ariën, Kevin K.; Kwong, Peter D.; Martin, Loïc; Kessler, Pascal

    2013-08-05

    Ligand affinities can be optimized by interfacial cavity filling. A hollow (Phe43 cavity) between HIV-1 surface glycoprotein (gp120) and cluster of differentiation 4 (CD4) receptor extends beyond residue phenylalanine 43 of CD4 and cannot be fully accessed by natural amino acids. To increase HIV-1 gp120 affinity for a family of CD4-mimetic miniproteins (miniCD4s), we targeted the gp120 Phe43 cavity with 11 non-natural phenylalanine derivatives, introduced into a miniCD4 named M48 (1). The best derivative, named M48U12 (13), bound HIV-1 YU2 gp120 with 8 pM affinity and showed potent HIV-1 neutralization. It contained a methylcyclohexyl derivative of 4-aminophenylalanine, and its cocrystal structure with gp120 revealed the cyclohexane ring buried within the gp120 hydrophobic core but able to assume multiple orientations in the binding pocket, and the aniline nitrogen potentially providing a focus for further improvement. Altogether, the results provide a framework for filling the interfacial Phe43 cavity to enhance miniCD4 affinity.

  8. Interfacial cavity filling to optimize CD4-mimetic miniprotein interactions with the HIV-1 surface protein

    PubMed Central

    Morellato-Castillo, Laurence; Acharya, Priyamvada; Combes, Olivier; Michiels, Johan; Descours, Anne; Ramos, Oscar H. P.; Yang, Yongping; Vanham, Guido; Ariën, Kevin K.; Kwong, Peter D.; Martin, Loïc; Kessler, Pascal

    2013-01-01

    Ligand affinities can be optimized by interfacial cavity filling. A hollow (Phe43 cavity) between HIV-1 surface protein (gp120) and cluster of differentiation 4 (CD4) receptor, extends beyond residue phenylalanine 43 of CD4 and cannot be fully accessed by natural amino acids. To increase HIV-1 gp120 affinity for a family of CD4-mimetic miniproteins (miniCD4s), we targeted the gp120 Phe43 cavity with eleven non-natural phenylalanine derivatives, introduced into a miniCD4 named M48 (1). The best derivative named M48U12 (13) binds HIV-1 YU2 gp120 with 8 pM affinity, and shows potent HIV-1 neutralization. It contained a methylcyclohexyl derivative of 4-aminophenylalanine and its co-crystal structure with gp120 revealed the cyclohexane ring buried within the gp120 hydrophobic core but able to assume multiple orientations in the binding pocket, and an aniline nitrogen potentially providing a focus for further improvement. Altogether, the results provide a framework for filling the interfacial Phe43 cavity to enhance miniCD4 affinity. PMID:23710622

  9. Recent Advances in Lentiviral Vaccines for HIV-1 Infection

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.

    2016-01-01

    The development of an effective HIV vaccine to prevent and/or cure HIV remains a global health priority. Given their central role in the initiation of adaptive immune responses, dendritic cell (DC)-based vaccines are being increasingly explored as immunotherapeutic strategies to enhance HIV-specific T cells in infected individuals and, thus, promote immune responses that may help facilitate a functional cure. HIV-1-based lentiviral (LV) vectors have inherent advantages as DC vaccine vectors due to their ability to transduce non-dividing cells and integrate into the target cell genomic DNA, allowing for expression of encoded antigens over the lifespan of the cell. Moreover, LV vectors may express additional immunostimulatory and immunoregulatory proteins that enhance DC function and direct antigen-specific T cells responses. Recent basic and clinical research efforts have broadened our understanding of LV vectors as DC-based vaccines. In this review, we provide an overview of the pre-clinical and clinical LV vector vaccine studies for treating HIV to date. We also discuss advances in LV vector designs that have enhanced DC transduction efficiency, target cell specificity, and immunogenicity, and address potential safety concerns regarding LV vector-based vaccines. PMID:27446074

  10. p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

    PubMed Central

    Yoon, Cheol-Hee; Kim, Sang-Yoon; Byeon, Se Eun; Jeong, Yideul; Lee, Jinjoo; Kim, Kwang Pyo; Park, Jinseu

    2015-01-01

    ABSTRACT Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53−/− and eIF2αCA cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat–Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCE HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53

  11. High resolution of Trypanosoma cruzi amastigote antigen in serodiagnosis of different clinical forms of Chagas' disease.

    PubMed Central

    Matsumoto, T K; Hoshino-Shimizu, S; Nakamura, P M; Andrade, H F; Umezawa, E S

    1993-01-01

    The serodiagnosis of Chagas' disease, a highly prevalent disorder in South American countries, is usually made by the detection of antibodies to Trypanosoma cruzi epimastigote antigen. In this study, we assess the diagnostic performance of the immunofluorescence test with T. cruzi (Y strain) amastigote antigen from an LLC-MK2-infected cell supernatant in comparison with a test with the conventional epimastigote antigen. A total of 238 serum samples from patients in the acute and chronic phases of the disease, with the chronic indeterminate, cardiac, and digestive forms, and from nonchagasic individuals were tested for the presence of immunoglobulin G (IgG), IgM, and IgA antibodies. The reactivity of the amastigote antigen in terms of geometric mean titers was 2 to 4 times higher than that of the epimastigote antigen. Clear-cut results were obtained with the amastigote antigen, with no overlapping of true and false positives. IgG antibodies to amastigotes were found in all patients with Chagas' disease, whereas all sera from nonchagasic patients were negative, except for those from patients with visceral leishmaniasis, in which 63% cross-reactivity was observed. IgM antibodies to amastigotes were detected in 100% of sera from patients with acute Chagas' disease and in 7.5% of sera from patients with chronic Chagas' disease, whereas IgA antibodies were found in 60% of sera from patients in the acute phase and in 33% of sera from patients in the chronic phase. Despite the cross-reactivity observed with sera from visceral leishmaniasis patients, the IgG immunofluorescence test with the amastigote antigen had the highest sensitivity, specificity, and efficiency. No relationship was observed between the class-specific antibodies or their titers and the clinical forms of patients in the chronic phase. Amastigotes from the cell culture supernatant proved to be useful as an alternative antigen to epimastigotes because of their high resolution in the serodiagnosis of Chagas

  12. Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

    PubMed Central

    2010-01-01

    Background The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Results Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. Conclusions The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology. PMID:20074347

  13. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin.

    PubMed

    Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2016-01-01

    HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312

  14. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin

    PubMed Central

    Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2016-01-01

    HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312

  15. TNPO3 Is Required for HIV-1 Replication after Nuclear Import but prior to Integration and Binds the HIV-1 Core

    PubMed Central

    Valle-Casuso, Jose Carlos; Di Nunzio, Francesca; Yang, Yang; Reszka, Natalia; Lienlaf, Maritza; Arhel, Nathalie; Perez, Patricio; Brass, Abraham L.

    2012-01-01

    TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import. PMID:22398280

  16. Anti-HIV-1 Activity of Flavonoid Myricetin on HIV-1 Infection in a Dual-Chamber In Vitro Model

    PubMed Central

    Pasetto, Silvana; Pardi, Vanessa; Murata, Ramiro Mendonça

    2014-01-01

    HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01–100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research. PMID:25546350

  17. Stochastic extinction dynamics of HIV-1

    NASA Astrophysics Data System (ADS)

    Schwartz, Ira; Forgoston, Eric; Weinberger, Leor

    2012-02-01

    We consider an HIV-1 within host model in which T cells are infected by the virus. Due to small numbers of molecules, stochastic effects play an important role in the dynamical outcomes in that two states are observed experimentally: a replication state in which the virus is active, or a dormant state leading to latency in which the virus becomes active after a delay. The two states are conjectured to be governed by the Tat gene protein transcription process, which does not possess two stable attractors. Rather, the active state is stable, while the dormant state is unstable. Therefore the dormant state can only be achieved through the dynamics of stochastic fluctuations in which noise organizes a path to dormancy. Here we use optimal path theory applied to a Tat gene stochastic model to show how random fluctuations generate the dormant state by deriving a path which optimizes the probability of achieving the dormant state. We explicitly show how the probability of achieving dormancy scales with the transition rate parameters.

  18. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

    PubMed

    Ogata, T; Higuchi, H; Mochida, S; Matsumoto, H; Kato, A; Endo, T; Kaji, A; Kaji, H

    1992-11-01

    An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system. PMID:1283310

  19. Identification of Siglec-1 null individuals infected with HIV-1

    PubMed Central

    Martinez-Picado, Javier; McLaren, Paul J.; Erkizia, Itziar; Martin, Maureen P.; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M.; Penugonda, Sudhir; Günthard, Huldrych F.; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-01-01

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals. PMID:27510803

  20. Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

    PubMed Central

    Koppensteiner, Herwig; Banning, Carina; Schneider, Carola; Hohenberg, Heinrich

    2012-01-01

    In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir. PMID:22205742

  1. Sex and gender differences in HIV-1 infection.

    PubMed

    Griesbeck, Morgane; Scully, Eileen; Altfeld, Marcus

    2016-08-01

    The major burden of the human immunodeficiency (HIV) type 1 pandemic is nowadays carried by women from sub-Saharan Africa. Differences in the manifestations of HIV-1 infection between women and men have been long reported, and might be due to both socio-economic (gender) and biological (sex) factors. Several studies have shown that women are more susceptible to HIV-1 acquisition than men. Following HIV-1 infection, women have lower viral loads during acute infection and exhibit stronger antiviral responses than men, which may contribute to differences in the size of viral reservoirs. Oestrogen receptor signalling could represent an important mediator of sex differences in HIV-1 reservoir size and may represent a potential therapeutic target. Furthermore, immune activation, a hallmark of HIV-1 infection, is generally higher in women than in men and could be a central mechanism in the sex difference observed in the speed of HIV-1 disease progression. Here, we review the literature regarding sex-based differences in HIV-1 infection and discuss how a better understanding of the underlying mechanisms could improve preventive and therapeutic strategies. PMID:27389589

  2. Identification of Siglec-1 null individuals infected with HIV-1.

    PubMed

    Martinez-Picado, Javier; McLaren, Paul J; Erkizia, Itziar; Martin, Maureen P; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M; Penugonda, Sudhir; Günthard, Huldrych F; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-01-01

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals. PMID:27510803

  3. Correlates of HIV-1 Genital Shedding in Tanzanian Women

    PubMed Central

    Tanton, Clare; Weiss, Helen A.; Le Goff, Jerome; Changalucha, John; Rusizoka, Mary; Baisley, Kathy; Everett, Dean; Ross, David A.; Belec, Laurent; Hayes, Richard J.; Watson-Jones, Deborah

    2011-01-01

    Background Understanding the correlates of HIV shedding is important to inform strategies to reduce HIV infectiousness. We examined correlates of genital HIV-1 RNA in women who were seropositive for both herpes simplex virus (HSV)-2 and HIV-1 and who were enrolled in a randomised controlled trial of HSV suppressive therapy (aciclovir 400 mg b.i.d vs. placebo) in Tanzania. Methodology Samples, including a cervico-vaginal lavage, were collected and tested for genital HIV-1 and HSV and reproductive tract infections (RTIs) at randomisation and 6, 12 and 24 months follow-up. Data from all women at randomisation and women in the placebo arm during follow-up were analysed using generalised estimating equations to determine the correlates of cervico-vaginal HIV-1 RNA detection and load. Principal Findings Cervico-vaginal HIV-1 RNA was detected at 52.0% of 971 visits among 482 women, and was independently associated with plasma viral load, presence of genital ulcers, pregnancy, bloody cervical or vaginal discharge, abnormal vaginal discharge, cervical ectopy, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, an intermediate bacterial vaginosis score and HSV DNA detection. Similar factors were associated with genital HIV-1 RNA load. Conclusions RTIs were associated with increased presence and quantity of genital HIV-1 RNA in this population. These results highlight the importance of integrating effective RTI treatment into HIV care services. PMID:21390251

  4. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

    PubMed

    Huang, Yaoxing; Yu, Jian; Lanzi, Anastasia; Yao, Xin; Andrews, Chasity D; Tsai, Lily; Gajjar, Mili R; Sun, Ming; Seaman, Michael S; Padte, Neal N; Ho, David D

    2016-06-16

    While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1. PMID:27315479

  5. RNA interference directed to CDK2 inhibits HIV-1 transcription.

    PubMed

    Ammosova, Tatyana; Berro, Reem; Kashanchi, Fatah; Nekhai, Sergei

    2005-10-25

    We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target. PMID:16085226

  6. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

    PubMed

    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection. PMID:27056720

  7. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  8. 6,7-Dihydroxy-1-oxoisoindoline-4-sulfonamide-containing HIV-1 Integrase Inhibitors

    PubMed Central

    Zhao, Xue Zhi; Maddali, Kasthuraiah; Smith, Steven J.; Métifiot, Mathieu; Johnson, Barry C.; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves; Burke, Terrence R.

    2012-01-01

    Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck’s raltegravir and Gilead’s elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5- positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant. PMID:23149229

  9. 6,7-Dihydroxy-1-oxoisoindoline-4-sulfonamide-containing HIV-1 integrase inhibitors.

    PubMed

    Zhao, Xue Zhi; Maddali, Kasthuraiah; Smith, Steven J; Métifiot, Mathieu; Johnson, Barry C; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves; Burke, Terrence R

    2012-12-15

    Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck's raltegravir and Gilead's elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5-positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant. PMID:23149229

  10. Extreme Genetic Fragility of the HIV-1 Capsid

    PubMed Central

    Rihn, Suzannah J.; Wilson, Sam J.; Loman, Nick J.; Alim, Mudathir; Bakker, Saskia E.; Bhella, David; Gifford, Robert J.; Rixon, Frazer J.; Bieniasz, Paul D.

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

  11. Extreme genetic fragility of the HIV-1 capsid.

    PubMed

    Rihn, Suzannah J; Wilson, Sam J; Loman, Nick J; Alim, Mudathir; Bakker, Saskia E; Bhella, David; Gifford, Robert J; Rixon, Frazer J; Bieniasz, Paul D

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

  12. T cell virological synapses and HIV-1 pathogenesis.

    PubMed

    Chen, Benjamin K

    2012-12-01

    Human immunodeficiency virus type 1 is the cause of a modern global pandemic associated with progressive acquired immune deficiency. The infection is characterized by the loss of the primary target of viral infection, the CD4+ T cell. The measurement of plasma viremia in patients can predict the rate of CD4+ cell decline; however, it is not clear whether this cell-free plasma virus represents the engine that drives viral spread. Active viral replication is mainly observed within lymphoid tissues that are hotbeds of cell-cell interactions that initiate and organize immune responses. It is well established that cell-cell interactions enhance viral spread in vitro. Dendritic cell-T cell interactions, which lie at the heart of adaptive immune responses, enhance viral infection in vitro. Interactions between infected and uninfected CD4+ T cells are a dominant route of viral spread in vitro and are likely to play a central role in viral dissemination in vivo. Future studies will test existing paradigms of HIV-1 dissemination to determine whether virus-transmitting contacts between infected and uninfected T cells called virological synapses are the dominant mode of viral spread in vivo. Here, we review the status of our understanding of this mode of infection with a focus on T cell-T cell interactions and examine how it may explain resistance to neutralizing antibodies and or the generation of genetic diversity of HIV. PMID:22477441

  13. Concordance between two phenotypic assays and ultradeep pyrosequencing for determining HIV-1 tropism.

    PubMed

    Saliou, Adrien; Delobel, Pierre; Dubois, Martine; Nicot, Florence; Raymond, Stéphanie; Calvez, Vincent; Masquelier, Bernard; Izopet, Jacques

    2011-06-01

    There have been few studies on the concordance between phenotypic assays for predicting human immunodeficiency virus type 1 (HIV-1) coreceptor usage. The sensitivity of ultradeep pyrosequencing combined with genotyping tools is similar to that of phenotypic assays for detecting minor CXCR4-using variants. We evaluated the agreement between two phenotypic assays, the Toulouse tropism test (TTT) and the Trofile assay, and ultradeep pyrosequencing for determining the tropism of HIV-1 quasispecies. The concordance between the TTT and Trofile assays was assessed for 181 samples successfully phenotyped by both assays. The TTT was 86% concordant with the standard Trofile assay and 91.7% with its enhanced-sensitivity version. The concordance between phenotypic characterization of HIV-1 tropism and ultradeep pyrosequencing genotypic prediction was further studied in selected samples. The HIV-1 tropism inferred from ultradeep pyrosequencing of 11 samples phenotyped as X4 and dualtropic and 12 phenotyped as R5-tropic agreed closely with the results of phenotyping. However, ultradeep pyrosequencing detected minor CXCR4-using variants in 3 of 12 samples phenotyped as R5-tropic. Ultradeep pyrosequencing also detected minor CXCR4-using variants that had been missed by direct sequencing in 6 of 9 samples phenotyped as X4-tropic but genotyped as R5-tropic by direct sequencing. Ultradeep pyrosequencing was 87% concordant with the Trofile and TTT phenotypic assays and was in the same range of sensitivity (0.4%) than these two phenotypic assays (0.3 to 0.5%) for detecting minor CXCR4-using variants. Ultradeep pyrosequencing provides a new way to improve the performance of genotypic prediction of HIV-1 tropism to match that of the phenotypic assays. PMID:21464245

  14. Concordance between Two Phenotypic Assays and Ultradeep Pyrosequencing for Determining HIV-1 Tropism▿†

    PubMed Central

    Saliou, Adrien; Delobel, Pierre; Dubois, Martine; Nicot, Florence; Raymond, Stéphanie; Calvez, Vincent; Masquelier, Bernard; Izopet, Jacques

    2011-01-01

    There have been few studies on the concordance between phenotypic assays for predicting human immunodeficiency virus type 1 (HIV-1) coreceptor usage. The sensitivity of ultradeep pyrosequencing combined with genotyping tools is similar to that of phenotypic assays for detecting minor CXCR4-using variants. We evaluated the agreement between two phenotypic assays, the Toulouse tropism test (TTT) and the Trofile assay, and ultradeep pyrosequencing for determining the tropism of HIV-1 quasispecies. The concordance between the TTT and Trofile assays was assessed for 181 samples successfully phenotyped by both assays. The TTT was 86% concordant with the standard Trofile assay and 91.7% with its enhanced-sensitivity version. The concordance between phenotypic characterization of HIV-1 tropism and ultradeep pyrosequencing genotypic prediction was further studied in selected samples. The HIV-1 tropism inferred from ultradeep pyrosequencing of 11 samples phenotyped as X4 and dualtropic and 12 phenotyped as R5-tropic agreed closely with the results of phenotyping. However, ultradeep pyrosequencing detected minor CXCR4-using variants in 3 of 12 samples phenotyped as R5-tropic. Ultradeep pyrosequencing also detected minor CXCR4-using variants that had been missed by direct sequencing in 6 of 9 samples phenotyped as X4-tropic but genotyped as R5-tropic by direct sequencing. Ultradeep pyrosequencing was 87% concordant with the Trofile and TTT phenotypic assays and was in the same range of sensitivity (0.4%) than these two phenotypic assays (0.3 to 0.5%) for detecting minor CXCR4-using variants. Ultradeep pyrosequencing provides a new way to improve the performance of genotypic prediction of HIV-1 tropism to match that of the phenotypic assays. PMID:21464245

  15. Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

    PubMed Central

    Warrilow, David; Warren, Kylie; Harrich, David

    2010-01-01

    Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis. PMID:20949087

  16. Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

    PubMed

    Rawson, Jonathan M O; Roth, Megan E; Xie, Jiashu; Daly, Michele B; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-06-01

    Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity. PMID:27117260

  17. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  18. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants.

    PubMed

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M; Sullivan, John L; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  19. Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

    PubMed Central

    Mouser, Emily E. I. M.; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William

    2016-01-01

    One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476

  20. Current Perspectives on HIV-1 Antiretroviral Drug Resistance

    PubMed Central

    Iyidogan, Pinar; Anderson, Karen S.

    2014-01-01

    Current advancements in antiretroviral therapy (ART) have turned HIV-1 infection into a chronic and manageable disease. However, treatment is only effective until HIV-1 develops resistance against the administered drugs. The most recent antiretroviral drugs have become superior at delaying the evolution of acquired drug resistance. In this review, the viral fitness and its correlation to HIV-1 mutation rates and drug resistance are discussed while emphasizing the concept of lethal mutagenesis as an alternative therapy. The development of resistance to the different classes of approved drugs and the importance of monitoring antiretroviral drug resistance are also summarized briefly. PMID:25341668

  1. Gelsolin activity controls efficient early HIV-1 infection

    PubMed Central

    2013-01-01

    Background HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events. Results Here we show that gelsolin restructures cortical F-actin during HIV-1 Env-gp120-mediated signalling, without affecting cell-surface expression of receptors or viral co-receptor signalling. Remarkably, efficient HIV-1 Env-mediated membrane fusion and infection of permissive lymphocytes were impaired when gelsolin was either overexpressed or silenced, which led to a loss or gain of cortical actin, respectively. Indeed, HIV-1 Env-gp120-induced F-actin reorganization and viral receptor capping were impaired under these experimental conditions. Moreover, gelsolin knockdown promoted HIV-1 Env-gp120-mediated aberrant pseudopodia formation. These perturbed-actin events are responsible for the inhibition of early HIV-1 infection. Conclusions For the first time we provide evidence that through its severing of cortical actin, and by controlling the amount of actin available for reorganization during HIV-1 Env-mediated viral fusion, entry and infection, gelsolin can constitute a barrier that restricts HIV-1 infection of CD4+ lymphocytes in a pre-fusion step. These findings provide important insights into the complex molecular and actin-associated dynamics events that underlie early viral infection. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection acting at a pre-fusion step

  2. Towards an HIV-1 cure: measuring the latent reservoir

    PubMed Central

    Bruner, Katherine M.; Hosmane, Nina N.; Siliciano, Robert F.

    2015-01-01

    The latent reservoir of HIV-1 in resting memory CD4+ T cells serves as a major barrier to curing HIV-1 infection. While many PCR- and culture-based assays have been used to measure the size of the latent reservoir, correlation between results of different assays is poor and recent studies indicate that no available assay provides an accurate measurement of reservoir size. The discrepancies between assays are a hurdle to clinical trials that aim to measure the efficacy of HIV-1 eradication strategies. Here we describe the advantages and disadvantages of various approaches to measure the latent reservoir. PMID:25747663

  3. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

    PubMed Central

    Guerrero, Santiago; Batisse, Julien; Libre, Camille; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe

    2015-01-01

    Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication. PMID:25606970

  4. HIV-1 replication and the cellular eukaryotic translation apparatus.

    PubMed

    Guerrero, Santiago; Batisse, Julien; Libre, Camille; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe

    2015-01-01

    Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication. PMID:25606970

  5. HIV-1 Vpr increases Env expression by preventing Env from endoplasmic reticulum-associated protein degradation (ERAD).

    PubMed

    Zhang, Xianfeng; Zhou, Tao; Frabutt, Dylan A; Zheng, Yong-Hui

    2016-09-01

    Vpr enhances HIV-1 replication in macrophages and dendritic cells, as well as the human CD4(+) CEM.NKR T cell line. Recently, Vpr was reported to increase HIV-1 Env expression in macrophages. Here, we report that Vpr also increases HIV-1 Env expression in dendritic cells and CEM.NKR cells. The Vpr activity depends on its N-terminal region, which was disrupted by a single A30L mutation. Env was rapidly degraded in the absence of Vpr, which was blocked by the ERAD pathway inhibitor kifunesine or the lysosome inhibitor Bafilomycin. As2O3 or PK11195, which reportedly enhances HIV-1 Env folding, also blocked the Env degradation in CEM.NKR cells. Thus, these results not only identify Env as a primary target for Vpr to boost HIV-1 replication, but also suggest that Vpr likely promotes Env folding in the ER, which is otherwise misfolded and targeted by the ERAD pathway to lysosomes for degradation. PMID:27343732

  6. Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes.

    PubMed

    Batista, Cassiano Martin; Kessler, Rafael Luis; Eger, Iriane; Soares, Maurilio José

    2015-01-01

    Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10(7) amastigotes--with typical shape and positive for the amastigote marker Ssp4--from 5x10(6) infected Vero cells (48 h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes. PMID:26057131

  7. Viremic HIV controllers exhibit high plasmacytoid dendritic cell\\reactive opsonophagocytic IgG antibody responses against HIV-1 p24 associated with greater antibody isotype diversification

    PubMed Central

    Tjiam, M. Christian; Taylor, James P. A.; Morshidi, Mazmah A.; Sariputra, Lucy; Burrows, Sally; Martin, Jeffrey N.; Deeks, Steven G.; Tan, Dino B.A.; Lee, Silvia; Fernandez, Sonia; French, Martyn A.

    2015-01-01

    Identifying the mechanisms of natural control of HIV-1 infection could lead to novel approaches to prevent or cure HIV infection. Several studies have associated natural control of HIV-1 infection with IgG antibodies against HIV-1 Gag proteins (e.g. p24) and/or production of IgG2 antibodies against HIV-1 proteins. These antibodies likely exert their effect by activating anti-viral effector cell responses rather than virus neutralization. We hypothesized that an opsonophagocytic IgG antibody response against HIV-1 p24 that activates plasmacytoid dendritic cells (pDCs) through FcγRIIa would be associated with control of HIV and that this would be enhanced by antibody isotype diversification. Using the Gen2.2 pDC cell line, we demonstrated that pDC-reactive opsonophagocytic IgG antibody responses against HIV-1 p24 were higher in HIV controllers (HIV RNA <2000 copies/mL) than non-controllers (HIV RNA >10,000 copies/mL) particularly in controllers with low but detectable viremia (HIV RNA 75–2000 copies/mL). Opsonophagocytic antibody responses correlated with plasma levels of IgG1 and IgG2 anti-HIV-1 p24 and notably, correlated inversely with plasma HIV RNA levels in viremic HIV patients. Phagocytosis of these antibodies was mediated via FcγRIIa. Isotype diversification (towards IgG2) was greatest in HIV controllers and depletion of IgG2 from immunoglobulin preparations indicated that IgG2 antibodies to HIV-1 p24 do not enhance phagocytosis, suggesting that they enhance other aspects of antibody function, such as antigen opsonization. Our findings emulate those for pDC-reactive opsonophagocytic antibody responses against coxsackie, picorna and influenza viruses and demonstrate a previously undefined immune correlate of HIV-1 control that may be relevant to HIV vaccine development. PMID:25911748

  8. HIV-1 propagates in human neuroblastoma cells.

    PubMed

    Shapshak, P; Sun, N C; Resnick, L; Thornthwaite, J T; Schiller, P; Yoshioka, M; Svenningsson, A; Tourtellotte, W W; Imagawa, D T

    1991-01-01

    A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS. PMID:1704060

  9. Persistent HIV-1 replication during antiretroviral therapy

    PubMed Central

    Martinez-Picado, Javier; Deeks, Steven G.

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on efficiently targeting these three aspects. The degree to which HIV replicates during ART remains controversial. Most studies have failed to find any evidence of HIV evolution in blood, even with samples collected over many years, although a recent very intensive study of three individuals suggested that the virus population does shift, at least during the first few months of therapy. Stronger but still not definitive evidence for replication comes from a series of studies in which standard regimens were intensified with an integration inhibitor, resulting in changes in episomal DNA (blood) and cell-associated RNA (tissue). Limited drug penetration within tissues and the presence of immune sanctuaries have been argued as potential mechanisms allowing HIV to spread during ART. Mathematical models suggest that HIV replication and evolution is possible even without the selection of fully drug-resistant variants. As persistent HIV replication could have clinical consequences and might limit the efficacy of curative interventions, determining if HIV replicates during ART and why, should remain a key focus of the HIV research community. Summary Residual viral replication likely persists in lymphoid tissues, at least in a subset of individuals. Abnormal levels of immune activation might contribute to sustain virus replication. PMID:27078619

  10. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives.

    PubMed

    Fernandes, Ana Paula; Coelho, Eduardo Antonio Ferraz; Machado-Coelho, George Luiz Lins; Grimaldi, Gabriel; Gazzinelli, Ricardo Tostes

    2012-08-01

    Visceral leishmaniasis is a major health problem in Latina America, as well as the Mediterranean region of Europe and Asia. We aimed to develop a vaccine against visceral leishmaniasis targeting the intracellular amastigotes, which is the parasite stage that persists throughout infections with Leishmania parasites. With this in mind, we identified an amastigote specific antigen (A2) that contains an immunogenic epitope for CD4+ T helper (Th) cells and multiple repetitive units encoding CD8+ cytotoxic T lymphocyte (CTL) epitopes. Vaccine formulations containing the recombinant A2 associated with saponin, alum and IL-12 or expressed by attenuated adenovirus were shown to be protective in mice, dogs and nonhuman-primates. We are currently identifying novel amastigote specific immunogenic proteins that could be aggregated to A2 to further improve the level of vaccine-induced cell-mediated immunity and protection against visceral leishmaniasis. PMID:22698479

  11. Leishmania amastigotes in the central nervous system of a naturally infected dog.

    PubMed

    Márquez, Merce; Pedregosa, José Raúl; López, Jesús; Marco-Salazar, Paola; Fondevila, Dolors; Pumarola, Martí

    2013-01-01

    A 4-year-old male Labrador Retriever dog was presented with a 10-day history of tetraplegia, depression, and absent postural reflexes. The cerebrospinal fluid was positive for Leishmania spp. DNA. At necropsy, a 2-cm long mass was observed adhered to C(7) and C(8) left spinal nerves. Microscopically, nerve fiber destruction together with mixed inflammatory infiltration was observed in the spinal nerves. Cervical spinal cord sections showed multifocal, diffuse granulomatous inflammation in the white matter. In the brain, perivascular infiltrates were observed in some areas together with subtle pallor of the parenchyma. Immunohistochemistry for Leishmania infantum confirmed the presence of amastigotes in the spinal nerves, spinal cord, brain parenchyma, and choroid plexuses. The current study describes the presence of Leishmania amastigotes in nervous tissue inciting radiculoneuritis, myelitis, and mild meningoencephalitis, suggesting a likely route by which L. infantum amastigotes reach and affect the central nervous system parenchyma. PMID:23166183

  12. Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects

    PubMed Central

    Roff, Shannon R; Sanou, Missa P; Rathore, Mobeen H; Levy, Jay A; Yamamoto, Janet K

    2015-01-01

    Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV+ groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2–4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy. PMID:25844718

  13. HIV-1 Vpr- and Reverse Transcription-Induced Apoptosis in Resting Peripheral Blood CD4 T Cells and Protection by Common Gamma-Chain Cytokines

    PubMed Central

    Trinité, Benjamin; Chan, Chi N.; Lee, Caroline S.

    2015-01-01

    ABSTRACT HIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms. In vivo, HIV-1 infects both activated and resting CD4 T cells, but in vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription (RT), resulting in abortive infection. Here, we show that in vitro HIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection. In vivo, however, various microenvironmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma-chain cytokines (CGCC), such as interleukin-7 (IL-7), render resting CD4 T cells permissible to HIV-1 infection without inducing T cell activation. Here, we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidenced by early release of cytochrome c and caspase 3/7 activation. Cell death is triggered both by products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1-induced cell death in this system. These results show that a major restriction on HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection. IMPORTANCE A major consequence of HIV-1 infection is the destruction of CD4 T cells. Here, we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in

  14. HIV-1 incidence and HIV-1 associated mortality in a cohort of urban factory workers in Tanzania.

    PubMed

    Borgdorff, M W; Barongo, L R; Klokke, A H; Newell, J N; Senkoro, K P; Velema, J P; Gabone, R M

    1995-08-01

    The objectives were to determine HIV-1 incidence and HIV-1 associated mortality in a prospective cohort study and to determine whether the cohort is suitable for studies attempting to determine the impact of interventions on HIV-1 incidence. The study population was a cohort of 2038 urban factory workers in northwest Tanzania of whom 1772 workers (1478 men or 87% and 294 women or 89%) had enrolled in the study during October 1991 to September 1993. 471 (27%) of the total study population were lost to follow-up by the end of the study period. Outcome measures were HIV-1 seroconversion and death. At intake, 153 of 1478 (10.4%) men and 52 of 294 (17.7%) women were infected with HIV-1. In the study period, 17 seroconversions took place in 1365.9 person years of follow-up giving an HIV-1 incidence rate of 1.2/100 person-years of follow-up. No association was found between seroconversion and age or sex. The crude annual mortality rate was 4.9/100 person-years in those with and 0.3/100 person-years in those without HIV-1 infection, giving an age- and sex-adjusted mortality ratio of 12.9. The age- and sex-adjusted population attributable risk was 0.5/100 person-years, and of all deaths, 62% were attributable to HIV-1 infection. Of the 14 HIV-1 infected people who died, 9 met the criteria of the 1987 revised Centers for Disease Control/World Health Organization AIDS case definition: one had cryptococcal meningitis and eight HIV wasting syndrome. Two others had had weight loss and fever, but the evidence was inadequate to make or reject the diagnosis of AIDS. The remaining three without an AIDS diagnosis had pulmonary tuberculosis, diarrhea, and pyomyositis, respectively. HIV-1 infection was a major cause of death in this adult population. At an HIV-1 incidence of 1.2/100 person-years, a large cohort size would be required to evaluate the impact of interventions on HIV-1 incidence. PMID:7590710

  15. Chemically Programmed Antibodies As HIV-1 Attachment Inhibitors

    PubMed Central

    2013-01-01

    Herein, we describe the design and application of two small-molecule anti-HIV compounds for the creation of chemically programmed antibodies. N-Acyl-β-lactam derivatives of two previously described molecules BMS-378806 and BMS-488043 that inhibit the interaction between HIV-1 gp120 and T-cells were synthesized and used to program the binding activity of aldolase antibody 38C2. Discovery of a successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1 gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel bispecific antibodies and topical microbicides. PMID:23750312

  16. Eradicating HIV-1 infection: seeking to clear a persistent pathogen

    PubMed Central

    Archin, Nancie M.; Sung, Julia Marsh; Garrido, Carolina; Soriano-Sarabia, Natalia; Margolis, David M.

    2015-01-01

    Effective antiretroviral therapy (ART) blunts viraemia, which enables HIV-1-infected individuals to control infection and live long, productive lives. However, HIV-1 infection remains incurable owing to the persistence of a viral reservoir that harbours integrated provirus within host cellular DNA. This latent infection is unaffected by ART and hidden from the immune system. Recent studies have focused on the development of therapies to disrupt latency. These efforts unmasked residual viral genomes and highlighted the need to enable the clearance of latently infected cells, perhaps via old and new strategies that improve the HIV-1-specific immune response. In this Review, we explore new approaches to eradicate established HIV-1 infection and avoid the burden of lifelong ART. PMID:25402363

  17. Evaluation of the Aptima(®) HIV-1 Quant Dx assay for HIV-1 RNA viral load detection and quantitation in plasma of HIV-1-infected individuals: A comparison with Abbott RealTime HIV-1 assay.

    PubMed

    Amendola, Alessandra; Pisciotta, Maria; Aleo, Loredana; Ferraioli, Valeria; Angeletti, Claudio; Capobianchi, Maria Rosaria

    2016-09-01

    The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P < 0.0001) and mean differences of measurement, conducted according to Bland-Altman method, was low (0.115 log10  copies/ml). The Aptima HIV quantified the WHO 3rd HIV-1 International Standard diluted from 2000 to 31 cp/ml (5,700-88 IU/ml) at expected values with excellent linearity (R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc. PMID:26864171

  18. High HIV-1 Genetic Diversity in Patients from Northern Brazil.

    PubMed

    da Costa, Carolina Marinho; Costa de Oliveira, Cintia Mara; Chehuan de Melo, Yonne Francis; Delatorre, Edson; Bello, Gonzalo; Couto-Fernandez, Jose Carlos

    2016-09-01

    The HIV-1 epidemic in Brazil is driven by subtypes B, F1, and C and recombinants forms among those subtypes. The distribution of HIV-1 subtypes, however, may vary across different Brazilian regions and the molecular epidemiologic profile in Northern Brazil remains poorly explored. HIV-1 pol sequences were obtained from 305 patients failing antiretroviral therapy followed at outpatient clinics from five Northern Brazilian states. The most prevalent HIV-1 clade observed in the Northern Brazilian region was subtype B (81%), followed by BF1 recombinants (10%), subtype F1 (4%), subtype C (3%), BC recombinants (2%), and BU recombinants (1%). Although HIV-1 subtype B was the predominant HIV-1 clade in Northern Brazil, its prevalence greatly varies among different states, ranging from 63% in Rondônia to 92% in Acre. Among the 37 HIV-1 recombinant sequences detected in the Northern Brazilian region, nine (24%) displayed a unique recombinant form structure, five (14%) a CRF28/29_BF-like structure, and four (11%) a CRF31_BC-like structure. Two other BF1 recombinant patterns were identified in 16 (43%) and three (8%) samples that may correspond to two potentially new CRFs_BF characteristic of the Northern region. This study reveals that despite the low spatial connectivity with other Brazilian regions, the genetic complexity of the HIV-1 epidemic in Northern Brazil is very high and that the molecular epidemiologic pattern may vary across different northern states, reflecting a complex epidemic with multiple independent viral introductions into this Brazilian region. PMID:27091699

  19. Imaging HIV-1 nuclear pre-integration complexes.

    PubMed

    Cereseto, Anna; Giacca, Mauro

    2014-01-01

    Advancements in fluorescent microscopy techniques now permit investigation of HIV-1 biology exploiting tools alternative to conventional molecular biology. Here we describe a novel, fluorescence-based method to visualize HIV-1 viral particles within intact nuclei of infected cells. This method allows investigating the localization of pre-integration complexes within the nuclear compartment with respect to the nuclear envelope and the chromatin territories. PMID:24158813

  20. Template-constrained cyclic sulfopeptide HIV-1 entry inhibitors†

    PubMed Central

    Rudick, Jonathan G.; Laakso, Meg M.; Schloss, Ashley C.; DeGrado, William F.

    2013-01-01

    Template-constrained cyclic sulfopeptides that inhibit HIV-1 entry were rationally designed based on a loop from monoclonal antibody (mAb) 412d. A focused set of sulfopeptides was synthesized using Fmoc-Tyr(SO3DCV)-OH (DCV = 2,2-dichlorovinyl). Three cyclic sulfopeptides that inhibit entry of HIV-1 and complement the activity of known CCR5 antagonists were identified. PMID:24065278

  1. Cell-free Assays for HIV-1 Uncoating

    PubMed Central

    Aiken, Christopher

    2013-01-01

    Summary/Abstract Uncoating is an essential step in the retrovirus life cycle about which little is known. Uncoating is defined as the specific dissociation of the capsid shell from the viral core in the host cell cytoplasm. In this chapter, biochemical assays for studying HIV-1 uncoating in vitro are described. These techniques have proven useful for characterizing HIV-1 mutants that exhibit defects in the uncoating step of infection. PMID:19020817

  2. Iron chelators ICL670 and 311 inhibit HIV-1 transcription.

    PubMed

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R; Ray, Patricio E; Gordeuk, Victor R; Nekhai, Sergei

    2007-10-25

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics. PMID:17631934

  3. HLA-C Downmodulation by HIV-1 Vpu.

    PubMed

    Barker, Edward; Evans, David T

    2016-05-11

    It is widely held that HIV-1 Nef downmodulates HLA-A and -B to protect infected cells from CD8(+) T cells but leaves HLA-C on the cell surface to inhibit NK cells. In this issue of Cell Host & Microbe, Apps et al. (2016) revise this model by showing that the Vpu protein of primary HIV-1 isolates downmodulate HLA-C. PMID:27173922

  4. Cell-free assays for HIV-1 uncoating.

    PubMed

    Aiken, Christopher

    2009-01-01

    Uncoating is an essential step in the retrovirus life cycle about which little is known. Uncoating is defined as the specific dissociation of the capsid shell from the viral core in the host cell cytoplasm. In this chapter, biochemical assays for studying HIV-1 uncoating in vitro are described. These techniques have proven useful for characterizing HIV-1 mutants that exhibit defects in the uncoating step of infection. PMID:19020817

  5. Evidence for Vpr-dependent HIV-1 Replication in Human CD4+ CEM.NKR T-Cells

    PubMed Central

    2012-01-01

    Background Vpr is exclusively expressed in primate lentiviruses and contributes to viral replication and disease progression in vivo. HIV-1 Vpr has two major activities in vitro: arrest of cell cycle in the G2 phase (G2 arrest), and enhancement of viral replication in macrophages. Previously, we reported a potent HIV-1 restriction in the human CD4+ CEM.NKR (NKR) T cells, where wild-type (WT) HIV-1 replication was inhibited by almost 1,000-fold. From the parental NKR cells, we isolated eight clones by limiting dilution. These clones showed three levels of resistance to the WT HIV-1 infection: non-permissive (NP), semi-permissive (SP), and permissive (P). Here, we compared the replication of WT, Vif-defective, Vpr-defective, and Vpu-defective viruses in these cells. Results Although both WT and Vpu-defective viruses could replicate in the permissive and semi-permissive clones, the replication of Vif-defective and Vpr-defective viruses was completely restricted. The expression of APOBEC3G (A3G) cytidine deaminase in NKR cells explains why Vif, but not Vpr, was required for HIV-1 replication. When the Vpr-defective virus life cycle was compared with the WT virus life cycle in the semi-permissive cells, it was found that the Vpr-defective virus could enter the cell and produce virions containing properly processed Gag and Env proteins, but these virions showed much less efficiency for reverse transcription during the next-round of infection. In addition, although viral replication was restricted in the non-permissive cells, treatment with arsenic trioxide (As2O3) could completely restore WT, but not Vpr-defective virus replication. Moreover, disruption of Vpr binding to its cofactor DCAF1 and/or induction of G2 arrest activity did not disrupt the Vpr activity in enhancing HIV-1 replication in NKR cells. Conclusions These results demonstrate that HIV-1 replication in NKR cells is Vpr-dependent. Vpr promotes HIV-1 replication from the 2nd cycle likely by overcoming a

  6. Phages and HIV-1: From Display to Interplay

    PubMed Central

    Delhalle, Sylvie; Schmit, Jean-Claude; Chevigné, Andy

    2012-01-01

    The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. PMID:22606007

  7. Prospects for a Globally Effective HIV-1 Vaccine.

    PubMed

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2015-12-01

    A globally effective vaccine strategy must cope with the broad genetic diversity of HIV and contend with multiple transmission modalities. Understanding correlates of protection and the role of diversity in limiting protective vaccines with those correlates is key. RV144 was the first HIV-1 vaccine trial to demonstrate efficacy against HIV-1 infection. A correlates analysis comparing vaccine-induced immune responses in vaccinated-infected and vaccinated-uninfected volunteers suggested that IgG specific for the V1V2 region of gp120 was associated with reduced risk of HIV-1 infection and that plasma Env IgA was directly correlated with infection risk. RV144 and recent non-human primate (NHP) challenge studies suggest that Env is essential and perhaps sufficient to induce protective antibody responses against mucosally acquired HIV-1. Whether RV144 immune correlates can apply to different HIV vaccines, to populations with different modes and intensity of transmission, or to divergent HIV-1 subtypes remains unknown. Newer prime-boost mosaic and conserved sequence immunization strategies aiming at inducing immune responses of greater breadth and depth as well as the development of immunogens inducing broadly neutralizing antibodies should be actively pursued. Efficacy trials are now planned in heterosexual populations in southern Africa and men who have sex with men in Thailand. Although NHP challenge studies may guide vaccine development, human efficacy trials remain key to answer the critical questions leading to the development of a global HIV-1 vaccine for licensure. PMID:26590431

  8. Prospects for a globally effective HIV-1 vaccine.

    PubMed

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2015-11-27

    A globally effective vaccine strategy must cope with the broad genetic diversity of HIV and contend with multiple transmission modalities. Understanding correlates of protection and the role of diversity in limiting protective vaccines with those correlates is key. RV144 was the first HIV-1 vaccine trial to demonstrate efficacy against HIV-1 infection. A correlates analysis compared vaccine-induced immune responses in vaccinated-infected and vaccinated-uninfected volunteers suggested that IgG specific for the V1V2 region of gp120 was associated with reduced risk of HIV-1 infection and that plasma Env IgA was directly correlated with infection risk. RV144 and recent NHP challenge studies suggest that Env is essential and perhaps sufficient to induce protective antibody responses against mucosally acquired HIV-1. Whether RV144 immune correlates can apply to different HIV vaccines, to populations with different modes and intensity of transmission, or to divergent HIV-1 subtypes remains unknown. Newer prime-boost mosaic and conserved sequence immunization strategies aiming at inducing immune responses of greater breadth and depth as well as the development of immunogens inducing broadly neutralizing antibodies should be actively pursued. Efficacy trials are now planned in heterosexual populations in southern Africa and MSM in Thailand. Although NHP challenge studies may guide vaccine development, human efficacy trials remain key to answer the critical questions leading to the development of a global HIV-1 vaccine for licensure. PMID:26100921

  9. HIV-1 increases TLR responses in human primary astrocytes

    PubMed Central

    Serramía, M Jesús; Muñoz-Fernández, M Ángeles; Álvarez, Susana

    2015-01-01

    Astrocytes are the major glial cell within the central nervous system and have a number of important physiological properties related to brain homeostasis. They provide trophic support to neurons and are immune cells with key roles during states-of-inflammation. The potential for production of proinflammatory cytokines and its consequences has been studied in the context of HIV-1 infection of normal human astrocytes (NHA). NHA express TLR3, TLR4, and TLR5. TLR3 ligation induced the strongest proinflammatory polarizing response, characterized by generation of high levels of TNF-α, IL-6, and IL-8. HIV-1 increased the transient production of key inflammatory mediators, and exposure to LPS of HIV-1-infected cells increased significantly the cytokine secretion. We confirmed that it is necessary viral gene expression from the moment of pretreatment with antiretrovirals inhibited totally HIV-1-induced TLR response. The higher response to LPS from HIV-1-infected cells did not correlate with TLR4 or MyD88 increased expression. LPS responsiveness of infected cells parallels MHC class II expression, but not CD14. HIV-1-infected NHA present increased sensitivity to the proinflammatory effects of LPS. If this phenomenon occurs in vivo, it will contribute to the immunopathogenesis of this disease and may ultimately offer novel targets for immunomodulatory therapy. PMID:26671458

  10. Engineering T Cells to Functionally Cure HIV-1 Infection

    PubMed Central

    Leibman, Rachel S; Riley, James L

    2015-01-01

    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. PMID:25896251

  11. 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

    2016-01-01

    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. PMID:27264714

  12. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  13. Viral piracy: HIV-1 targets dendritic cells for transmission.

    PubMed

    Lekkerkerker, Annemarie N; van Kooyk, Yvette; Geijtenbeek, Teunis B H

    2006-04-01

    Dendritic cells (DCs), the professional antigen presenting cells, are critical for host immunity by inducing specific immune responses against a broad variety of pathogens. Remarkably the human immunodeficiency virus-1 (HIV-1) subverts DC function leading to spread of the virus. At an early phase of HIV-1 transmission, DCs capture HIV-1 at mucosal surfaces and transmit the virus to T cells in secondary lymphoid tissues. Capture of the virus on DCs takes place via C-type lectins of which the dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN) is the best studied. DC-SIGN-captured HIV-1 particles accumulate in CD81(+) multivesicular bodies (MVBs) in DCs and are subsequently transmitted to CD4+ T cells resulting in infection of T cells. The viral cell-to-cell transmission takes place at the DC-T cell interface termed the infectious synapse. Recent studies demonstrate that direct infection of DCs contributes to the transmission to T cells at a later phase. Moreover, the infected DCs may function as cellular reservoirs for HIV-1. This review discusses the different processes that govern viral piracy of DCs by HIV-1, emphasizing the intracellular routing of the virus from capture on the cell surface to egress in the infectious synapse. PMID:16611055

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

    PubMed Central

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

    2016-01-01

    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. PMID:27264714

  15. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  16. HIV-1 Vaccine Trials: Evolving Concepts and Designs

    PubMed Central

    Sanou, Missa P; De Groot, Anne S; Murphey-Corb, Michael; Levy, Jay A; Yamamoto, Janet K

    2012-01-01

    An effective prophylactic HIV-1 vaccine is needed to eradicate the HIV/AIDS pandemic but designing such a vaccine is a challenge. Despite many advances in vaccine technology and approaches to generate both humoral and cellular immune responses, major phase-II and -III vaccine trials against HIV/AIDS have resulted in only moderate successes. The modest achievement of the phase-III RV144 prime-boost trial in Thailand re-emphasized the importance of generating robust humoral and cellular responses against HIV. While antibody-directed approaches are being pursued by some groups, others are attempting to develop vaccines targeting cell-mediated immunity, since evidence show CTLs to be important for the control of HIV replication. Phase-I and -IIa multi-epitope vaccine trials have already been conducted with vaccine immunogens consisting of known CTL epitopes conserved across HIV subtypes, but have so far fallen short of inducing robust and consistent anti-HIV CTL responses. The concepts leading to the development of T-cell epitope-based vaccines, the outcomes of related clinical vaccine trials and efforts to enhance the immunogenicity of cell-mediated approaches are summarized in this review. Moreover, we describe a novel approach based on the identification of SIV and FIV antigens which contain conserved HIV-specific T-cell epitopes and represent an alternative method for developing an effective HIV vaccine against global HIV isolates. PMID:23289052

  17. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  18. Adenosine deaminase regulates Treg expression in autologous T cell-dendritic cell cocultures from patients infected with HIV-1.

    PubMed

    Naval-Macabuhay, Isaac; Casanova, Víctor; Navarro, Gemma; García, Felipe; León, Agathe; Miralles, Laia; Rovira, Cristina; Martinez-Navio, José M; Gallart, Teresa; Mallol, Josefa; Gatell, José M; Lluís, Carme; Franco, Rafael; McCormick, Peter J; Climent, Núria

    2016-02-01

    Regulatory T cells have an important role in immune suppression during HIV-1 infection. As regulatory T cells produce the immunomodulatory molecule adenosine, our aim here was to assess the potential of adenosine removal to revert the suppression of anti-HIV responses exerted by regulatory T cells. The experimental setup consisted of ex vivo cocultures of T and dendritic cells, to which adenosine deaminase, an enzyme that hydrolyzes adenosine, was added. In cells from healthy individuals, adenosine hydrolysis decreased CD4(+)CD25(hi) regulatory T cells. Addition of 5'-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, significantly decreased CD4(+)CD25(lo) cells, confirming a modulatory role of adenosine acting via adenosine receptors. In autologous cocultures of T cells with HIV-1-pulsed dendritic cells, addition of adenosine deaminase led to a significant decrease of HIV-1-induced CD4(+)CD25(hi) forkhead box p3(+) cells and to a significant enhancement of the HIV-1-specific CD4(+) responder T cells. An increase in the effector response was confirmed by the enhanced production of CD4(+) and CD8(+) CD25(-)CD45RO(+) memory cell generation and secretion of Th1 cytokines, including IFN-γ and IL-15 and chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5. These ex vivo results show, in a physiologically relevant model, that adenosine deaminase is able to enhance HIV-1 effector responses markedly. The possibility to revert regulatory T cell-mediated inhibition of immune responses by use of adenosine deaminase, an enzyme that hydrolyzes adenosine, merits attention for restoring T lymphocyte function in HIV-1 infection. PMID:26310829

  19. A Nonfucosylated Variant of the anti-HIV-1 Monoclonal Antibody b12 Has Enhanced FcγRIIIa-Mediated Antiviral Activity In Vitro but Does Not Improve Protection against Mucosal SHIV Challenge in Macaques

    PubMed Central

    Moldt, Brian; Shibata-Koyama, Mami; Rakasz, Eva G.; Schultz, Niccole; Kanda, Yutaka; Dunlop, D. Cameron; Finstad, Samantha L.; Jin, Chenggang; Landucci, Gary; Alpert, Michael D.; Dugast, Anne-Sophie; Parren, Paul W. H. I.; Nimmerjahn, Falk; Evans, David T.; Alter, Galit; Forthal, Donald N.; Schmitz, Jörn E.; Iida, Shigeru; Poignard, Pascal; Watkins, David I.

    2012-01-01

    Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge. PMID:22457527

  20. Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs

    PubMed Central

    Malatinkova, Eva; Spiegelaere, Ward De; Bonczkowski, Pawel; Kiselinova, Maja; Vervisch, Karen; Trypsteen, Wim; Johnson, Margaret; Verhofstede, Chris; de Looze, Danny; Murray, Charles; Loes, Sabine Kinloch-de; Vandekerckhove, Linos

    2015-01-01

    Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. DOI: http://dx.doi.org/10.7554/eLife.09115.001 PMID:26439007

  1. Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs.

    PubMed

    Malatinkova, Eva; De Spiegelaere, Ward; Bonczkowski, Pawel; Kiselinova, Maja; Vervisch, Karen; Trypsteen, Wim; Johnson, Margaret; Verhofstede, Chris; de Looze, Danny; Murray, Charles; Kinloch-de Loes, Sabine; Vandekerckhove, Linos

    2015-01-01

    Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. PMID:26439007

  2. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  3. Design, synthesis and anti-HIV-1 evaluation of hydrazide-based peptidomimetics as selective gelatinase inhibitors.

    PubMed

    Yang, Liang; Wang, Ping; Wu, Ji-Feng; Yang, Liu-Meng; Wang, Rui-Rui; Pang, Wei; Li, Yong-Gang; Shen, Yue-Mao; Zheng, Yong-Tang; Li, Xun

    2016-05-01

    As our ongoing work on research of gelatinase inhibitors, an array of hydrazide-containing peptidomimetic derivatives bearing quinoxalinone as well as spiro-heterocyclic backbones were designed, synthesized, and assayed for their in vitro enzymatic inhibitory effects. The results demonstrated that both the quinoxalinone (series I and II) and 1,4-dithia-7-azaspiro[4,4]nonane-based hydrazide peptidomimetics (series III) displayed remarkably selectivity towards gelatinase A as compared to APN, with IC50 values in the micromole range. Structure-activity relationships were herein briefly discussed. Given evidences have validated that gelatinase inhibition may be contributable to the therapy of HIV-1 infection, all the target compounds were also submitted to the preliminary in vitro anti-HIV-1 evaluation. It resulted that gelatinase inhibition really has positive correlation with anti-HIV-1 activity, especially compounds 4m and 7h, which gave enhanced gelatinase inhibition in comparison with the positive control LY52, and also decent anti-HIV-1 potencies. The FlexX docking results provided a straightforward insight into the binding pattern between inhibitors and gelatinase, as well as the selective inhibition towards gelatinase over APN. Collectively, our research encouraged potent gelatinase inhibitors might be used in the development of anti-HIV-1 agents. And else, compounds 4m and 7h might be promising candidates to be considered for further chemical optimization. PMID:27039251

  4. Inhibiting early-stage events in HIV-1 replication by small-molecule targeting of the HIV-1 capsid.

    PubMed

    Kortagere, Sandhya; Madani, Navid; Mankowski, Marie K; Schön, Arne; Zentner, Isaac; Swaminathan, Gokul; Princiotto, Amy; Anthony, Kevin; Oza, Apara; Sierra, Luz-Jeannette; Passic, Shendra R; Wang, Xiaozhao; Jones, David M; Stavale, Eric; Krebs, Fred C; Martín-García, Julio; Freire, Ernesto; Ptak, Roger G; Sodroski, Joseph; Cocklin, Simon; Smith, Amos B

    2012-08-01

    The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4'-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity. PMID:22647699

  5. Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes

    PubMed Central

    Eger, Iriane; Soares, Maurilio José

    2015-01-01

    Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x107 amastigotes—with typical shape and positive for the amastigote marker Ssp4—from 5x106 infected Vero cells (48h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes. PMID:26057131

  6. Innate Invariant NKT Cell Recognition of HIV-1–Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion

    PubMed Central

    Paquin-Proulx, Dominic; Gibbs, Anna; Bächle, Susanna M.; Checa, Antonio; Introini, Andrea; Leeansyah, Edwin; Wheelock, Craig E.; Nixon, Douglas F.; Broliden, Kristina; Tjernlund, Annelie; Moll, Markus

    2016-01-01

    Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms. PMID:27481843

  7. Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion.

    PubMed

    Paquin-Proulx, Dominic; Gibbs, Anna; Bächle, Susanna M; Checa, Antonio; Introini, Andrea; Leeansyah, Edwin; Wheelock, Craig E; Nixon, Douglas F; Broliden, Kristina; Tjernlund, Annelie; Moll, Markus; Sandberg, Johan K

    2016-09-01

    Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell-mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms. PMID:27481843

  8. CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs.

    PubMed

    Zhang, Yonggang; Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Wensheng; Kaminski, Rafal; Fagan, Philip Regis; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui

    2015-01-01

    Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a "shock and kill" strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs. PMID:26538064

  9. HIV-1 Infection-Induced Suppression of the Let-7i/IL-2 Axis Contributes to CD4+ T Cell Death

    PubMed Central

    Zhang, Yijun; Yin, Yue; Zhang, Shaoying; Luo, Haihua; Zhang, Hui

    2016-01-01

    The mechanisms underlying HIV-1-mediated CD4+ T cell depletion are highly complicated. Interleukin-2 (IL-2) is a key cytokine that maintains the survival and proliferation of activated CD4+ T cells. IL-2 levels are disturbed during HIV-1 infection, but the underlying mechanism(s) requires further investigation. We have reported that cellular microRNA (miRNA) let-7i upregulates IL-2 expression by targeting the promoter TATA-box region, which functions as a positive regulator. In this study, we found that HIV-1 infection decreases the expression of let-7i in CD4+ T cells by attenuating its promoter activity. The reduced let-7i miRNA expression led to a decline in IL-2 levels. A let-7i mimic increased IL-2 expression and subsequently enhanced the resistance of CD4+ T cells to HIV-1-induced apoptosis. By contrast, the blockage of let-7i with a specific inhibitor resulted in elevated CD4+ T cell apoptosis during HIV-1 infection. Furthermore, by knocking down the expression of IL-2, we found that the let-7i-mediated CD4+ T cell resistance to apoptosis during HIV-1 infection was dependent on IL-2 signaling rather than an alternative CD95-mediated cell-death pathway. Taken together, our findings reveal a novel pathway for HIV-1-induced dysregulation of IL-2 cytokines and depletion of CD4+ T-lymphocytes. PMID:27145859

  10. CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

    PubMed Central

    Zhang, Yonggang; Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Wensheng; Kaminski, Rafal; Fagan, Philip Regis; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui

    2015-01-01

    Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a “shock and kill” strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs. PMID:26538064

  11. Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4{sup +} T cell proliferation

    SciTech Connect

    Liu, Wan; Qin, Yan; Bai, Lei; Lan, Ke; Wang, Jian-Hua

    2013-06-05

    Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4{sup +} T cells.

  12. Clinical performance of the Multispot HIV-1/HIV-2 rapid test to correctly differentiate HIV-2 from HIV-1 infection in screening algorithms using third and fourth generation assays and to identify cross reactivity with the HIV-1 Western Blot

    PubMed Central

    Ramos, Eric M.; Harb, Socorro; Dragavon, Joan; Coombs, Robert W.

    2014-01-01

    Background An accurate and rapid serologic method to differentiate HIV-2 from HIV-1 infection is required since the confirmatory HIV-1 Western Blot (WB) may demonstrate cross-reactivity with HIV-2 antibodies. Objectives To evaluate the performance of the Bio-Rad Multispot HIV-1/HIV-2 rapid assay as a supplemental test to correctly identify HIV-2 infection and identify HIV-1 WB cross-reactivity with HIV-2 in clinical samples tested at an academic medical center. Study design Between August 2008 and July 2012, clinical samples were screened for HIV using either 3rd-or 4th-generation HIV-1/2 antibody or combination antibody and HIV-1 p24 antigen assays, respectively. All repeatedly reactive samples were reflexed for Multispot rapid testing. Multispot HIV-2 and HIV-1 and HIV-2-reactive samples were further tested using an HIV-2 immunoblot assay and HIV-1 or HIV-2 RNA assays when possible. The HIV-1 WB was performed routinely for additional confirmation and to assess for HIV-2 antibody cross-reactivity. Results Of 46,061 samples screened, 890 (89.6%) of 993 repeatedly reactive samples were also Multispot-reactive: 882 for HIV-1; three for only HIV-2; and five for both HIV-1 and HIV-2. All three HIV-2-only Multispot-positives along with a single dually reactive HIV-1/2 Multispot-positive were also HIV-2 immunoblot-positive; the latter was HIV-1 RNA negative and HIV-2 RNA positive. Conclusions The Multispot rapid test performed well as a supplemental test for HIV-1/2 diagnostic testing. Four new HIV-2 infections (0.45%) were identified from among 890 Multispot-reactive tests. The use of HIV-1 WB alone to confirm HIV-1/2 screening assays may underestimate the true prevalence of HIV-2 infection in the United States. PMID:24342468

  13. 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

    SciTech Connect

    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

    2015-09-15

    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. - 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.

  14. Increase of HIV-1 subtype A in Central African Republic.

    PubMed

    Müller-Trutwin, M C; Chaix, M L; Letourneur, F; Bégaud, E; Beaumont, D; Deslandres, A; You, B; Morvan, J; Mathiot, C; Barré-Sinoussi, F; Saragosti, S

    1999-06-01

    The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants. PMID:10360809

  15. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

    PubMed Central

    Benko, Zsigmond; Elder, Robert T.; Li, Ge; Liang, Dong; Zhao, Richard Y.

    2016-01-01

    Background 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. Results 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. Conclusions 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. PMID:26982200

  16. Impact of HIV-1 Membrane Cholesterol on Cell-Independent Lytic Inactivation and Cellular Infectivity.

    PubMed

    Kalyana Sundaram, Ramalingam Venkat; Li, Huiyuan; Bailey, Lauren; Rashad, Adel A; Aneja, Rachna; Weiss, Karl; Huynh, James; Bastian, Arangaserry Rosemary; Papazoglou, Elisabeth; Abrams, Cameron; Wrenn, Steven; Chaiken, Irwin

    2016-01-26

    Peptide triazole thiols (PTTs) have been found previously to bind to HIV-1 Env spike gp120 and cause irreversible virus inactivation by shedding gp120 and lytically releasing luminal capsid protein p24. Since the virions remain visually intact, lysis appears to occur via limited membrane destabilization. To better understand the PTT-triggered membrane transformation involved, we investigated the role of envelope cholesterol on p24 release by measuring the effect of cholesterol depletion using methyl beta-cyclodextrin (MβCD). An unexpected bell-shaped response of PTT-induced lysis to [MβCD] was observed, involving lysis enhancement at low [MβCD] vs loss of function at high [MβCD]. The impact of cholesterol depletion on PTT-induced lysis was reversed by adding exogenous cholesterol and other sterols that support membrane rafts, while sterols that do not support rafts induced only limited reversal. Cholesterol depletion appears to cause a reduced energy barrier to lysis as judged by decreased temperature dependence with MβCD. Enhancement/replenishment responses to [MβCD] also were observed for HIV-1 infectivity, consistent with a similar energy barrier effect in the membrane transformation of virus cell fusion. Overall, the results argue that cholesterol in the HIV-1 envelope is important for balancing virus stability and membrane transformation, and that partial depletion, while increasing infectivity, also makes the virus more fragile. The results also reinforce the argument that the lytic inactivation and infectivity processes are mechanistically related and that membrane transformations occurring during lysis can provide an experimental window to investigate membrane and protein factors important for HIV-1 cell entry. PMID:26713837

  17. [Sensitivity of the COBAS AmpliScreen™ HIV-1 test v1.5 for HIV-1 detection].

    PubMed

    Gomez, Lucía P; Balangero, Marcos C; Castro, Gonzalo; Kademian, Silvia; Mangeaud, Arnaldo; Barbas, María G; Cudolá, Analía; de León, Juan F; Carrizo, Horacio; Gallego, Sandra V

    2014-01-01

    The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants. PMID:25444127

  18. Regulatory Variation in HIV-1 Dependency Factor ZNRD1 Associates with Host Resistance to HIV-1 Acquisition

    PubMed Central

    An, Ping; Goedert, James J.; Donfield, Sharyne; Buchbinder, Susan; Kirk, Gregory D.; Detels, Roger; Winkler, Cheryl A.

    2014-01-01

    Background. ZNRD1 was identified as a host protein required for the completion of the human immunodeficiency virus (HIV) lifecycle in a genome-wide screen using small interfering RNA gene silencing. Subsequently, a genome-wide association study (GWAS) of host determinants for HIV-1 disease identified an association of single nucleotide polymorphisms (SNPs) in the ZNRD1 region with CD4+ T-cell depletion. Methods. We investigated the effects of SNPs in the ZNRD1 region on human immunodeficiency virus type 1 (HIV-1) infection and progression to clinical outcomes in 5 US-based HIV-1 longitudinal cohorts consisting of men who have sex with men, males with hemophilia, and injection drug users (IDUs) (n = 1865). SNP function was evaluated by electrophoretic mobility shift assay and promoter luciferase assay. Results. A haplotype in the ZNRD1 gene showed significant association with a 35% decreased risk of HIV-1 acquisition (OR = 0.65, 95% CI, .47–.89), independent of HLA-C rs9264942, in European Americans. The SNP rs3132130 tagging this haplotype, located in the ZNRD1 5′ upstream region, caused a loss of nuclear factor binding and decrease in ZNRD1 promoter activity. ZNRD1 variants also affected HIV-1 disease progression in European- and African-American cohorts. Conclusions. This study provides novel evidence that ZNRD1 polymorphism may confer host resistance to HIV-1 acquisition. PMID:24842830

  19. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible and inhibits HIV-1 infectivity

    PubMed Central

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S.; Morris, Kevin V.; Burnett, John; Rossi, John

    2015-01-01

    SUMMARY The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T-cells and macrophages that serves as a co-receptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here we combine the live cell-based SELEX with high throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as siRNA delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5 expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4+ T cells with a nanomolar IC50. G-3 was also capable of transferring functional siRNAs to CCR5 expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties. PMID:25754473

  20. ZNF10 inhibits HIV-1 LTR activity through interaction with NF-κB and Sp1 binding motifs.

    PubMed

    Nishitsuji, Hironori; Sawada, Leila; Sugiyama, Ryuichi; Takaku, Hiroshi

    2015-07-01

    Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed ⩾50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense. PMID:26096782

  1. The strength of the HIV-1 3' splice sites affects Rev function

    PubMed Central

    Kammler, Susanne; Otte, Marianne; Hauber, Ilona; Kjems, Jørgen; Hauber, Joachim; Schaal, Heiner

    2006-01-01

    Background The HIV-1 Rev protein is a key component in the early to late switch in HIV-1 splicing from early intronless (e.g. tat, rev) to late intron-containing Rev-dependent (e.g. gag, vif, env) transcripts. Previous results suggested that cis-acting sequences and inefficient 5' and 3' splice sites are a prerequisite for Rev function. However, we and other groups have shown that two of the HIV-1 5' splice sites, D1 and D4, are efficiently used in vitro and in vivo. Here, we focus on the efficiency of the HIV-1 3' splice sites taking into consideration to what extent their intrinsic efficiencies are modulated by their downstream cis-acting exonic sequences. Furthermore, we delineate their role in RNA stabilization and Rev function. Results In the presence of an efficient upstream 5' splice site the integrity of the 3' splice site is not essential for Rev function whereas an efficient 3' splice site impairs Rev function. The detrimental effect of a strong 3' splice site on the amount of Rev-dependent intron-containing HIV-1 glycoprotein coding (env) mRNA is not compensatable by weakening the strength of the upstream 5' splice site. Swapping the HIV-1 3' splice sites in an RRE-containing minigene, we found a 3' splice site usage which was variably dependent on the presence of the usual downstream exonic sequence. The most evident activation of 3' splice site usage by its usual downstream exonic sequence was observed for 3' splice site A1 which was turned from an intrinsic very weak 3' splice site into the most active 3' splice site, even abolishing Rev activity. Performing pull-down experiments with nuclear extracts of HeLa cells we identified a novel ASF/SF2-dependent exonic splicing enhancer (ESE) within HIV-1 exon 2 consisting of a heptameric sequence motif occurring twice (M1 and M2) within this short non-coding leader exon. Single point mutation of M1 within an infectious molecular clone is detrimental for HIV-1 exon 2 recognition without affecting Rev

  2. HIV-1, methamphetamine and astrocytes at neuroinflammatory Crossroads

    PubMed Central

    Borgmann, Kathleen; Ghorpade, Anuja

    2015-01-01

    As a popular psychostimulant, methamphetamine (METH) use leads to long-lasting, strong euphoric effects. While METH abuse is common in the general population, between 10 and 15% of human immunodeficiency virus-1 (HIV-1) patients report having abused METH. METH exacerbates the severity and onset of HIV-1-associated neurocognitive disorders (HAND) through direct and indirect mechanisms. Repetitive METH use impedes adherence to antiretroviral drug regimens, increasing the likelihood of HIV-1 disease progression toward AIDS. METH exposure also directly affects both innate and adaptive immunity, altering lymphocyte numbers and activity, cytokine signaling, phagocytic function and infiltration through the blood brain barrier. Further, METH triggers the dopamine reward pathway and leads to impaired neuronal activity and direct toxicity. Concurrently, METH and HIV-1 alter the neuroimmune balance and induce neuroinflammation, which modulates a wide range of brain functions including neuronal signaling and activity, glial activation, viral infection, oxidative stress, and excitotoxicity. Pathologically, reactive gliosis is a hallmark of both HIV-1- and METH-associated neuroinflammation. Significant commonality exists in the neurotoxic mechanisms for both METH and HAND; however, the pathways dysregulated in astroglia during METH exposure are less clear. Thus, this review highlights alterations in astrocyte intracellular signaling pathways, gene expression and function during METH and HIV-1 comorbidity, with special emphasis on HAND-associated neuroinflammation. Importantly, this review carefully evaluates interventions targeting astrocytes in HAND and METH as potential novel therapeutic approaches. This comprehensive overview indicates, without a doubt, that during HIV-1 infection and METH abuse, a complex dialog between all neural cells is orchestrated through astrocyte regulated neuroinflammation. PMID:26579077

  3. HIV-1 evades innate immune recognition through specific cofactor recruitment

    NASA Astrophysics Data System (ADS)

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

    2013-11-01

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

  4. Identification of primary HIV-1C infection in Botswana.

    PubMed

    Novitsky, V; Woldegabriel, E; Wester, C; McDonald, E; Rossenkhan, R; Ketunuti, M; Makhema, J; Seage, G R; Essex, M

    2008-08-01

    Methods for identification of primary HIV infections seem increasingly important to understand pathogenesis, and to prevent transmission, which is particularly efficient during acute infection. Most current algorithms for HIV testing are based on detection of HIV antibodies and are unable to identify early infections before seroconversion. The efficiency of prospective cohorts, which is a standard approach for identifying primary HIV-1 infection, depends on a variety of epidemiological and cultural factors including HIV incidence and stigma and, not surprisingly, varies significantly in different geographical areas. We report a voluntary counseling and testing (VCT)-based approach to identifying primary HIV-1C infection that was developed as part of a primary HIV-1 subtype C infection study in Botswana. The referral strategy was based on: (1) collaboration with VCT centers at city clinics operated by the Ministry of Health; (2) partnering with the busiest non-government VCT center; (3) educating healthcare workers and the community about primary HIV infection; and (4) pairing with diverse VCT providers, including NGOs and private-sector organizations. Acute HIV-1 infections were defined by a negative HIV-1 serology combined with a positive HIV-1 RT-PCR test. Recent HIV-1 infections were identified by detuned EIA testing according to the classic STARTH algorithm. The VCT-based referral strategy resulted in the successful identification of 57 cases of acute and early HIV infection. A referral strategy of expanded VCT with viral RNA (Ribonucleic acid) testing to a national program in Botswana may be a promising approach for identification of primary HIV infections on a countrywide level. The program should offer VCT with viral RNA testing to the general public, facilitate proper counseling and risk reduction, and allow initiation of early HAART, and may reduce new viral transmissions. PMID:18608056

  5. HIV-1 evades innate immune recognition through specific cofactor recruitment.

    PubMed

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J; Price, Amanda J; Blondeau, Caroline; Hilditch, Laura; Jacques, David A; Selwood, David L; James, Leo C; Noursadeghi, Mahdad; Towers, Greg J

    2013-11-21

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages. PMID:24196705

  6. HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    PubMed Central

    Mesquita, Milene; Fintelman-Rodrigues, Natalia; Sacramento, Carolina Q.; Abrantes, Juliana L.; Costa, Eduardo; Temerozo, Jairo R.; Siqueira, Marilda M.; Bou-Habib, Dumith Chequer; Souza, Thiago Moreno L.

    2014-01-01

    HIV-1-infected patients co-infected with A(H1N1)pdm09 surprisingly presented benign clinical outcome. The knowledge that HIV-1 changes the host homeostatic equilibrium, which may favor the patient resistance to some co-pathogens, prompted us to investigate whether HIV-1 infection could influence A(H1N1)pdm09 life cycle in vitro. We show here that exposure of A(H1N1)pdm09-infected epithelial cells to HIV-1 viral particles or its gp120 enhanced by 25% the IFITM3 content, resulting in a decrease in influenza replication. This event was dependent on toll-like receptor 2 and 4. Moreover, knockdown of IFITM3 prevented HIV-1 ability to inhibit A(H1N1)pdm09 replication. HIV-1 infection also increased IFITM3 levels in human primary macrophages by almost 100%. Consequently, the arrival of influenza ribonucleoproteins (RNPs) to nucleus of macrophages was inhibited, as evaluated by different approaches. Reduction of influenza RNPs entry into the nucleus tolled A(H1N1)pdm09 life cycle in macrophages earlier than usual, limiting influenza's ability to induce TNF-α. As judged by analysis of the influenza hemagglutin (HA) gene from in vitro experiments and from samples of HIV-1/A(H1N1)pdm09 co-infected individuals, the HIV-1-induced reduction of influenza replication resulted in delayed viral evolution. Our results may provide insights on the mechanisms that may have attenuated the clinical course of Influenza in HIV-1/A(H1N1)pdm09 co-infected patients during the recent influenza form 2009/2010. PMID:24978204

  7. Large-scale analysis of the prevalence and geographic distribution of HIV-1 non-B variants in the United States.

    PubMed

    Pyne, Michael T; Hackett, John; Holzmayer, Vera; Hillyard, David R

    2013-08-01

    The genetic diversity of human immunodeficiency virus type 1 (HIV-1) has significant implications for diagnosis, vaccine development, and clinical management of patients. Although HIV-1 subtype B is predominant in the United States, factors such as global travel, immigration, and military deployment have the potential to increase the proportion of non-subtype B infections. Limited data are available on the prevalence and distribution of non-B HIV-1 strains in the United States. We sought to retrospectively examine the prevalence, geographic distribution, diversity, and temporal trends of HIV-1 non-B infections in samples obtained by ARUP Laboratories, a national reference laboratory, from all regions of the United States. HIV-1 pol sequences from 24,386 specimens collected from 46 states between 2004 and September 2011 for drug resistance genotyping were analyzed using the REGA HIV-1 Subtyping Tool, version 2.0. Sequences refractory to subtype determination or reported as non-subtype B by this tool were analyzed by PHYLIP version 3.5 and Simplot version 3.5.1. Non-subtype B strains accounted for 3.27% (798/24,386) of specimens. The 798 non-B specimens were received from 37 states and included 5 subtypes, 23 different circulating recombinant forms (CRFs), and 39 unique recombinant forms (URFs). The non-subtype B prevalence varied from 0% in 2004 (0/54) to 4.12% in 2011 (201/4,884). This large-scale analysis reveals that the diversity of HIV-1 in the United States is high, with multiple subtypes, CRFs, and URFs circulating. Moreover, the geographic distribution of non-B variants is widespread. Data from HIV-1 drug resistance testing have the potential to significantly enhance the surveillance of HIV-1 variants in the United States. PMID:23761148

  8. Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.

    PubMed

    Schweitzer, Cameron J; Matthews, John M; Madson, Christian J; Donnellan, Meghan R; Cerny, Ronald L; Belshan, Michael

    2012-01-01

    HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more

  9. Diversity of in-vivo assembled HIV-1 capsids

    NASA Astrophysics Data System (ADS)

    Lee, Se Il; Nguyen, Toan

    2008-03-01

    Understanding the capsid assembly process of Human Immunodeficiency Virus (HIV), the causative agent of Acute Immuno Deficiency Syndrom (AIDS), is very important because of recent intense interest in capsid-oriented viral therapy. The unique conical shapes of mature HIV-1 capsid have drawn significant interests in the biological community and started to attract attention from the physics community. Previous studies showed that in a free assembly process, the HIV-1 conical shape is not thermodynamically stable. However, if the volume of the capsid is constrained during assembly and the capsid protein shell has high spontaneous curvature, the conical shape is stable. In this work, we focus on in-vivo HIV-1 capsid assembly. For this case, the viral envelope membrane present during assembly imposes constraint on the length of the capsid. We use an elastic continuum shell theory to approximate the energies of various HIV-1 capsid shapes (spherical, cylindrical and conical). We show that for certain range of viral membrane diameter, the conical and cylindrical shapes are both thermodynamically stable. This result is supported by experimental observation that in-vivo assembled HIV-1 capsids are very heterogeneous in shapes and sizes. Numerical calculation is also performed to improve theoretical approximation.

  10. Structure and immune recognition of trimeric prefusion HIV-1 Env

    PubMed Central

    Pancera, Marie; Zhou, Tongqing; Druz, Aliaksandr; Georgiev, Ivelin S.; Soto, Cinque; Gorman, Jason; Huang, Jinghe; Acharya, Priyamvada; Chuang, Gwo-Yu; Ofek, Gilad; Stewart-Jones, Guillaume B. E.; Stuckey, Jonathan; Bailer, Robert T.; Joyce, M. Gordon; Louder, Mark K.; Tumba, Nancy; Yang, Yongping; Zhang, Baoshan; Cohen, Myron S.; Haynes, Barton F.; Mascola, John R.; Morris, Lynn; Munro, James B.; Blanchard, Scott C.; Mothes, Walther; Connors, Mark; Kwong, Peter D.

    2015-01-01

    The HIV-1-envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a postfusion state. As the sole viral antigen on the HIV-1-virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5-Å resolution for an HIV-1-Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the prefusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Prefusion gp41 encircles N- and C-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry likely involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the prefusion closed spike: we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation. PMID:25296255

  11. HLA-C and HIV-1: friends or foes?

    PubMed Central

    2012-01-01

    The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins. PMID:22571741

  12. Antiretroviral Therapy and Central Nervous System HIV-1 Infection

    PubMed Central

    Price, Richard W.; Spudich, Serena

    2008-01-01

    Central nervous system (CNS) HIV-1 infection begins during primary viremia and continues throughout the course of untreated systemic infection. While frequently accompanied by local inflammatory reactions detectable in cerebrospinal fluid (CSF), CNS HIV-1 infection is not usually clinically apparent. In a minority of patients, CNS HIV-1 infection evolves late in the course of systemic infection into encephalitis, which compromises brain function and presents clinically as AIDS dementia complex (ADC). Combination highly active antiretroviral therapy (HAART) has had a major impact on all aspects of HIV-1 CNS infection and disease. In those with asymptomatic infection, HAART usually effectively suppresses CSF HIV-1 and markedly reduces the incidence of symptomatic ADC. In those presenting with ADC, HAART characteristically prevents neurological progression and leads to variable, and at times substantial, recovery. Treatment has similarly reduced CNS opportunistic infections. With better control of these severe disorders, attention has turned to the possible consequences of chronic silent infection, and the issue of whether indolent, low-grade brain injury might require earlier treatment intervention. PMID:18447615

  13. Characterization of HIV-1 Resistance to Tenofovir Alafenamide In Vitro.

    PubMed

    Margot, Nicolas A; Johnson, Audun; Miller, Michael D; Callebaut, Christian

    2015-10-01

    Tenofovir alafenamide (TAF) is an investigational prodrug of the HIV-1 nucleotide reverse transcriptase (RT) inhibitor (NtRTI) tenofovir (TFV), with improved potency and drug delivery properties over the current prodrug, tenofovir disoproxil fumarate (TDF). TAF is currently in phase 3 clinical studies for the treatment of HIV-1 infection, in combination with other antiretroviral agents. Phase 1 and 2 studies have shown that TAF was associated with increased peripheral blood mononuclear cell (PBMC) drug loading and increased suppression of HIV-1 replication compared to treatment with TDF. In this study, selection of in vitro resistance to both TAF and the parent compound, TFV, led to the emergence of HIV-1 with the K65R amino acid substitution in RT with 6.5-fold-reduced susceptibility to TAF. Although TAF is more potent than TFV in vitro, the antiviral susceptibilities to TAF and TFV of a large panel of nucleoside/nucleotide RT inhibitor (NRTI)-resistant mutants were highly correlated (R(2) = 0.97), indicating that the two compounds have virtually the same resistance profile when assessed as fold change from the wild type. TAF showed full antiviral activity in PBMCs against primary HIV-1 isolates with protease inhibitor, nonnucleoside RT inhibitor (NNRTI), or integrase strand transfer inhibitor resistance but reduced activity against isolates with extensive NRTI resistance amino acid substitutions. However, the increased cell loading of TFV with TAF versus TDF observed in vivo suggests that TAF may retain activity against TDF-resistant mutant viruses. PMID:26149983

  14. The triple threat of HIV-1 protease inhibitors.

    PubMed

    Potempa, Marc; Lee, Sook-Kyung; Wolfenden, Richard; Swanstrom, Ronald

    2015-01-01

    Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy. PMID:25778681

  15. The HIV-1 transgenic rat model of neuroHIV

    PubMed Central

    Vigorito, Michael; Connaghan, Kaitlyn P.; Chang, Sulie L.

    2016-01-01

    Despite the ability of current combination anti-retroviral therapy (cART) to limit the progression of HIV-1 to AIDS, HIV-positive individuals continue to experience neuroHIV in the form of HIV-associated neurological disorders (HAND), which can range from subtle to substantial neurocognitive impairment. NeuroHIV may also influence substance use, abuse, and dependence in HIV-positive individuals. Because of the nature of the virus, variables such as mental health co-morbidities make it difficult to study the interaction between HIV and substance abuse in human populations. Several rodent models have been developed in an attempt to study the transmission and pathogenesis of the HIV-1 virus. The HIV-1 transgenic (HIV-1Tg) rat is a reliable model of neuroHIV because it mimics the condition of HIV-infected patients on cART. Research using this model supports the hypothesis that the presence of HIV-1 viral proteins in the central nervous system increases the sensitivity and susceptibility of HIV-positive individuals to substance abuse. PMID:25733103

  16. HIV-1 subtype B: Traces of a pandemic.

    PubMed

    Junqueira, Dennis Maletich; Almeida, Sabrina Esteves de Matos

    2016-08-01

    Human migration is a major process that shaped the origin and dissemination of HIV. Within HIV-1, subtype B (HIV-1B) is the most disseminated variant and it is assumed to be the causative agent in approximately 11% of all cases of HIV worldwide. Phylogenetic studies have revealed that HIV-1B emerged in Kinshasa (Africa) and was introduced into the Caribbean region via Haiti in or around 1966 by human migration. After localized dispersion, the virus was brought to the United States of America via homosexual/bisexual contact around 1969. Inside USA, the incidence of HIV-1B infection increased exponentially and it became established in the population, affecting not only homosexual individuals but also heterosexual individuals and injecting drug users. Soon after, the virus was disseminated and became established in other regions, including Europe, Asia, Latin America, and Australia. Recent studies suggest that, in addition to this pandemic clade, several lineages have emerged from Haiti and reached other Caribbean and Latin American countries via short-distance dissemination. Different subtype B genetic variants have also been detected in these epidemics. Four genetic variants have been described to date: subtype B', which mainly circulates in Thailand and other Asian countries; a specific variant mainly found in Trinidad and Tobago; the GPGS variant, which is primarily detected in Korea; and the GWGR variant, which is mainly detected in Brazil. This paper reviews the evolution of HIV-1B and its impact on the human population. PMID:27228177

  17. Role and modulation of drug transporters in HIV-1 therapy.

    PubMed

    Alam, Camille; Whyte-Allman, Sana-Kay; Omeragic, Amila; Bendayan, Reina

    2016-08-01

    Current treatment of human immunodeficiency virus type-1 (HIV-1) infection involves a combination of antiretroviral drugs (ARVs) that target different stages of the HIV-1 life cycle. This strategy is commonly referred to as highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART). Membrane-associated drug transporters expressed ubiquitously in mammalian systems play a crucial role in modulating ARV disposition during HIV-1 infection. Members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter superfamilies have been shown to interact with ARVs, including those that are used as part of first-line treatment regimens. As a result, the functional expression of drug transporters can influence the distribution of ARVs at specific sites of infection. In addition, pathological factors related to HIV</