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Sample records for hiv-1 vif interacts

  1. Structural Insights into HIV-1 Vif-APOBEC3F Interaction

    PubMed Central

    Nakashima, Masaaki; Ode, Hirotaka; Kawamura, Takashi; Kitamura, Shingo; Naganawa, Yuriko; Awazu, Hiroaki; Tsuzuki, Shinya; Matsuoka, Kazuhiro; Nemoto, Michiko; Hachiya, Atsuko; Sugiura, Wataru; Yokomaku, Yoshiyuki; Watanabe, Nobuhisa

    2015-01-01

    ABSTRACT The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCE HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction

  2. Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis

    PubMed Central

    Lavens, Delphine; Peelman, Frank; Van der Heyden, José; Uyttendaele, Isabel; Catteeuw, Dominiek; Verhee, Annick; Van Schoubroeck, Bertrand; Kurth, Julia; Hallenberger, Sabine; Clayton, Reginald; Tavernier, Jan

    2010-01-01

    The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G–Apobec3G and the Apobec3G–Vif interactions, the mammalian protein–protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G–Apobec3G interaction. All mutations that inhibit Apobec3G–Apobec3G binding also inhibit the Apobec3G–Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G–Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer. PMID:20015971

  3. Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis.

    PubMed

    Lavens, Delphine; Peelman, Frank; Van der Heyden, José; Uyttendaele, Isabel; Catteeuw, Dominiek; Verhee, Annick; Van Schoubroeck, Bertrand; Kurth, Julia; Hallenberger, Sabine; Clayton, Reginald; Tavernier, Jan

    2010-04-01

    The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G-Apobec3G and the Apobec3G-Vif interactions, the mammalian protein-protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G-Apobec3G interaction. All mutations that inhibit Apobec3G-Apobec3G binding also inhibit the Apobec3G-Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G-Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer. PMID:20015971

  4. The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex.

    PubMed

    Bergeron, Julien R C; Huthoff, Hendrik; Veselkov, Dennis A; Beavil, Rebecca L; Simpson, Peter J; Matthews, Stephen J; Malim, Michael H; Sanderson, Mark R

    2010-01-01

    The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.

  5. Molecular Characterization of Mexican HIV-1 Vif Sequences.

    PubMed

    Guerra-Palomares, Sandra E; Hernandez-Sanchez, Pedro G; Esparza-Perez, Mario A; Arguello, J Rafael; Noyola, Daniel E; Garcia-Sepulveda, Christian A

    2016-03-01

    The viral infectivity factor (Vif) is an HIV accessory protein that counteracts host antiviral proteins of the APOBEC3 family. Accumulating evidence highlights the pivotal role that accessory HIV proteins have on disease pathogenesis, a fact that has made them targets of interest for novel therapeutic and preventive strategies. Little is known about Vif sequence diversity outside of African or white populations. Mexico is home to Americas' third largest HIV-affected population and Mexican Hispanics represent an ever-increasing U.S. minority. This study provides a detailed analysis of the diversity seen in 77 Mexican Vif protein sequences. Phylogenetic analysis shows that most sequences cluster with HIV-1 subtype B, while less than 10% exhibit greater similarity to subtype D and A subtypes. Although most functional motifs are conserved among the Mexican sequences, substantial diversity was seen in some APOBEC binding sites, the nuclear localization inhibitory signal, and the CBFβ interaction sites.

  6. Determinants of Efficient Degradation of APOBEC3 Restriction Factors by HIV-1 Vif

    PubMed Central

    Baig, Tayyba T.; Feng, Yuqing

    2014-01-01

    ABSTRACT The APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces 48K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in 63K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation. IMPORTANCE APOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used

  7. Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

    PubMed

    Freisz, Séverine; Mezher, Joelle; Hafirassou, Lamine; Wolff, Philippe; Nominé, Yves; Romier, Christophe; Dumas, Philippe; Ennifar, Eric

    2012-07-01

    The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

  8. Evolution of HIV-1 Isolates that Use a Novel Vif-independent Mechanism to Resist Restriction by Human APOBEC3G

    PubMed Central

    Haché, Guylaine; Shindo, Keisuke; Albin, John S.; Harris, Reuben S.

    2008-01-01

    Summary The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cDNA cytosines to uracils, leading to viral genomic strand G-to-A hypermutations [1–4]. However, the HIV-1 Vif protein triggers APOBEC3G degradation, helping explain why this innate defense does not protect patients [5]. The APOBEC3G-Vif interaction is a promising therapeutic target, but the benefit of enabling HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to ask whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants would emerge that resist restriction. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for over 5 weeks, but replication was eventually detected in 3 cultures. Virus growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient. Rather, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of anti-retrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication. PMID:18501607

  9. APOBEC3H Haplotypes and HIV-1 Pro-Viral vif DNA Sequence Diversity in Early Untreated HIV-1 Infection

    PubMed Central

    Gourraud, PA; Karaouni, A; Woo, JM; Schmidt, T; Oksenberg, JR; Hecht, FM; Liegler, TJ; Barbour, JD

    2011-01-01

    We examined single nucleotide polymorphisms (SNP) in the APOBEC3 locus on chromosome 22, paired to population sequences of pro-viral HIV-1 vif of peripheral blood mononuclear cells (PBMC), from 96 recently HIV-1 infected treatment naïve adults. We found evidence for the existence of an APOBEC3H linkage disequilibrium (LD) block associated with variation in GA->AA, or APOBEC3F signature, sequence changes in pro-viral HIV-1 vif sequence (top significant 10 SNPs with a top-significant p=4.8×10−3). We identified a common 5 position risk haplotype distal to APOBEC3H (A3Hrh). These markers were in high LD (D′ = 1; r2=0.98) to a previously described A3H ‘RED’ haplotype containing a variant (E121) with enhanced susceptibility to HIV-1 Vif (Zhen et al 2009 [1]). This association is confirmed by a haplotype analysis: Homozygote carriers of the A3Hrh had lower GA->AA (A3F) sequence editing on pro-viral HIV-1 vif sequence (p = 0.01), and lower HIV-1 RNA levels over time during early, untreated HIV-1 infection, (p = 0.015 mixed effects model). This effect may be due to enhanced susceptibility of A3H forms to HIV-1 Vif mediated viral suppression of sequence editing activity, slowing viral diversification and escape from immune responses. PMID:21167246

  10. HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

    PubMed

    Mercenne, Gaëlle; Bernacchi, Serena; Richer, Delphine; Bec, Guillaume; Henriet, Simon; Paillart, Jean-Christophe; Marquet, Roland

    2010-01-01

    The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

  11. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

    SciTech Connect

    Okumura, Atsushi; Alce, Tim; Lubyova, Barbora; Ezelle, Heather; Strebel, Klaus; Pitha, Paula M.

    2008-03-30

    The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.

  12. Mapping the Vif-A3G interaction using peptide arrays: a basis for anti-HIV lead peptides.

    PubMed

    Reingewertz, Tali H; Britan-Rosich, Elena; Rotem-Bamberger, Shahar; Viard, Mathias; Jacobs, Amy; Miller, Abigail; Lee, Ji Youn; Hwang, Jeeseong; Blumenthal, Robert; Kotler, Moshe; Friedler, Assaf

    2013-06-15

    Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3G (A3G) is a cytidine deaminase that restricts retroviruses, endogenous retro-elements and DNA viruses. A3G plays a key role in the anti-HIV-1 innate cellular immunity. The HIV-1 Vif protein counteracts A3G mainly by leading A3G towards the proteosomal machinery and by direct inhibition of its enzymatic activity. Both activities involve direct interaction between Vif and A3G. Disrupting the interaction between A3G and Vif may rescue A3G antiviral activity and inhibit HIV-1 propagation. Here, mapping the interaction sites between A3G and Vif by peptide array screening revealed distinct regions in Vif important for A3G binding, including the N-terminal domain (NTD), C-terminal domain (CTD) and residues 83-99. The Vif-binding sites in A3G included 12 different peptides that showed strong binding to either full-length Vif, Vif CTD or both. Sequence similarity was found between Vif-binding peptides from the A3G CTD and NTD. A3G peptides were synthesized and tested for their ability to counteract Vif action. A3G 211-225 inhibited HIV-1 replication in cell culture and impaired Vif dependent A3G degradation. In vivo co-localization of full-length Vif with A3G 211-225 was demonstrated by use of FRET. This peptide has the potential to serve as an anti-HIV-1 lead compound. Our results suggest a complex interaction between Vif and A3G that is mediated by discontinuous binding regions with different affinities.

  13. Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

    PubMed

    Friedler, A; Zakai, N; Karni, O; Friedler, D; Gilon, C; Loyter, A

    1999-06-11

    The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

  14. An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

    PubMed Central

    Widera, Marek; Erkelenz, Steffen; Hillebrand, Frank; Krikoni, Aikaterini; Widera, Darius; Kaisers, Wolfgang; Deenen, René; Gombert, Michael; Dellen, Rafael; Pfeiffer, Tanya; Kaltschmidt, Barbara; Münk, Carsten; Bosch, Valerie; Köhrer, Karl

    2013-01-01

    Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3′ splice site (3′ss) A1 but lack splicing at 5′ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3′ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3′ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5′ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5′ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. PMID:23255806

  15. Genetic and functional characterization of HIV-1 Vif on APOBEC3G degradation: First report of emergence of B/C recombinants from North India

    PubMed Central

    Ronsard, Larance; Raja, Rameez; Panwar, Vaishali; Saini, Sanjesh; Mohankumar, Kumaravel; Sridharan, Subhashree; Padmapriya, Ramamoorthy; Chaudhuri, Suhnrita; Ramachandran, Vishnampettai G; Banerjea, Akhil C

    2015-01-01

    HIV-1 is characterized by high genetic heterogeneity which is a challenge for developing therapeutics. Therefore, it is necessary to understand the extent of genetic variations that HIV is undergoing in North India. The objective of this study was to determine the role of genetic and functional role of Vif on APOBEC3G degradation. Vif is an accessory protein involved in counteracting APOBEC3/F proteins. Genetic analysis of Vif variants revealed that Vif C variants were closely related to South African Vif C whereas Vif B variants and Vif B/C showed distinct geographic locations. This is the first report to show the emergence of Vif B/C in our population. The functional domains, motifs and phosphorylation sites were well conserved. Vif C variants differed in APOBEC3G degradation from Vif B variants. Vif B/C revealed similar levels of APOBEC3G degradation to Vif C confirming the presence of genetic determinants in C-terminal region. High genetic diversity was observed in Vif variants which may cause the emergence of more complex and divergent strains. These results reveal the genetic determinants of Vif in mediating APOBEC3G degradation and highlight the genetic information for the development of anti-viral drugs against HIV. Importance: Vif is an accessory HIV-1 protein which plays significant role in the degradation of human DNA-editing factor APOBEC3G, thereby impeding the antiretroviral activity of APOBEC3G. It is known that certain natural polymorphisms in Vif could degrade APOBEC3G relatively higher rate, suggesting its role in HIV-1 pathogenesis. This is the first report from North India showcasing genetic variations and novel polymorphisms in Vif gene. Subtype C is prevalent in India, but for the first time we observed putative B/C recombinants with a little high ability to degrade APOBEC3G indicating adaptation and evolving nature of virus in our population. Indian Vif C variants were able to degrade APOBEC3G well in comparison to Vif B variants. These

  16. Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage

    PubMed Central

    Alteri, Claudia; Surdo, Matteo; Bellocchi, Maria Concetta; Saccomandi, Patrizia; Continenza, Fabio; Armenia, Daniele; Parrotta, Lucia; Carioti, Luca; Costa, Giosuè; Fourati, Slim; Di Santo, Fabiola; Scutari, Rossana; Barbaliscia, Silvia; Fedele, Valentina; Carta, Stefania; Balestra, Emanuela; Alcaro, Stefano; Marcelin, Anne Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Artese, Anna

    2015-01-01

    Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e−7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, −40.1 kcal/mol; G24E, −510 kcal/mol; E25K, −522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression. PMID:26055363

  17. Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage.

    PubMed

    Alteri, Claudia; Surdo, Matteo; Bellocchi, Maria Concetta; Saccomandi, Patrizia; Continenza, Fabio; Armenia, Daniele; Parrotta, Lucia; Carioti, Luca; Costa, Giosuè; Fourati, Slim; Di Santo, Fabiola; Scutari, Rossana; Barbaliscia, Silvia; Fedele, Valentina; Carta, Stefania; Balestra, Emanuela; Alcaro, Stefano; Marcelin, Anne Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Artese, Anna; Perno, Carlo Federico; Svicher, Valentina

    2015-08-01

    Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression.

  18. Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels

    PubMed Central

    Nomaguchi, Masako; Doi, Naoya; Sakai, Yosuke; Ode, Hirotaka; Iwatani, Yasumasa; Matsumoto, Yui; Miyazaki, Yasuyuki; Masuda, Takao

    2016-01-01

    ABSTRACT We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. IMPORTANCE While human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving

  19. Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection.

    PubMed

    Mlcochova, Petra; Apolonia, Luis; Kluge, Silvia F; Sridharan, Aishwarya; Kirchhoff, Frank; Malim, Michael H; Sauter, Daniel; Gupta, Ravindra K

    2015-08-01

    Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection.

  20. HIV-1 Vif Versus the APOBEC3 Cytidine Deaminases: An Intracellular Duel Between Pathogen and Host Restriction Factors

    PubMed Central

    Wissing, Silke; Galloway, Nicole L. K.; Greene, Warner C.

    2010-01-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  1. HIV-1 Vif versus the APOBEC3 cytidine deaminases: an intracellular duel between pathogen and host restriction factors.

    PubMed

    Wissing, Silke; Galloway, Nicole L K; Greene, Warner C

    2010-10-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  2. A computational analysis of the structural determinants of APOBEC3's catalytic activity and vulnerability to HIV-1 Vif

    PubMed Central

    Shandilya, M.D. Shivender; Bohn, Markus-Frederik; Schiffer, Celia A.

    2016-01-01

    APOBEC3s (A3) are Zn2+ dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by 'hypermutating' the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn2+ coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design. PMID:25461536

  3. A computational analysis of the structural determinants of APOBEC3's catalytic activity and vulnerability to HIV-1 Vif.

    PubMed

    Shandilya, Shivender M D; Bohn, Markus-Frederik; Schiffer, Celia A

    2014-12-01

    APOBEC3s (A3) are Zn(2+) dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by 'hypermutating' the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn(2+) coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design. PMID:25461536

  4. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    PubMed

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-04-19

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  5. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach

    PubMed Central

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-01-01

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application. PMID:27093170

  6. Tertiary Element Interaction in HIV-1 TAR.

    PubMed

    Krawczyk, Konrad; Sim, Adelene Y L; Knapp, Bernhard; Deane, Charlotte M; Minary, Peter

    2016-09-26

    HIV-1 replication requires binding to occur between Trans-activation Response Element (TAR) RNA and the TAT protein. This TAR-TAT binding depends on the conformation of TAR, and therapeutic development has attempted to exploit this dynamic behavior. Here we simulate TAR dynamics in the context of mutations inhibiting TAR binding. We find that two tertiary elements, the apical loop and the bulge, can interact directly, and this interaction may be linked to the affinity of TAR for TAT. PMID:27500460

  7. Zinc binding to the HCCH motif of HIV-1 virion infectivity factor induces a conformational change that mediates protein-protein interactions.

    PubMed

    Paul, Indrani; Cui, Jian; Maynard, Ernest L

    2006-12-01

    Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4(+) T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)(5)-C-(Xaa)(17-18)-C-(Xaa)(3-5)-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif-Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif, as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein-protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation.

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

    PubMed

    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.

  9. 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

  10. MicroRNAs and HIV-1: Complex Interactions*

    PubMed Central

    Klase, Zachary; Houzet, Laurent; Jeang, Kuan-Teh

    2012-01-01

    RNAi plays important roles in many biological processes, including cellular defense against viral infection. Components of the RNAi machinery are widely conserved in plants and animals. In mammals, microRNAs (miRNAs) represent an abundant class of cell encoded small noncoding RNAs that participate in RNAi-mediated gene silencing. Here, findings that HIV-1 replication in cells can be regulated by miRNAs and that HIV-1 infection of cells can alter cellular miRNA expression are reviewed. Lessons learned from and questions outstanding about the complex interactions between HIV-1 and cellular miRNAs are discussed. PMID:23043098

  11. The Complex Interaction between Methamphetamine Abuse and HIV-1 pathogenesis

    PubMed Central

    Passaro, Ryan Colby; Pandhare, Jui; Qian, Han-Zhu; Dash, Chandravanu

    2016-01-01

    The global HIV/AIDS pandemic has claimed the lives of an estimated 35 million people. A significant barrier for combating this global pandemic is substance use since it is associated with HIV transmission, delayed diagnosis/initiation of therapy, and poor adherence to therapy. Clinical studies also suggest a link between substance use and HIV-disease progression/AIDS-associated mortality. Methamphetamine (METH) use is one of the fastest-growing substance use problems in the world. METH use enhances high-risk sexual behaviors, therefore increases the likelihood of HIV-1 acquisition. METH use is also associated with higher viral loads, immune dysfunction, and antiretroviral resistance. Moreover, METH use has also been correlated with rapid progression to AIDS. However, direct effects of METH on HIV-1 disease progression remains poorly understood because use of METH and other illicit drugs is often associated with reduced/non adherence to ART. Nevertheless, in vitro studies demonstrate that METH increases HIV-1 replication in cell cultures and animal models. Thus, it has been proposed that METH’s potentiating effects on HIV-1 replication may in part contribute to the worsening of HIV-1 pathogenesis. However, our recent data demonstrate that METH inhibits HIV-1 replication in CD4+ T cells and challenges this paradigm. Thus, the goal of this review is to systematically examine the published literature to better understand the complex interaction between METH abuse and HIV-1 disease progression. PMID:25850893

  12. On the solution conformation and dynamics of the HIV-1 Viral Infectivity Factor

    PubMed Central

    Marcsisin, Sean R.; Narute, Purushottam S.; Emert-Sedlak, Lori A.; Kloczewiak, Marek; Smithgall, Thomas E.; Engen, John R.

    2011-01-01

    SUMMARY HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host-cell enzymes. The HIV-1 virion infectivity factor, one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and down-regulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution including the APOBEC3G/F binding site and HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif. PMID:21763503

  13. HIV-1 Tat interacts with LIS1 protein

    PubMed Central

    Epie, Nicolas; Ammosova, Tatyana; Sapir, Tamar; Voloshin, Yaroslav; Lane, William S; Turner, Willie; Reiner, Orly; Nekhai, Sergei

    2005-01-01

    Background HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization. Results We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system. Conclusion Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation. PMID:15698475

  14. Replication potentials of HIV-1/HSIV in PBMCs from northern pig-tailed macaque (Macaca leonina).

    PubMed

    Lei, Ai-Hua; Zhang, Gao-Hong; Tian, Ren-Rong; Zhu, Jia-Wu; Zheng, Hong-Yi; Pang, Wei; Zheng, Yong-Tang

    2014-05-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.

  15. Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque (Macaca leonina)

    PubMed Central

    LEI, Ai-Hua; ZHANG, Gao-Hong; TIAN, Ren-Rong; ZHU, Jia-Wu; ZHENG, Hong-Yi; PANG, Wei; ZHENG, Yong-Tang

    2014-01-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS. PMID:24866489

  16. Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.

    PubMed

    Fisher, Robert J; Fivash, Matthew J; Stephen, Andrew G; Hagan, Nathan A; Shenoy, Shilpa R; Medaglia, Maxine V; Smith, Lindsey R; Worthy, Karen M; Simpson, John T; Shoemaker, Robert; McNitt, Karen L; Johnson, Donald G; Hixson, Catherine V; Gorelick, Robert J; Fabris, Daniele; Henderson, Louis E; Rein, Alan

    2006-01-01

    The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

  17. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  18. Interactions between HIV-1 and the Cell-Autonomous Innate Immune System

    PubMed Central

    Towers, Greg J.; Noursadeghi, Mahdad

    2014-01-01

    HIV-1 was recognized as the cause of AIDS in humans in 1984. Despite 30 years of intensive research, we are still unraveling the molecular details of the host-pathogen interactions that enable this virus to escape immune clearance and cause immunodeficiency. Here we explore a series of recent studies that consider how HIV-1 interacts with the cell-autonomous innate immune system as it navigates its way in and out of host cells. We discuss how these studies improve our knowledge of HIV-1 and host biology as well as increase our understanding of transmission, persistence, and immunodeficiency and the potential for therapeutic or prophylactic interventions. PMID:25011104

  19. Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection

    SciTech Connect

    Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M.

    2014-01-20

    The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R{sub 0}, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis.

  20. Inhibition of human immunodeficiency virus type 1 (HIV-1) nuclear import via Vpr-Importin {alpha} interactions as a novel HIV-1 therapy

    SciTech Connect

    Suzuki, Tatsunori; Yamamoto, Norio; Nonaka, Mizuho; Hashimoto, Yoshie; Matsuda, Go; Takeshima, Shin-nosuke; Matsuyama, Megumi; Igarashi, Tatsuhiko; Miura, Tomoyuki; Tanaka, Rie; Kato, Shingo; Aida, Yoko

    2009-03-20

    The development of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus (HIV) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. One such target is the interaction between Vpr, one of the accessory gene products of HIV-1 and Importin {alpha}, which is crucial, not only for the nuclear import of Vpr, but also for HIV-1 replication in macrophages. We have identified a potential parent compound, hematoxylin, which suppresses Vpr-Importin {alpha} interaction, thereby inhibiting HIV-1 replication in a Vpr-dependent manner. Analysis by real-time PCR demonstrated that hematoxylin specifically inhibited nuclear import step of pre-integration complex. Thus, hematoxylin is a new anti-HIV-1 inhibitor that targets the nuclear import of HIV-1 via the Vpr-Importin {alpha} interaction, suggesting that a specific inhibitor of the interaction between viral protein and the cellular factor may provide a new strategy for HIV-1 therapy.

  1. Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

    PubMed

    Uyttendaele, Isabel; Lavens, Delphine; Catteeuw, Dominiek; Lemmens, Irma; Bovijn, Celia; Tavernier, Jan; Peelman, Frank

    2012-01-01

    The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization. PMID:22970171

  2. Random Mutagenesis MAPPIT Analysis Identifies Binding Sites for Vif and Gag in Both Cytidine Deaminase Domains of Apobec3G

    PubMed Central

    Uyttendaele, Isabel; Lavens, Delphine; Catteeuw, Dominiek; Lemmens, Irma; Bovijn, Celia

    2012-01-01

    The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultanously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization. PMID:22970171

  3. Interaction of the HIV-1 frameshift signal with the ribosome

    PubMed Central

    Mazauric, Marie-Hélène; Seol, Yeonee; Yoshizawa, Satoko; Visscher, Koen; Fourmy, Dominique

    2009-01-01

    Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5′ direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome. PMID:19812214

  4. Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

    PubMed Central

    Dirk, Brennan S.; Van Nynatten, Logan R.; Dikeakos, Jimmy D.

    2016-01-01

    Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle. PMID:27775563

  5. The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.

    PubMed Central

    Hottiger, M; Gramatikoff, K; Georgiev, O; Chaponnier, C; Schaffner, W; Hübscher, U

    1995-01-01

    HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV-1 virions. Images PMID:7535922

  6. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    PubMed

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

  7. Potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) by 5-ethyl-6-phenylthiouracil derivatives through their interaction with the HIV-1 reverse transcriptase.

    PubMed Central

    Baba, M; De Clercq, E; Tanaka, H; Ubasawa, M; Takashima, H; Sekiya, K; Nitta, I; Umezu, K; Nakashima, H; Mori, S

    1991-01-01

    In the search for 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT) derivatives, we have found several 5-ethyl-6-(phenylthio)uracil analogues to be highly potent and selective inhibitors of human immunodeficiency virus (HIV) type 1. 1-Benzyloxymethyl-5-ethyl-6-phenylthiouracil, the most potent congener of the series, inhibits HIV-1 replication in a variety of cell systems, including peripheral blood lymphocytes, at a concentration of 1.5-7.0 nM, which is lower by a factor of 10(3) than the 50% antivirally effective concentration of the parent compound HEPT. The 5-ethyl-6-(phenylthio)uracil analogues, like HEPT itself, do not inhibit HIV-2 replication but do inhibit replication of 3'-azido-3'-deoxythymidine-resistant mutants of HIV-1. 1-Benzyloxy-methyl-5-ethyl-6-phenylthiouracil and its congeners are targeted at the HIV-1 reverse transcriptase (RT). They do not inhibit HIV-2 RT. They do not need to be metabolized to exert their inhibitory effect on HIV-1 RT. Yet this inhibitory effect is competitive with the natural substrate dTTP. The HEPT derivatives represent a group of RT inhibitors with a unique mode of interaction with HIV-1 RT. PMID:1706522

  8. LILRB2 interaction with HLA class I correlates with control of HIV-1 infection.

    PubMed

    Bashirova, Arman A; Martin-Gayo, Enrique; Jones, Des C; Qi, Ying; Apps, Richard; Gao, Xiaojiang; Burke, Patrick S; Taylor, Craig J; Rogich, Jerome; Wolinsky, Steven; Bream, Jay H; Duggal, Priya; Hussain, Shehnaz; Martinson, Jeremy; Weintrob, Amy; Kirk, Gregory D; Fellay, Jacques; Buchbinder, Susan P; Goedert, James J; Deeks, Steven G; Pereyra, Florencia; Trowsdale, John; Lichterfeld, Mathias; Telenti, Amalio; Walker, Bruce D; Allen, Rachel L; Carrington, Mary; Yu, Xu G

    2014-03-01

    Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10(-2)). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10(-11)-10(-9)) and African (p = 10(-5)-10(-3)) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement. PMID:24603468

  9. Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate.

    PubMed

    Mbemba, E; Gluckman, J C; Gattegno, L

    1994-02-01

    Envelope glycoproteins of human immunodeficiency viruses (HIV-1 and HIV-2) can interact with high-mannose glycans and with the mannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidic structures. HIV-1 glycoproteins also specifically bind sulphated polysaccharides such as dextran sulphate (DS) and heparin. Here, we show that the latter property is shared by HIV-2 recombinant gp140 (rgp140) precursor glycoprotein. Binding of rgp140 and of corresponding rgp160 of HIV-1 to heparin- and DS-substituted (sulphated dextran beads; SDB) affinity matrices was inhibited by the soluble specific ligand and also by fetuin, asialofetuin or the anionic simple carbohydrate derivative mannose-6-phosphate (M6P). Interaction of HIV-1 rgp120 subunit with the two affinity matrices was also inhibited by M6P, but only rgp120 binding to heparin-agarose, and not that to SDB, was affected by fetuin and asialofetuin. These results suggest that HIV-1 and HIV-2 envelope glycoproteins presumably display different sulphated polysaccharide and carbohydrate recognition sites. Some of these may be common or in close proximity: with respect to rgp160, for example, the sites may be common on the gp41 moiety and/or in a region of gp120 which would be more accessible when expressed on rgp160 than on processed gp120, while they may be distinct on the cleaved gp120 subunit. Finally, because M6P is a marker of lysosomal enzymes, we verified that HIV-1 and HIV-2 envelope glycoproteins could specifically bind in a M6P-inhibitable manner to a representative lysosomal enzyme, bovine liver beta-glucuronidase coupled to agarose, suggesting that they may possibly interfere with lysosomal enzyme sorting in HIV-infected cells.

  10. LILRB2 Interaction with HLA Class I Correlates with Control of HIV-1 Infection

    PubMed Central

    Qi, Ying; Apps, Richard; Gao, Xiaojiang; Burke, Patrick S.; Taylor, Craig J.; Rogich, Jerome; Wolinsky, Steven; Bream, Jay H.; Duggal, Priya; Hussain, Shehnaz; Martinson, Jeremy; Weintrob, Amy; Kirk, Gregory D.; Fellay, Jacques; Buchbinder, Susan P.; Goedert, James J.; Deeks, Steven G.; Pereyra, Florencia; Trowsdale, John; Lichterfeld, Mathias; Telenti, Amalio; Walker, Bruce D.; Allen, Rachel L.; Carrington, Mary; Yu, Xu G.

    2014-01-01

    Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10−2). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10−11–10−9) and African (p = 10−5–10−3) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement. PMID:24603468

  11. The evolution of HIV-1 interactions with coreceptors and mannose C-type lectin receptors.

    PubMed

    Borggren, Marie; Jansson, Marianne

    2015-01-01

    The phenotype of human immunodeficiency virus type 1 (HIV-1) commonly evolves between and within infected individuals, at virus transmission, and during disease progression. This evolution includes altered interactions between the virus and its coreceptors, i.e., chemokine receptors, as well as mannose C-type lectin receptors (CLRs). Transmitted/founder viruses are predominantly restricted to CCR5, whereas the subsequent intrapatient evolution of HIV-1 coreceptor use during progressive disease can be subdivided into two distinct pathways. Accordingly, the CCR5-restricted virus population is either gradually replaced by virus variants able to use CXCR4 or evolves toward an altered, more flexible use of CCR5. Despite a strong dependency on these coreceptors for host cell entry, HIV-1 also interacts with other cell surface molecules during target cell attachment, including the CLRs. The virus interaction with the CLRs may result either in the efficient transfer of virus to CD4(+) T cells or in the degradation of the virus in endosomal compartments. The determinants of the diverse outcomes depend on which CLR is engaged and also on the glycan makeup of the envelope glycoproteins, which may evolve with the strength of the immune pressure during the disease course. With the current clinical introduction of CCR5 antagonists and the development of additional entry inhibitors, knowledge on the evolution and baseline characteristics of HIV-1 interactions with coreceptor and CLR interactions may play important roles for individualized and optimized treatment strategies. This review summarizes our current understanding of the evolution of HIV-1 interactions with these receptors. PMID:25595802

  12. Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Thammaporn, Ratsupa; Ishii, Kentaro; Yagi-Utsumi, Maho; Uchiyama, Susumu; Hannongbua, Supa; Kato, Koichi

    2016-01-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have been developed for the treatment of acquired immunodeficiency syndrome. HIV-1 RT binding to NNRTIs has been characterized by various biophysical techniques. However, these techniques are often hampered by the low water solubility of the inhibitors, such as the current promising diarylpyrimidine-based inhibitors rilpivirine and etravirine. Hence, a conventional and rapid method that requires small sample amounts is desirable for studying NNRTIs with low water solubility. Here we successfully applied a recently developed mass spectrometric technique under non-denaturing conditions to characterize the interactions between the heterodimeric HIV-1 RT enzyme and NNRTIs with different inhibitory activities. Our data demonstrate that mass spectrometry serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low and small amounts of samples, offering a new high-throughput screening tool for identifying novel RT inhibitors as anti-HIV drugs. PMID:26934936

  13. HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes

    PubMed Central

    Brinzevich, Daria; Young, George R.; Sebra, Robert; Ayllon, Juan; Maio, Susan M.; Deikus, Gintaras; Chen, Benjamin K.; Fernandez-Sesma, Ana; Simon, Viviana

    2014-01-01

    ABSTRACT Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication. IMPORTANCE Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins

  14. Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

    NASA Astrophysics Data System (ADS)

    Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

    2012-02-01

    Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

  15. 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.

  16. Interaction of small molecule inhibitors of HIV-1 entry with CCR5

    SciTech Connect

    Seibert, Christoph . E-mail: seiberc@mail.rockefeller.edu; Ying Weiwen; Gavrilov, Svetlana; Tsamis, Fotini; Kuhmann, Shawn E.; Palani, Anandan; Tagat, Jayaram R.; Clader, John W.; McCombie, Stuart W.; Baroudy, Bahige M.; Smith, Steven O.; Dragic, Tatjana; Moore, John P.; Sakmar, Thomas P.

    2006-05-25

    The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands.

  17. APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

    PubMed

    Sato, Kei; Takeuchi, Junko S; Misawa, Naoko; Izumi, Taisuke; Kobayashi, Tomoko; Kimura, Yuichi; Iwami, Shingo; Takaori-Kondo, Akifumi; Hu, Wei-Shau; Aihara, Kazuyuki; Ito, Mamoru; An, Dong Sung; Pathak, Vinay K; Koyanagi, Yoshio

    2014-10-01

    Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

  18. APOBEC3D and APOBEC3F Potently Promote HIV-1 Diversification and Evolution in Humanized Mouse Model

    PubMed Central

    Misawa, Naoko; Izumi, Taisuke; Kobayashi, Tomoko; Kimura, Yuichi; Iwami, Shingo; Takaori-Kondo, Akifumi; Hu, Wei-Shau; Aihara, Kazuyuki; Ito, Mamoru; An, Dong Sung; Pathak, Vinay K.; Koyanagi, Yoshio

    2014-01-01

    Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo. PMID:25330146

  19. Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator.

    PubMed Central

    Desai-Yajnik, V; Hadzic, E; Modlinger, P; Malhotra, S; Gechlik, G; Samuels, H H

    1995-01-01

    Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes. PMID:7609079

  20. Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

    PubMed Central

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

  1. Morphine and gp120 toxic interactions in striatal neurons are dependent on HIV-1 strain

    PubMed Central

    Podhaizer, Elizabeth M.; Zou, Shiping; Fitting, Sylvia; Samano, Kimberly L.; El-Hage, Nazira; Knapp, Pamela E.; Hauser, Kurt F.

    2011-01-01

    A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed μ-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120IIIB, R5-tropic gp120ADA, or dual-tropic gp120MN was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120IIIB-induced neuronal death; however, in combination with gp120MN, morphine caused sustained increases in the rate of neuronal death compared to gp120MN alone that were prevented by naloxone. Alternatively, gp120ADA significantly increased the rate of neuron death, which was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120IIIB were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs. PMID:22101471

  2. Efficient Interaction of HIV-1 with Purified Dendritic Cells via Multiple Chemokine Coreceptors

    PubMed Central

    Granelli-Piperno, Angela; Moser, Bernhard; Pope, Melissa; Chen, Dongling; Wei, Yang; Isdell, Frank; O'Doherty, Una; Paxton, William; Koup, Richard; Mojsov, Svetlana; Bhardwaj, Nina; Clark-Lewis, Ian; Baggiolini, Marco; Steinman, Ralph M.

    1996-01-01

    HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37°C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37°C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus. PMID:8976200

  3. Effect on HIV-1 Gene Expression, Tat-Vpr Interaction and Cell Apoptosis by Natural Variants of HIV-1 Tat Exon 1 and Vpr from Northern India

    PubMed Central

    Lata, Sneh; Ronsard, Larance; Sood, Vikas; Dar, Sajad A.; Ramachandran, Vishnampettai G.; Das, Shukla; Banerjea, Akhil C.

    2013-01-01

    Background Since HIV-1 Tat and Vpr genes are involved in promoter transactivation, apoptosis, etc, we carried out studies to find out nature and extent of natural variation in the two genes from seropositive patients from Northern India and determined their functional implications. Methods HIV-1 tat exon 1 and vpr were amplified from the genomic DNA isolated from the blood of HIV-1 infected individuals using specific primers by Polymerase Chain reaction (PCR) and subjected to extensive genetic analysis (CLUSTAL W, Simplot etc). Their expression was monitored by generating myc fusion clones. Tat exon 1 and Vpr variants were co-transfected with the reporter gene construct (LTR-luc) and their transactivation potential was monitored by measuring luciferase activity. Apoptosis and cell cycle analysis was done by Propidium Iodide (PI) staining followed by FACS. Results Exon 1 of tat was amplified from 21 samples and vpr was amplified from 16 samples. One of the Tat exon 1 variants showed phylogenetic relatedness to subtype B & C and turned out to be a unique recombinant. Two of the Vpr variants were B/C/D recombinants. These natural variations were found to have no impact on the stability of Tat and Vpr. These variants differed in their ability to transactivate B LTR and C LTR promoters. B/C recombinant Tat showed better co-operative interaction with Vpr. B/C/D recombination in Vpr was found to have no effect on its co-operativity with Tat. Recombinant Tat (B/C) induced more apoptosis than wild type B and C Tat. The B/C/D recombination in Vpr did not affect its G2 arrest induction potential but reduced its apoptosis induction ability. Conclusions Extensive sequence and region-specific variations were observed in Tat and Vpr genes from HIV-1 infected individuals from Northern India. These variations have functional implications & therefore important for the pathogenicity of virus. PMID:24367500

  4. Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection.

    PubMed Central

    Purohit, P; Dupont, S; Stevenson, M; Green, M R

    2001-01-01

    The human immunodeficiency virus type-1 matrix protein (HIV-1 MA) is a multifunctional structural protein synthesized as part of the Pr55 gag polyprotein. We have used in vitro genetic selection to identify an RNA consensus sequence that specifically interacts with MA (Kd = 5 x 10(-7) M). This 13-nt MA binding consensus sequence bears a high degree of homology (77%) to a region (nt 1433-1446) within the POL open reading frame of the HIV-1 genome (consensus sequence from 38 HIV-1 strains). Chemical interference experiments identified the nucleotides within the MA binding consensus sequence involved in direct contact with MA. We further demonstrate that this RNA-protein interaction is mediated through a stretch of basic amino acids within MA. Mutations that disrupt the interaction between MA and its RNA binding site within the HIV-1 genome resulted in a measurable decrease in viral replication. PMID:11345436

  5. Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

    NASA Astrophysics Data System (ADS)

    Beltram, Fabio

    2002-03-01

    The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

  6. Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core

    PubMed Central

    Öhagen, Åsa; Gabuzda, Dana

    2000-01-01

    The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription. vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry. PMID:11070000

  7. 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

  8. Investigating the Cellular Distribution and Interactions of HIV-1 Nucleocapsid Protein by Quantitative Fluorescence Microscopy

    PubMed Central

    Anton, Halina; Taha, Nedal; Boutant, Emmanuel; Richert, Ludovic; Khatter, Heena; Klaholz, Bruno; Rondé, Philippe; Réal, Eléonore; de Rocquigny, Hugues; Mély, Yves

    2015-01-01

    The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection. PMID:25723396

  9. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif

    PubMed Central

    Land, Allison M.; Wang, Jiayi; Law, Emily K.; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E.; Harris, Reuben S.

    2015-01-01

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy. PMID:26544511

  10. Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

    PubMed Central

    Hendrix, Jelle; Baumgärtel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kräusslich, Hans-Georg; Müller, Barbara

    2015-01-01

    Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly. PMID:26283800

  11. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    PubMed Central

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-01-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates. PMID:27405089

  12. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    NASA Astrophysics Data System (ADS)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  13. HIV-1 Induced Nuclear Factor I-B (NF-IB) Expression Negatively Regulates HIV-1 Replication through Interaction with the Long Terminal Repeat Region

    PubMed Central

    Vemula, Sai Vikram; Veerasamy, Ravichandran; Ragupathy, Viswanath; Biswas, Santanu; Devadas, Krishnakumar; Hewlett, Indira

    2015-01-01

    Background: Retroviruses rely on host factors for cell entry, replication, transcription, and other major steps during their life cycle. Human Immunodeficiency Virus-1 (HIV-1) is well known for utilizing a plethora of strategies to evade the host immune response, including the establishment of latent infection within a subpopulation of susceptible cells. HIV-1 also manipulates cellular factors in latently infected cells and persists for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). Results: In this study we demonstrate that Nuclear Factor-IB (NF-IB) is induced during HIV-1 infection and its expression negatively impacts viral replication. During HIV-1 infection in peripheral blood mononuclear cells (PBMCs), and the T cell line, Jurkat or during induction of virus replication in latently infected cells, ACH2 and J1.1, we observed a time-dependent alteration in NF-IB expression pattern that correlated with HIV-1 viral expression. Using the Chip assay, we observed an association of NF-IB with the long terminal repeat region of HIV-1 (LTR) (-386 to -453 nt), and this association negatively correlated with HIV-1 transcription. Furthermore, knock-down of NF-IB levels in J1.1 cells resulted in an increase of HIV-1 levels. Knock-down of NF-IB levels in J-Lat-Tat-GFP (A1), (a Jurkat cell GFP reporter model for latent HIV-1 infection) resulted in an increase in GFP levels, indicating a potential negative regulatory role of NF-IB in HIV-1 replication. Conclusion: Overall, our results suggest that NF-IB may play a role in intrinsic antiretroviral defenses against HIV-1. These observations may offer new insights into the correlation of the latently infected host cell types and HIV-1, and help to define new therapeutic approaches for triggering the switch from latency to active replication thereby eliminating HIV-1 latent infection. PMID:25664610

  14. The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

    PubMed Central

    Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

    2014-01-01

    HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

  15. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Arachchilage, Madara Hetti; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  16. Interaction between Nef and INI1/SMARCB1 augments replicability of HIV-1 in resting human peripheral blood mononuclear cells.

    PubMed

    Pyeon, Dohun; Park, In-Woo

    2015-03-01

    A central feature of HIV-1 infection is the inability of entering virus to integrate into chromosomes of resting T lymphocytes unless they are mitogenically activated. In contrast, SIVpbj1.9 replicates in initially resting T lymphocytes by activating infected cells. Previous reports have shown that a difference in Nef-mediated T cell activation between HIV-1 and SIVpbj1.9 plays a critical role in the differing abilities of these viruses to replicate in resting lymphocytes. However, the molecular details of these differences are still unclear. Here, we show that infection with a chimeric virus, HSIVnef, which harbors the 5' 308 nucleotides of SIVpbj1.9 nef in place of the 5' 221 nucleotides of HIV-1 nef in the HIV-1 proviral backbone, resulted in integration of the provirus into host chromosomes without mitogenic activation and thereby replication in resting human PBMCs (hPBMCs). These results indicate that Nef is an essential viral determinant for the integration of provirus into host chromosomes in resting T cells. Using the yeast two-hybrid system, we identified integrase interactor-1 (INI1/SMARCB1) as a cellular factor that is involved in the integration process via interaction with Nef. Although INI1 interacted with both SIVpbj1.9 and HIV-1 Nefs, SIVpbj1.9 Nef, but not HIV-1 Nef, enhanced proviral integration into host DNA. Furthermore, mutational analysis revealed that the basic-amino-acid-rich amino-terminal domain in SIVpbj1.9 Nef is crucial for interaction with INI1 and virus replication in resting hPBMCs. Taken together, these data indicate that Nef is a critical viral protein for incorporating nascent proviral DNA into host chromosomes in resting PBMCs and that this occurs through interaction with INI1. This elucidates the basis for replication of the integrated provirus when the host cell is in a resting state.

  17. LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

    PubMed

    Jones, R Brad; Song, Haihan; Xu, Yang; Garrison, Keith E; Buzdin, Anton A; Anwar, Naveed; Hunter, Diana V; Mujib, Shariq; Mihajlovic, Vesna; Martin, Eric; Lee, Erika; Kuciak, Monika; Raposo, Rui André Saraiva; Bozorgzad, Ardalan; Meiklejohn, Duncan A; Ndhlovu, Lishomwa C; Nixon, Douglas F; Ostrowski, Mario A

    2013-12-01

    Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.

  18. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.

    PubMed

    Chen, Kuan-Ming; Harjes, Elena; Gross, Phillip J; Fahmy, Amr; Lu, Yongjian; Shindo, Keisuke; Harris, Reuben S; Matsuo, Hiroshi

    2008-03-01

    The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

  19. Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)*

    PubMed Central

    Larue, Ross; Gupta, Kushol; Wuensch, Christiane; Shkriabai, Nikolozi; Kessl, Jacques J.; Danhart, Eric; Feng, Lei; Taltynov, Oliver; Christ, Frauke; Van Duyne, Gregory D.; Debyser, Zeger; Foster, Mark P.; Kvaratskhelia, Mamuka

    2012-01-01

    Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3 either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering, and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the capsid cores. Instead, TNPO3 effectively bound to the functional intasome but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication. PMID:22872640

  20. Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

    PubMed Central

    Maubert, Monique E.; Pirrone, Vanessa; Rivera, Nina T.; Wigdahl, Brian; Nonnemacher, Michael R.

    2016-01-01

    In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS). Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART), CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND). A number of HIV-1 proteins (Tat, gp120, Nef, Vpr) have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients. PMID:26793168

  1. Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

    PubMed Central

    2013-01-01

    Background We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. Results KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. Conclusions The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion. PMID:24330857

  2. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

    PubMed Central

    Barat, C; Lullien, V; Schatz, O; Keith, G; Nugeyre, M T; Grüninger-Leitch, F; Barré-Sinoussi, F; LeGrice, S F; Darlix, J L

    1989-01-01

    The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3. Images PMID:2479543

  3. Relationships between structure and interaction kinetics for HIV-1 protease inhibitors.

    PubMed

    Markgren, Per-Olof; Schaal, Wesley; Hämäläinen, Markku; Karlén, Anders; Hallberg, Anders; Samuelsson, Bertil; Danielson, U Helena

    2002-12-01

    The interaction between HIV-1 protease and 58 structurally diverse transition-state analogue inhibitors has been analyzed by a surface plasmon resonance based biosensor. Association and dissociation rate constants and affinities were determined and displayed as k(on)-k(off)-K(D) maps. It was shown that different classes of inhibitors fall into distinct clusters in these maps. Significant changes in association and dissociation rates were found as a result of modifying the P1/P1' or P2/P2' side chains of a linear lead compound. Similarly, cyclic urea and cyclic sulfamide inhibitors displayed different kinetic features and the affinities of both classes of cyclic compounds were limited by fast dissociation rates. These results confirm that association and dissociation rates are important features of drug-target interactions and indicate that optimization of inhibitor efficacy may be guided by aiming for high association and low dissociation rates rather than high affinity alone. The present approach thus provides a new tool for structure-interaction kinetic analysis and drug discovery. PMID:12459011

  4. The study of interaction of HIV-1 surface GP120 protein with CD4 cell receptor by EXAFS spectroscopy

    NASA Astrophysics Data System (ADS)

    Lukashev, Vitaly Alexeevich; Bausk, Nikolay Vladimirovich; Mazalov, Lev Nikolaevich; Kaurov, Oleg Alexeevich; Kolobov, Alexandr Alexandrovich; Naumochkin, Andrey Nikolaevich; Kulichkov, Vladimir Anatolevich

    1995-02-01

    This work is aimed at the study of the interaction between gp120 protein (HIV-1) and peptides, which are similar to CD4 protein receptors from T4 lymphocytes. The preliminary results demonstrated that peptide structural information could be obtained from fluorescent EXAFS.

  5. Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection.

    PubMed

    Thangavelu, Pulari U; Gupta, Vipul; Dixit, Narendra M

    2014-01-20

    The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G-Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ~0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R0, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G-Vif axis.

  6. 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.

  7. HIC1 controls cellular- and HIV-1- gene transcription via interactions with CTIP2 and HMGA1

    PubMed Central

    Le Douce, Valentin; Forouzanfar, Faezeh; Eilebrecht, Sebastian; Van Driessche, Benoit; Ait-Ammar, Amina; Verdikt, Roxane; Kurashige, Yoshihito; Marban, Céline; Gautier, Virginie; Candolfi, Ermanno; Benecke, Arndt G.; Van Lint, Carine; Rohr, Olivier; Schwartz, Christian

    2016-01-01

    Among many cellular transcriptional regulators, Bcl11b/CTIP2 and HGMA1 have been described to control the establishment and the persistence of HIV-1 latency in microglial cells, the main viral reservoir in the brain. In this present work, we identify and characterize a transcription factor i.e. HIC1, which physically interacts with both Bcl11b/CTIP2 and HMGA1 to co-regulate specific subsets of cellular genes and the viral HIV-1 gene. Our results suggest that HIC1 represses Tat dependent HIV-1 transcription. Interestingly, this repression of Tat function is linked to HIC1 K314 acetylation status and to SIRT1 deacetylase activity. Finally, we show that HIC1 interacts and cooperates with HGMA1 to regulate Tat dependent HIV-1 transcription. Our results also suggest that HIC1 repression of Tat function happens in a TAR dependent manner and that this TAR element may serve as HIC1 reservoir at the viral promoter to facilitate HIC1/TAT interaction. PMID:27725726

  8. Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

    PubMed

    Jayaraman, Bhargavi; Mavor, David; Gross, John D; Frankel, Alan D

    2015-10-27

    The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function. PMID:26422686

  9. Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

    PubMed

    Jayaraman, Bhargavi; Mavor, David; Gross, John D; Frankel, Alan D

    2015-10-27

    The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.

  10. Particular interaction between efavirenz and the HIV-1 reverse transcriptase binding site as explained by the ONIOM2 method

    NASA Astrophysics Data System (ADS)

    Nunrium, Peerapol; Kuno, Mayuso; Saen-oon, Suwipa; Hannongbua, Supa

    2005-03-01

    Particular interaction between efavirenz and the HIV-1 reverse transcriptase binding site was investigated, based on the B3LYP/6-31G(d,p) and ONIOM2 methods. The interaction between efavirenz and Lys101 was found to be the strongest interaction, typically, -11.29 kcal/mol. The stability of this complex system leads to the foundation of the estimated binding energy of approximately -22.66 kcal/mol. Moreover, two hydrogen bonds between benzoxazin-2-one, and the backbone carbonyl oxygen and the backbone amino hydrogen of Lys101 were observed. These hydrogen bond interactions play an important role in the bound efavirenz/HIV-1 RT complex.

  11. Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter.

    PubMed

    el Kharroubi, A; Verdin, E

    1994-08-01

    We have examined by in vitro footprinting a region located downstream of the human immunodeficiency virus, type 1 (HIV-1) promoter found to be hypersensitive to DNase I digestion in vivo. Recognition sites for several constitutive or inducible DNA binding factors were identified. Three AP-1 binding sites and an AP-3-like motif were situated within the R-U5 region of the long terminal repeat. A novel purine-rich motif (5'-GAAAGC-GAAAGDD-3' (D represents G, A, or T residues)), which interacts with a nuclear factor designated downstream binding factor 1 (DBF1), and two juxtaposed Sp-1 binding sites were located in the untranslated sequence immediately downstream of the 5'-long terminal repeat. Genomic footprinting of these sequence elements in the HIV-1 chronically infected cell lines revealed that the DBF1 and Sp-1 sites are occupied in vivo. Furthermore, transient transfection assays showed that point mutations in the DBF1 binding site decreased significantly the HIV-1 basal promoter activity. Taken together, these results suggest that the DBF1 play a role in the HIV-1 transcription regulation. PMID:8051074

  12. GM-3 Lactone Mimetic Interacts with CD4 and HIV-1 Env Proteins, Hampering HIV-1 Infection without Inducing a Histopathological Alteration.

    PubMed

    Richichi, Barbara; Pastori, Claudia; Gherardi, Stefano; Venuti, Assunta; Cerreto, Antonella; Sanvito, Francesca; Toma, Lucio; Lopalco, Lucia; Nativi, Cristina

    2016-08-12

    Glycosphingolipids (GSLs) are involved in HIV-1 entry. GM-3 ganglioside, a widespread GSL, affects HIV entry and infection in different ways, depending on the concentration, through its anchoring activity in lipid rafts. This explains why the induction of an altered GSLs metabolism was a tempting approach to reducing HIV-1 cell infection. This study assayed the biological properties of a synthetic GM-3 lactone mimetic, 1, aimed at blocking HIV-1 infection without inducing the adverse events expected by an altered metabolism of GLSs in vivo. The mimetic, conjugated to immunogenic protein ovalbumin and multivalently presented, was able to bind the CD4 molecule with high affinity and block its engagement with gp120, thus inhibiting virus entry. Elicited antimimetic antibodies were also able to block HIV-1 infection in vitro, with activity complementary to that observed for 1. These preliminary results show that the use of GSLs mimetics can be a novel promising mode to block HIV-1 infection and that 1 and other GSL mimetics deserve further attention. PMID:27626296

  13. Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1.

    PubMed

    Abbott, Karen L; Archambault, Jacques; Xiao, Hua; Nguyen, Bao D; Roeder, Robert G; Greenblatt, Jack; Omichinski, James G; Legault, Pascale

    2005-03-01

    FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HIV-1 Tat protein, CK2, TFIIB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1(562)(-)(738)). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCP1 are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74(436)(-)(517) interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif. HIV-1 Tat inhibits the binding of RAP74(436)(-)(517) to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1(562)(-)(619) by CK2 at this site increases binding to RAP74(436)(-)(517), but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions. PMID:15723517

  14. MicroRNAs in HIV-1 infection: an integration of viral and cellular interaction at the genomic level

    PubMed Central

    Tan Gana, Neil H.; Onuki, Tomohiro; Victoriano, Ann Florence B.; Okamoto, Takashi

    2012-01-01

    The microRNA pathways govern complex interactions of the host and virus at the transcripts level that regulate cellular responses, viral replication and viral pathogenesis. As a group of single-stranded short non-coding ribonucleotides (ncRNAs), the microRNAs complement their messenger RNA (mRNA) targets to effect post-transcriptional or translational gene silencing. Previous studies showed the ability of human immunodeficiency virus 1 (HIV-1) to encode microRNAs which modify cellular defence mechanisms thus creating an environment favorable for viral invasion and replication. In corollary, cellular microRNAs were linked to the alteration of HIV-1 infection at different stages of replication and latency. As evidences further establish the regulatory involvement of both cellular and viral microRNA in HIV-1-host interactions, there is a necessity to organize this information. This paper would present current and emerging knowledge on these multi-dimensional interactions that may facilitate the design of microRNAs as effective antiretroviral reagents. PMID:22936931

  15. Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups.

    PubMed

    Vincent, Nadine; Genin, Christian; Malvoisin, Etienne

    2002-12-23

    A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.

  16. Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 Gag PTAP Interaction

    SciTech Connect

    Im, Young Jun; Kuo, Lillian; Ren, Xuefeng; Burgos, Patricia V.; Zhao, Xue Zhi; Liu, Fa; Burke, Jr., Terrence R.; Bonifacino, Juan S.; Freed, Eric O.; Hurley, James H.

    2010-12-03

    Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.41.6 {angstrom} structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions.

  17. 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

  18. 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.

  19. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env.

    PubMed

    Kwon, Young Do; Pancera, Marie; Acharya, Priyamvada; Georgiev, Ivelin S; Crooks, Emma T; Gorman, Jason; Joyce, M Gordon; Guttman, Miklos; Ma, Xiaochu; Narpala, Sandeep; Soto, Cinque; Terry, Daniel S; Yang, Yongping; Zhou, Tongqing; Ahlsen, Goran; Bailer, Robert T; Chambers, Michael; Chuang, Gwo-Yu; Doria-Rose, Nicole A; Druz, Aliaksandr; Hallen, Mark A; Harned, Adam; Kirys, Tatsiana; Louder, Mark K; O'Dell, Sijy; Ofek, Gilad; Osawa, Keiko; Prabhakaran, Madhu; Sastry, Mallika; Stewart-Jones, Guillaume B E; Stuckey, Jonathan; Thomas, Paul V; Tittley, Tishina; Williams, Constance; Zhang, Baoshan; Zhao, Hong; Zhou, Zhou; Donald, Bruce R; Lee, Lawrence K; Zolla-Pazner, Susan; Baxa, Ulrich; Schön, Arne; Freire, Ernesto; Shapiro, Lawrence; Lee, Kelly K; Arthos, James; Munro, James B; Blanchard, Scott C; Mothes, Walther; Binley, James M; McDermott, Adrian B; Mascola, John R; Kwong, Peter D

    2015-07-01

    As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.

  20. HIV-1 gp120 and drugs of abuse: interactions in the central nervous system.

    PubMed

    Silverstein, Peter S; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D P; Kumar, Anil

    2012-07-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs.

  1. HIV-1 gp120 and Drugs of Abuse: Interactions in the Central Nervous System

    PubMed Central

    Silverstein, Peter S.; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D.P.; Kumar, Anil

    2014-01-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs. PMID:22591361

  2. HIV-1 gp120 and drugs of abuse: interactions in the central nervous system.

    PubMed

    Silverstein, Peter S; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D P; Kumar, Anil

    2012-07-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs. PMID:22591361

  3. Does the HIV-1 primer activation signal interact with tRNA3Lys during the initiation of reverse transcription?

    PubMed Central

    Goldschmidt, Valérie; Ehresmann, Chantal; Ehresmann, Bernard; Marquet, Roland

    2003-01-01

    Reverse transcription of HIV-1 RNA is primed by a tRNA3Lys molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3Lys and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TΨC stem of tRNA3Lys and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3Lys or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3Lys or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3Lys. These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3Lys does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs. PMID:12560480

  4. Four-tiered {pi} interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity

    SciTech Connect

    Al-Mawsawi, Laith Q.; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

    2008-08-01

    HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive {pi} electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact {pi} electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact {pi} electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished {pi} interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the {pi} interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this {pi}-{pi} interaction should lead to powerful anti-retroviral drugs.

  5. Adaptive interactions between HLA and HIV-1: Highly divergent selection imposed by HLA class I molecules with common supertype motifs1

    PubMed Central

    John, Mina; Heckerman, David; James, Ian; Park, Lawrence P.; Carlson, Jonathan M.; Chopra, Abha; Gaudieri, Silvana; Nolan, David; Haas, David W.; Riddler, Sharon A.; Haubrich, Richard; Mallal, Simon

    2010-01-01

    Currently 1.1 million individuals in the United States of America are living with HIV-1 infection. While this is a relatively small proportion of the global pandemic, the remarkable mix of ancestries in the U.S.A, drawn together over the past two centuries of continuous population migrations, provides an important and unique perspective on adaptive interactions between HIV-1 and human genetic diversity. HIV-1 is a rapidly adaptable organism and mutates within or near immune epitopes which are determined by the human leukocyte antigen (HLA) class I genotype of the infected host. We characterized HLA-associated polymorphisms across the full HIV-1 proteome in a large, ethnically diverse, national U.S cohort of HIV-1 infected individuals. We found a striking divergence in the immunoselection patterns associated with HLA variants which have very similar or identical peptide binding specificities but are differentially distributed among racial/ethnic groups. Though their similarity in peptide binding functionally clusters these HLA variants into supertypes, their differences at sites within the peptide binding groove contributes to ‘race-specific’ selection effects on circulating HIV-1 viruses. This suggests that the interactions between the HLA/HIV peptide complex and the T cell receptor (TCR) varies significantly within HLA supertype groups, which in turn, influences HIV-1 evolution. PMID:20231689

  6. Gene loss and adaptation to hominids underlie the ancient origin of HIV-1.

    PubMed

    Etienne, Lucie; Hahn, Beatrice H; Sharp, Paul M; Matsen, Frederick A; Emerman, Michael

    2013-07-17

    HIV-1 resulted from cross-species transmission of SIVcpz, a simian immunodeficiency virus that naturally infects chimpanzees. SIVcpz, in turn, is a recombinant between two SIV lineages from Old World monkeys. Lentiviral interspecies transmissions are partly driven by the evolution and capacity of viral accessory genes, such as vpx, vpr, and vif, to antagonize host antiviral factors, such as SAMHD1 and the APOBEC3 proteins. We show that vpx, which in other lentiviruses antagonizes SAMHD1, was deleted during the creation of SIVcpz. This genomic deletion resulted in the reconstruction of the overlapping vif gene by "overprinting," creating a unique vif that overlaps in its 3' end with the vpr gene and can antagonize hominid APOBEC3s. Moreover, passage of SIVs through chimpanzees facilitated the subsequent adaptation of HIV-1 to humans. Thus, HIV-1 originated through a series of gene loss and adaptation events that generated its chimpanzee precursor and lowered the species barrier to human infection.

  7. Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1

    PubMed Central

    Costa, Luciana J; Chen, Nan; Lopes, Adriana; Aguiar, Renato S; Tanuri, Amilcar; Plemenitas, Ana; Peterlin, B Matija

    2006-01-01

    Background Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1. Results To this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, these functions of Nef were reproduced in primary macrophages, where the wild type but not mutant Nef proteins led to increased release of new viral particles from infected cells. Conclusion We conclude that by binding GagPol and AIP1, Nef not only proliferates MVBs but also contributes to the egress of viral particles from infected cells. PMID:16764724

  8. Advances in targeting nucleocapsid-nucleic acid interactions in HIV-1 therapy

    PubMed Central

    Garg, Divita; Torbett, Bruce E.

    2014-01-01

    The continuing challenge of HIV-1 treatment resistance in patients creates a need for the development of new antiretroviral inhibitors. The HIV nucleocapsid (NC) protein is a potential therapeutic target. NC is necessary for viral RNA packaging and in the early stages of viral infection. The high level of NC amino acid conservation among all HIV-1 clades suggests a low tolerance for mutations. Thus, NC mutations that could arise during inhibitor treatment to provide resistance may render the virus less fit. Disruption of NC function provides a unique opportunity to strongly dampen replication at multiple points during the viral life cycle with a single inhibitor. Although NC exhibits desirable features for a potential antiviral target, the structural flexibility, size, and the presence of two zinc fingers makes small molecule targeting of NC a challenging task. In this review, we discuss the recent advances in strategies to develop inhibitors of NC function and present a perspective on potential novel approaches that may help to overcome some of the current challenges in the field. PMID:25026536

  9. Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition.

    PubMed

    Galatola, R; Cruz, A; Gómara, M J; Prat, J; Alsina, M A; Haro, I; Pujol, M

    2015-02-01

    The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m(-1)) and the HIV-1 FP (35 mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

  10. Topological Layers in the HIV-1 gp120 Inner Domain Regulate gp41 Interaction and CD4-Triggered Conformational Transitions

    PubMed Central

    Finzi, Andrés; Xiang, Shi-Hua; Pacheco, Beatriz; Wang, Liping; Haight, Jessica; Kassa, Aemro; Danek, Brenda; Pancera, Marie; Kwong, Peter D.; Sodroski, Joseph

    2010-01-01

    SUMMARY The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (“Layers 1 and 2”) in the gp120 inner domain. Both layers apparently contribute to the non-covalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, Layer 1-Layer 2 interactions strengthen gp120-CD4 binding by reducing the off-rate. Layer 1-Layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41. PMID:20227370

  11. HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

    PubMed Central

    Huang, Chih-Yang; Chiang, Shu-Fen; Lin, Tze-Yi; Chiou, Shiow-Her; Chow, Kuan-Chih

    2012-01-01

    Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4+ T lymphoblast cell line SupT1, or human primary CD4+ T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1. PMID:22438978

  12. Short Communication: HIV-1 Variants That Use Mouse CCR5 Reveal Critical Interactions of gp120's V3 Crown with CCR5 Extracellular Loop 1.

    PubMed

    Platt, Emily J; Durnin, James P; Kabat, David

    2015-10-01

    The CCR5 coreceptor amino terminus and extracellular (ECL) loops 1 and 2 have been implicated in HIV-1 infections, with species differences in these regions inhibiting zoonoses. Interactions of gp120 with CD4 and CCR5 reduce constraints on metastable envelope subunit gp41, enabling gp41 conformational changes needed for infection. We previously selected HIV-1JRCSF variants that efficiently use CCR5(Δ18) with a deleted amino terminus or CCR5(HHMH) with ECL2 from an NIH/Swiss mouse. Unexpectedly, the adaptive gp120 mutations were nearly identical, suggesting that they function by weakening gp120's grip on gp41 and/or by increasing interactions with ECL1. To analyze this and further wean HIV-1 from human CCR5, we selected variants using CCR5(HMMH) with murine ECL1 and 2 sequences. HIV-1JRCSF mutations adaptive for CCR5(Δ18) and CCR5(HHMH) were generally maladaptive for CCR5(HMMH), whereas the converse was true for CCR5(HMMH) adaptations. The HIV-1JRCSF variant adapted to CCR5(HMMH) also weakly used intact NIH/Swiss mouse CCR5. Our results strongly suggest that HIV-1JRCSF makes functionally critical contacts with human ECL1 and that adaptation to murine ECL1 requires multiple mutations in the crown of gp120's V3 loop. PMID:26114311

  13. Short Communication: HIV-1 Variants That Use Mouse CCR5 Reveal Critical Interactions of gp120's V3 Crown with CCR5 Extracellular Loop 1.

    PubMed

    Platt, Emily J; Durnin, James P; Kabat, David

    2015-10-01

    The CCR5 coreceptor amino terminus and extracellular (ECL) loops 1 and 2 have been implicated in HIV-1 infections, with species differences in these regions inhibiting zoonoses. Interactions of gp120 with CD4 and CCR5 reduce constraints on metastable envelope subunit gp41, enabling gp41 conformational changes needed for infection. We previously selected HIV-1JRCSF variants that efficiently use CCR5(Δ18) with a deleted amino terminus or CCR5(HHMH) with ECL2 from an NIH/Swiss mouse. Unexpectedly, the adaptive gp120 mutations were nearly identical, suggesting that they function by weakening gp120's grip on gp41 and/or by increasing interactions with ECL1. To analyze this and further wean HIV-1 from human CCR5, we selected variants using CCR5(HMMH) with murine ECL1 and 2 sequences. HIV-1JRCSF mutations adaptive for CCR5(Δ18) and CCR5(HHMH) were generally maladaptive for CCR5(HMMH), whereas the converse was true for CCR5(HMMH) adaptations. The HIV-1JRCSF variant adapted to CCR5(HMMH) also weakly used intact NIH/Swiss mouse CCR5. Our results strongly suggest that HIV-1JRCSF makes functionally critical contacts with human ECL1 and that adaptation to murine ECL1 requires multiple mutations in the crown of gp120's V3 loop.

  14. Secretion Modification Region-Derived Peptide Disrupts HIV-1 Nef's Interaction with Mortalin and Blocks Virus and Nef Exosome Release

    PubMed Central

    Shelton, Martin N.; Huang, Ming-Bo; Ali, Syed A.; Powell, Michael D.

    2012-01-01

    Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4+ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties. PMID:22013042

  15. Crystal structure, conformational fixation, and entry-related interactions of mature ligand-free HIV-1 Env

    PubMed Central

    Kwon, Young Do; Pancera, Marie; Acharya, Priyamvada; Georgiev, Ivelin S.; Crooks, Emma T.; Gorman, Jason; Joyce, M. Gordon; Guttman, Miklos; Ma, Xiaochu; Narpala, Sandeep; Soto, Cinque; Terry, Daniel S.; Yang, Yongping; Zhou, Tongqing; Ahlsen, Goran; Bailer, Robert T.; Chambers, Michael; Chuang, Gwo-Yu; Doria-Rose, Nicole A.; Druz, Aliaksandr; Hallen, Mark A.; Harned, Adam; Kirys, Tatsiana; Louder, Mark K.; O’Dell, Sijy; Ofek, Gilad; Osawa, Keiko; Prabhakaran, Madhu; Sastry, Mallika; Stewart-Jones, Guillaume B.E.; Stuckey, Jonathan; Thomas, Paul V.; Tittley, Tishina; Williams, Constance; Zhang, Baoshan; Zhao, Hong; Zhou, Zhou; Donald, Bruce R.; Lee, Lawrence K.; Zolla-Pazner, Susan; Baxa, Ulrich; Schön, Arne; Freire, Ernesto; Shapiro, Lawrence; Lee, Kelly K.; Arthos, James; Munro, James B.; Blanchard, Scott C.; Mothes, Walther; Binley, James M.; McDermott, Adrian B.; Mascola, John R.; Kwong, Peter D.

    2016-01-01

    As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation, and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies, but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C-433C (DS) variant, specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like particle and soluble formats providing a new generation of vaccine antigens. PMID:26098315

  16. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

    PubMed Central

    2011-01-01

    Background MicroRNA (miRNA)-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution) or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured lymphocytes as a tractable model

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

    NASA Astrophysics Data System (ADS)

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

    2003-03-01

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

  18. APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

    PubMed Central

    Krisko, John F.; Begum, Nurjahan; Baker, Caroline E.

    2016-01-01

    ABSTRACT The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF (JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA. IMPORTANCE Vif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly

  19. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

    SciTech Connect

    Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Kwon, Young Do; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E.; Schief, William R.; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D.

    2010-04-15

    The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded {beta}-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate - and structurally plastic - layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated {beta}-sandwich and providing for conformational diversity used in immune evasion. A 'layered' gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a {beta}-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

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

    PubMed

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

    2013-08-01

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

  1. D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

    SciTech Connect

    Du Li; Zhao Yaxue; Chen, Jing; Yang Liumeng; Zheng Yongtang; Tang Yun Shen Xu Jiang Hualiang

    2008-10-10

    Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. Since LEDGF/p75 plays an important role in HIV integration, disruption of the LEDGF/p75 interaction with IN has provided a special interest for anti-HIV agent discovery. In this work, we reported that a benzoic acid derivative, 4-[(5-bromo-4-{l_brace}[2,4-dioxo-3-(2-oxo-2-phenylethyl) -1,3-thiazolidin-5-ylidene]methyl{r_brace}-2-ethoxyphenoxy)methyl]benzoic acid (D77) could potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution thus exhibiting antiretroviral activity. Molecular docking with site-directed mutagenesis analysis and surface plasmon resonance (SPR) binding assays has clarified possible binding mode of D77 against HIV-1 integrase. As the firstly discovered small molecular compound targeting HIV-1 integrase interaction with LEDGF/p75, D77 might supply useful structural information for further anti-HIV agent discovery.

  2. HLA Class II Antigens and Their Interactive Effect on Perinatal Mother-To-Child HIV-1 Transmission

    PubMed Central

    Luo, Ma; Embree, Joanne; Ramdahin, Suzie; Bielawny, Thomas; Laycock, Tyler; Tuff, Jeffrey; Haber, Darren; Plummer, Mariel; Plummer, Francis A.

    2015-01-01

    HLA class II antigens are central in initiating antigen-specific CD4+ T cell responses to HIV-1. Specific alleles have been associated with differential responses to HIV-1 infection and disease among adults. This study aims to determine the influence of HLA class II genes and their interactive effect on mother-child perinatal transmission in a drug naïve, Mother-Child HIV transmission cohort established in Kenya, Africa in 1986. Our study showed that DRB concordance between mother and child increased risk of perinatal HIV transmission by three fold (P = 0.00035/Pc = 0.0014, OR: 3.09, 95%CI, 1.64-5.83). Whereas, DPA1, DPB1 and DQB1 concordance between mother and child had no significant influence on perinatal HIV transmission. In addition, stratified analysis showed that DRB1*15:03+ phenotype (mother or child) significantly increases the risk of perinatal HIV-1 transmission. Without DRB1*15:03, DRB1 discordance between mother and child provided 5 fold protection (P = 0.00008, OR: 0.186, 95%CI: 0.081-0.427). However, the protective effect of DRB discordance was diminished if either the mother or the child was DRB1*15:03+ phenotype (P = 0.49-0.98, OR: 0.7-0.99, 95%CI: 0.246-2.956). DRB3+ children were less likely to be infected perinatally (P = 0.0006, Pc = 0.014; OR:0.343, 95%CI:0.183-0.642). However, there is a 4 fold increase in risk of being infected at birth if DRB3+ children were born to DRB1*15:03+ mother compared to those with DRB1*15:03- mother. Our study showed that DRB concordance/discordance, DRB1*15:03, children’s DRB3 phenotype and their interactions play an important role in perinatal HIV transmission. Identification of genetic factors associated with protection or increased risk in perinatal transmission will help develop alternative prevention and treatment methods in the event of increases in drug resistance of ARV. PMID:25945792

  3. Fluorescence energy transfer monitoring of protein-protein interaction in human cells: the Cyclin T1-HIV1 Tat case.

    NASA Astrophysics Data System (ADS)

    Ferrari, Aldo; Cinelli, Riccardo A. G.; Pellegrini, Vittorio; Beltram, Fabio; Marcello, Alessandro; Tyagi, Mudit; Giacca, Mauro

    2001-03-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein promotes transcriptional elongation of viral RNAs. Here we show that human Cyclin T1 directly binds Tat in cultured cells. By mapping fluorescence resonance energy transfer (FRET) in different cellular compartments we shall present a quantitative analysis of this interaction. The matched tagging pair consists of two optically matched variants of the green fluorescent protein: the enhanced GFP and the blue fluorescent protein. Strong energy transfer was observed between Cyclin T1 and Tat both in the cytoplasm and in specific subnuclear regions. We shall argue that such high-resolution optical studies can provide significant new insight in molecular processes and demonstrate that, for the specific case-study presented, they lead to a model by which Tat recruits Cyclin T1 out of the nuclear compartments where the protein resides to promote transcriptional activation.

  4. Strategies for inhibiting function of HIV-1 accessory proteins: a necessary route to AIDS therapy?

    PubMed

    Richter, S N; Frasson, I; Palù, G

    2009-01-01

    The Human Immunodeficiency Virus (HIV) genome encodes three major structural proteins common to all retroviruses (Gag, Pol and Env), two regulatory proteins (Tat and Rev) that are essential for viral replication, and four accessory proteins (Nef, Vif, Vpu, Vpr). While accessory proteins were initially reported to be unnecessary for viral growth, their importance as virulence factors is now being more and more appreciated: they can dramatically alter the course and severity of viral infection, replication and disease progression. None of the HIV accessory proteins display enzymatic activity: they rather act altering cellular pathways via multiple protein-protein interactions with a number of host cell factors. All currently approved anti-HIV drugs target pol and env encoded proteins. Therefore, widening the molecular targets of HIV therapy by additionally targeting accessory proteins may expand treatment options, resulting in high impact effective new therapy. In this review we present the state of the art of compounds that target HIV accessory proteins. Most of the research has focused on the inhibition of specific accessory proteins/host cell partner interactions. Promising compounds have been found within different classes of molecules: small natural and synthetic molecules, peptides and proteins, oligonucleotides, in particular those used as RNA interference (RNAi) tools. With the assortment of compounds available, especially against Nef and Vif functions, the demonstration of the clinical efficacy of the new anti-HIV-1 drugs targeting accessory proteins is next challenge.

  5. Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

    SciTech Connect

    Stanley,B.; Ehrlich, E.; Short, L.; Yu, Y.; Xiao, Z.; Yu, X.; Xiong, Y.

    2008-01-01

    Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

  6. Surfactant Protein D Inhibits HIV-1 Infection of Target Cells via Interference with gp120-CD4 Interaction and Modulates Pro-Inflammatory Cytokine Production

    PubMed Central

    Pandit, Hrishikesh; Gopal, Sandhya; Sonawani, Archana; Yadav, Ajit Kumar; Qaseem, Asif S.; Warke, Himangi; Patil, Anushree; Gajbhiye, Rahul; Kulkarni, Vijay; Al-Mozaini, Maha Ahmed; Idicula-Thomas, Susan; Kishore, Uday; Madan, Taruna

    2014-01-01

    Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection. PMID:25036364

  7. Probing Multidrug-Resistance and Protein-Ligand Interactions with Oxatricyclic Designed Ligands in HIV-1 Protease Inhibitors

    SciTech Connect

    Ghosh, Arun K.; Xu, Chun-Xiao; Rao, Kalapala V.; Baldridge, Abigail; Agniswamy, Johnson; Wang, Yuan-Fang; Weber, Irene T.; Aoki, Manabu; Miguel, Salcedo Pedro; Amano, Masayuki; Mitsuya, Hiroaki

    2010-10-29

    We report the design, synthesis, biological evaluation, and X-ray crystallographic analysis of a new class of HIV-1 protease inhibitors. Compound 4 proved to be an extremely potent inhibitor toward various multidrug-resistant HIV-1 variants, representing a near 10-fold improvement over darunavir (DRV). Compound 4 also blocked protease dimerization with at least 10-fold greater potency than DRV.

  8. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    PubMed Central

    2012-01-01

    Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting. PMID:22420777

  9. Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia.

    PubMed

    An, Ping; Penugonda, Sudhir; Thorball, Christian W; Bartha, Istvan; Goedert, James J; Donfield, Sharyne; Buchbinder, Susan; Binns-Roemer, Elizabeth; Kirk, Gregory D; Zhang, Wenyan; Fellay, Jacques; Yu, Xiao-Fang; Winkler, Cheryl A

    2016-03-01

    Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37-0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression.

  10. Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia.

    PubMed

    An, Ping; Penugonda, Sudhir; Thorball, Christian W; Bartha, Istvan; Goedert, James J; Donfield, Sharyne; Buchbinder, Susan; Binns-Roemer, Elizabeth; Kirk, Gregory D; Zhang, Wenyan; Fellay, Jacques; Yu, Xiao-Fang; Winkler, Cheryl A

    2016-03-01

    Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37-0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression. PMID:26942578

  11. Role of APOBEC3F Gene Variation in HIV-1 Disease Progression and Pneumocystis Pneumonia

    PubMed Central

    An, Ping; Penugonda, Sudhir; Thorball, Christian W.; Bartha, Istvan; Goedert, James J.; Donfield, Sharyne; Buchbinder, Susan; Binns-Roemer, Elizabeth; Kirk, Gregory D.; Zhang, Wenyan; Fellay, Jacques; Yu, Xiao-Fang; Winkler, Cheryl A.

    2016-01-01

    Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37–0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression. PMID:26942578

  12. Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target

    PubMed Central

    Rawle, Daniel J.; Qin, Fangyun; Wang, Rui; Soares, Dinesh C.; Jin, Hongping; Sivakumaran, Haran; Lin, Min-Hsuan; Spann, Kirsten; Abbott, Catherine M.; Harrich, David

    2015-01-01

    Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3–4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and

  13. Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target.

    PubMed

    Li, Dongsheng; Wei, Ting; Rawle, Daniel J; Qin, Fangyun; Wang, Rui; Soares, Dinesh C; Jin, Hongping; Sivakumaran, Haran; Lin, Min-Hsuan; Spann, Kirsten; Abbott, Catherine M; Harrich, David

    2015-12-01

    Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and

  14. The interaction of HIV-1 Tat(32-72) with its target RNA: a fluorescence and nuclear magnetic resonance study.

    PubMed

    Metzger, A U; Bayer, P; Willbold, D; Hoffmann, S; Frank, R W; Goody, R S; Rösch, P

    1997-12-01

    We performed intrinsic peptide fluorescence experiments to characterize the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. Kd values for wild-type BP1 and cysteine-modified BP1 were found to be in the range of 60 to 70 nM for both peptides, indicating that free sulfhydryl groups of the cysteines within the peptide are not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1SW) was used to further investigate peptide RNA interaction by fluorescence studies. Titration of BP1SW with TAR resulted in a dissociation constant (Kd = 9 nM) nearly an order of magnitude lower than that of the wild-type peptide. The change of the BP1SW fluorescence intensity on TAR binding was used to investigate the kinetics of this interaction by stopped-flow experiments. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement step (k approximately 60 s-1) in order to form the specific BP1SW-TAR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1SW and BP1SW bound to TAR shows that only resonances from amino acid residues of the core and basic sequence regions are shifted on TAR binding.

  15. Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction.

    PubMed Central

    Esposito, D; Craigie, R

    1998-01-01

    HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction. PMID:9755183

  16. Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by quaternary interactions and variable loops

    SciTech Connect

    Kwon, Young Do; Finzi, Andrés; Wu, Xueling; Dogo-Isonagie, Cajetan; Lee, Lawrence K.; Moore, Lucas R.; Schmidt, Stephen D.; Stuckey, Jonathan; Yang, Yongping; Zhou, Tongqing; Zhu, Jiang; Vicic, David A.; Debnath, Asim K.; Shapiro, Lawrence; Bewley, Carole A.; Mascola, John R.; Sodroski, Joseph G.; Kwong, Peter D.

    2013-03-04

    The HIV-1 envelope (Env) spike (gp120{sub 3}/gp41{sub 3}) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a 'ground state' for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from 'snapping' into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.

  17. In-Depth Analysis of the Interaction of HIV-1 with Cellular microRNA Biogenesis and Effector Mechanisms

    PubMed Central

    Whisnant, Adam W.; Bogerd, Hal P.; Flores, Omar; Ho, Phong; Powers, Jason G.; Sharova, Natalia; Stevenson, Mario; Chen, Chin-Ho; Cullen, Bryan R.

    2013-01-01

    ABSTRACT The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. PMID:23592263

  18. Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

    PubMed

    Dinh, Minh H; Anderson, Meegan R; McRaven, Michael D; Cianci, Gianguido C; McCoombe, Scott G; Kelley, Z L; Gioia, Casey J; Fought, Angela J; Rademaker, Alfred W; Veazey, Ronald S; Hope, Thomas J

    2015-03-01

    To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV

  19. 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

  20. Probability weighted ensemble transfer learning for predicting interactions between HIV-1 and human proteins.

    PubMed

    Mei, Suyu

    2013-01-01

    Reconstruction of host-pathogen protein interaction networks is of great significance to reveal the underlying microbic pathogenesis. However, the current experimentally-derived networks are generally small and should be augmented by computational methods for less-biased biological inference. From the point of view of computational modelling, data scarcity, data unavailability and negative data sampling are the three major problems for host-pathogen protein interaction networks reconstruction. In this work, we are motivated to address the three concerns and propose a probability weighted ensemble transfer learning model for HIV-human protein interaction prediction (PWEN-TLM), where support vector machine (SVM) is adopted as the individual classifier of the ensemble model. In the model, data scarcity and data unavailability are tackled by homolog knowledge transfer. The importance of homolog knowledge is measured by the ROC-AUC metric of the individual classifiers, whose outputs are probability weighted to yield the final decision. In addition, we further validate the assumption that only the homolog knowledge is sufficient to train a satisfactory model for host-pathogen protein interaction prediction. Thus the model is more robust against data unavailability with less demanding data constraint. As regards with negative data construction, experiments show that exclusiveness of subcellular co-localized proteins is unbiased and more reliable than random sampling. Last, we conduct analysis of overlapped predictions between our model and the existing models, and apply the model to novel host-pathogen PPIs recognition for further biological research.

  1. Interactions of HIV-1 inhibitory peptide T20 with the gp41 N-HR coiled coil.

    PubMed

    Champagne, Kelly; Shishido, Akira; Root, Michael J

    2009-02-01

    Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K(D) = 30 nm), close to its IC50 concentration (approximately 3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K(D) = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF --> ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012-21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K(D) = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher

  2. Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

    NASA Astrophysics Data System (ADS)

    Serena, Michela; Giorgetti, Alejandro; Busato, Mirko; Gasparini, Francesca; Diani, Erica; Romanelli, Maria Grazia; Zipeto, Donato

    2016-03-01

    HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

  3. Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120

    PubMed Central

    Madani, Navid; Schön, Arne; Princiotto, Amy M.; LaLonde, Judith M.; Courter, Joel R.; Soeta, Takahiro; Ng, Danny; Wang, Liping; Brower, Evan T.; Xiang, Shi-Hua; Kwon, Young Do; Huang, Chih-chin; Wyatt, Richard; Kwong, Peter D.; Freire, Ernesto; Smith, Amos B.; Sodroski, Joseph

    2008-01-01

    Summary Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells, and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogues and gp120 mutants suggest that: 1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4-bound conformation; 2) the NBD-556 phenyl ring projects into the Phe 43 cavity; 3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and 4) increased affinity of NBD-556 analogues for gp120 improves antiviral potency during infection of CD4-expressing cells. PMID:19000821

  4. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    SciTech Connect

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-02-20

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  5. CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

    PubMed Central

    Greenwood, Alexander I.; Pan, Jianjun; Mills, Thalia T.; Nagle, John F.; Epand, Richard M.; Tristram-Nagle, Stephanie

    2008-01-01

    This study uses low-angle (LAXS) and wide-angle (WAXS) x-ray synchrotron scattering, volume measurements and thin layer chromatography to determine structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by ~3 Å, and cause the area/unit cell (peptide+SOPC) to increase by ~9 Å2 from the area/lipid of SOPC at 30 °C (67.0 ± 0.9 Å2). Model fitting suggests that LWYIK’s average position is slightly closer to the bilayer center than IWYIK’s, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease KC, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily. PMID:18262490

  6. Unbiased proteomic analysis of proteins interacting with the HIV-1 5′LTR sequence: role of the transcription factor Meis

    PubMed Central

    Tacheny, A.; Michel, S.; Dieu, M.; Payen, L.; Arnould, T.; Renard, P.

    2012-01-01

    To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5′long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5′LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5′LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches. PMID:22904091

  7. 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

  8. Tail-interacting protein TIP47 is a connector between Gag and Env and is required for Env incorporation into HIV-1 virions

    PubMed Central

    Lopez-Vergès, Sandra; Camus, Grégory; Blot, Guillaume; Beauvoir, Roxane; Benarous, Richard; Berlioz-Torrent, Clarisse

    2006-01-01

    The presence of the envelope glycoprotein Env in HIV-1 virions is essential for infectivity. To date, the molecular mechanism by which Env is packaged into virions has been largely unknown. Here, we show that TIP47 (tail-interacting protein of 47 kDa), which has been shown to interact with Env, also binds the MA (matrix) domain of HIV-1 Gag protein and that these three proteins form a ternary complex. Mutations in Gag that abrogate interaction with TIP47 inhibit Env incorporation and virion infectivity as well as colocalization between Gag and Env. We also show that TIP47 silencing impairs Env incorporation and infectivity and abolishes coimmunoprecipitation of Gag with Env. In contrast, overexpression of TIP47 increases Env packaging. Last, we demonstrate that TIP47 can interact simultaneously with Env and Gag. Taken together, our results show that TIP47 is a cellular cofactor that plays an essential role in Env incorporation, allowing the encounter and the physical association between HIV-1 Gag and Env proteins during the viral assembly process. PMID:17003132

  9. Tail-interacting protein TIP47 is a connector between Gag and Env and is required for Env incorporation into HIV-1 virions.

    PubMed

    Lopez-Vergès, Sandra; Camus, Grégory; Blot, Guillaume; Beauvoir, Roxane; Benarous, Richard; Berlioz-Torrent, Clarisse

    2006-10-01

    The presence of the envelope glycoprotein Env in HIV-1 virions is essential for infectivity. To date, the molecular mechanism by which Env is packaged into virions has been largely unknown. Here, we show that TIP47 (tail-interacting protein of 47 kDa), which has been shown to interact with Env, also binds the MA (matrix) domain of HIV-1 Gag protein and that these three proteins form a ternary complex. Mutations in Gag that abrogate interaction with TIP47 inhibit Env incorporation and virion infectivity as well as colocalization between Gag and Env. We also show that TIP47 silencing impairs Env incorporation and infectivity and abolishes coimmunoprecipitation of Gag with Env. In contrast, overexpression of TIP47 increases Env packaging. Last, we demonstrate that TIP47 can interact simultaneously with Env and Gag. Taken together, our results show that TIP47 is a cellular cofactor that plays an essential role in Env incorporation, allowing the encounter and the physical association between HIV-1 Gag and Env proteins during the viral assembly process.

  10. Naphthalenedisulfonic acid derivatives inhibit HIV-1-induced cytopathogenesis, syncytia formation and virus-cell binding by interaction with the viral envelope glycoprotein

    SciTech Connect

    Mohan, P.; Schols, D.; De Clercq, E.; Shigeta, S.; Baba, M.

    1993-12-31

    Bis naphthalenedisulfonic acid analogs with biphenyl spacers have exhibited potent and selective inhibition of HIV-1 replication and giant cell formation. FACS analysis has revealed that these agents also inhibit viral binding to the target cell. Further mechanism of action studies by the FACA method demonstrate that the sulfonic acid analogs inhibit binding of anti-gp120 monoclonal antibody to the viral envelope of glycoprotein, gp120. Binding of OKT4A/Leu3a monoclonal antibody to the target cell CD4 receptor is not affected by these compounds. This investigation suggests that these naphthalenedisulfonic acid derivatives exert their anti-HIV-1 activity by inhibiting the gp120-CD4 interaction through binding of these agents to the viral gp120 antigen.

  11. Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event.

    PubMed Central

    De Clercq, E; Yamamoto, N; Pauwels, R; Baba, M; Schols, D; Nakashima, H; Balzarini, J; Debyser, Z; Murrer, B A; Schwartz, D

    1992-01-01

    A series of bicyclams have been shown to be potent and selective inhibitors of human immunodeficiency virus (HIV). The compounds are inhibitory to the replication of various HIV-1 and HIV-2 strains in various human T-cell systems, including peripheral blood lymphocytes, at 0.14-1.4 microM, without being toxic to the host cells at 2.2 mM. The bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant HIV-1 strains and acts additively with AZT. Mechanism of action studies revealed that the bicyclams (i.e., JM2763) interact with an early event of the retrovirus replicative cycle, which could be tentatively identified as a viral uncoating event. Images PMID:1608936

  12. Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75.

    PubMed

    Li, Bo-Wen; Zhang, Feng-Hua; Serrao, Erik; Chen, Huan; Sanchez, Tino W; Yang, Liu-Meng; Neamati, Nouri; Zheng, Yong-Tang; Wang, Hui; Long, Ya-Qiu

    2014-06-15

    HIV integrase (IN) is an essential enzyme for the viral replication. Currently, three IN inhibitors have been approved for treating HIV-1 infection. All three drugs selectively inhibit the strand transfer reaction by chelating a divalent metal ion in the enzyme active site. Flavonoids are a well-known class of natural products endowed with versatile biological activities. Their β-ketoenol or catechol structures can serve as a metal chelation motif and be exploited for the design of novel IN inhibitors. Using the metal chelation as a common pharmacophore, we introduced appropriate hydrophobic moieties into the flavonol core to design natural product-based novel IN inhibitors. We developed selective and efficient syntheses to generate a series of mono 3/5/7/3'/4'-substituted flavonoid derivatives. Most of these new compounds showed excellent HIV-1 IN inhibitory activity in enzyme-based assays and protected against HIV-1 infection in cell-based assays. The 7-morpholino substituted 7c showed effective antiviral activity (EC50=0.826 μg/mL) and high therapeutic index (TI>242). More significantly, these hydroxyflavones block the IN-LEDGF/p75 interaction with low- to sub-micromolar IC50 values and represent a novel scaffold to design new generation of drugs simultaneously targeting the catalytic site as well as protein-protein interaction domains.

  13. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters.

    PubMed

    Ziegler, André; Seelig, Joachim

    2004-01-01

    The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal

  14. Primary human epithelial cell culture system for studying interactions between female upper genital tract and sexually transmitted viruses, HSV-2 and HIV-1.

    PubMed

    Kaushic, Charu; Nazli, Aisha; Ferreira, Victor H; Kafka, Jessica K

    2011-10-01

    Evidence from clinical and epidemiological studies indicates that women are disproportionately susceptible to sexually transmitted viral infections. To understand the underlying biological basis for this increased susceptibility, more studies are needed to examine the acute events in the female reproductive tract following exposure to viruses during sexual transmission. The epithelial lining of the female reproductive tract is the primary barrier that sexually transmitted viruses, such as HIV-1 and HSV-2 need to infect or traverse, in order to initiate and establish productive infection. We have established an ex-vivo primary culture system to grow genital epithelial cells from upper reproductive tract tissues of women. Using these cultures, we have extensively examined the interactions between epithelial cells of the female genital tract and HSV-2 and HIV-1. In this review, we describe in detail the experimental protocol to grow these cultures, monitor their differentiation and inoculate with HSV-2 and HIV-1. Prospective use of these cultures to re-create the microenvironment in the reproductive tract is discussed.

  15. Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1.

    PubMed

    Jain, Niyati; Morgan, Christopher E; Rife, Brittany D; Salemi, Marco; Tolbert, Blanton S

    2016-01-29

    Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein. PMID:26607354

  16. 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

  17. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

    PubMed

    Kenyon, Julia C; Prestwood, Liam J; Lever, Andrew M L

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

  18. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader

    PubMed Central

    Kenyon, Julia C.; Prestwood, Liam J.; Lever, Andrew M. L.

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome. PMID:26449409

  19. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

    PubMed

    Kenyon, Julia C; Prestwood, Liam J; Lever, Andrew M L

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome. PMID:26449409

  20. Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

    PubMed Central

    Kassler, Kristin; Meier, Julia; Eichler, Jutta; Sticht, Heinrich

    2011-01-01

    The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120. PMID:22312332

  1. Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

    PubMed Central

    Friew, Yeshitila N; Boyko, Vitaly; Hu, Wei-Shau; Pathak, Vinay K

    2009-01-01

    Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules. PMID:19497112

  2. 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…

  3. Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

    PubMed

    Wynn, Jessica E; Zhang, Wenyu; Tebit, Denis M; Gray, Laurie R; Hammarskjold, Marie-Louise; Rekosh, David; Santos, Webster L

    2016-09-01

    A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding. PMID:27091070

  4. Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

    PubMed Central

    Yuan, Yaxia; Quizon, Pamela M.; Sun, Wei-Lun; Yao, Jianzhuang; Zhu, Jun; Zhan, Chang-Guo

    2016-01-01

    HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND. PMID:27250920

  5. Specific interaction of CXCR4 with CD4 and CD8{alpha}: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

    SciTech Connect

    Basmaciogullari, Stephane . E-mail: basmaciogullari@cochin.inserm.fr; Pacheco, Beatriz; Bour, Stephan; Sodroski, Joseph

    2006-09-15

    We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8{alpha} in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8{alpha}/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8{alpha} molecules.

  6. 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.

  7. HIV-1 Capsid: The Multifaceted Key Player in HIV-1 infection

    PubMed Central

    Campbell, Edward M.; Hope, Thomas J.

    2016-01-01

    In a mature, infectious HIV-1 virion, the viral genome is housed within a conical capsid core comprised of the viral capsid (CA) protein. The CA protein, and the structure into which it assembles, facilitate virtually every step of infection through a series of interactions with multiple host cell factors. This review describes our understanding of the interactions between the viral capsid core and several cellular factors that enable efficient HIV-1 genome replication, timely core disassembly, nuclear import and the integration of the viral genome into the genome of the target cell. We then discuss how elucidating these interactions can reveal new targets for therapeutic interactions against HIV-1. PMID:26179359

  8. HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2

    PubMed Central

    Fernández-Oliva, Alberto; Finzi, Andrés; Haim, Hillel; Menéndez-Arias, Luis; Sodroski, Joseph

    2015-01-01

    ABSTRACT Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the

  9. INTERACTIONS OF DIFFERENT INHIBITORS WITH ACTIVE-SITE ASPARTYL RESIDUES OF HIV-1 PROTEASE AND POSSIBLE RELEVANCE TO PEPSINS

    PubMed Central

    Sayer, Jane M.; Louis, John M.

    2008-01-01

    The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors (symmetric cyclic urea inhibitor DMP323, non-hydrolysable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)) was assessed by differential scanning calorimetry. ΔTm values, defined as the difference in Tm for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 °C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by ΔTm ratios [ΔTm(PR)/ΔTm(PRD25N)] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (ΔTm ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (ΔTm(PR)/ΔTm(PRD29N) ≥ 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (ΔTm ratios of 1.4-2.2). Of the 9 FDA approved clinical HIV-1 protease inhibitors screened, APV, RTV and DRV competitively inhibit porcine pepsin with Ki values of 0.3, 0.6 and 2.14 μM, respectively. DSC results were consistent with this relatively weak binding of APV (ΔTm 2.7 °C) compared with the tight binding of AcPEP (ΔTm ≥17 °C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32 and Ser219 in pepsin, equivalent to Asp25, Asp25′ and Asp29 in PR, in the binding and stabilization of the pepsin/APV complex. PMID:18951411

  10. Effects of the protonation state in the interaction of an HIV-1 reverse transcriptase (RT) amino acid, Lys101, and a non nucleoside RT inhibitor, GW420867X.

    PubMed

    Galembeck, Sérgio E; Bickelhaupt, F Matthias; Fonseca Guerra, Célia; Galembeck, Eduardo

    2014-07-01

    Interactions between an inhibitor and amino acids from a binding pocket could help not only to understand the nature of these interactions, but also to support the design of new inhibitors. In this paper, we explore the key interaction between a second generation non-nucleoside reverse transcriptase inhibitor (NNRTI), GW420867X, and HIV-1 RT amino acid Lys101 (K101), by quantum mechanical methods. The neutral, protonated, and zwitterionic complexes of GW420867X-K101 were studied. The interaction energies were determined by SCS-MP2/def2-cc-pVQZ, and the electron density was analyzed by natural bond orbital (NBO), atoms in molecules (AIM) and reduced gradient analysis. A large increase in the interaction was observed with the tautomerization of neutral or neutral protonated species. The monomers interact by two medium-strength hydrogen bonds, one partially covalent and another noncovalent. There are some van der Waals intramolecular interactions that are topologically unstable. The nature of the intermolecular interactions was also analyzed using quantitative molecular orbital (MO) theory in combination with an energy decomposition analysis (EDA) based on dispersion-corrected density functional theory (DFT) at BLYP-D/TZ2P. PMID:24965933

  11. Cell-cell interactions regulate dendritic cell-dependent HIV-1 production in CD4+ T lymphocytes.

    PubMed

    Pinchuk, L M; Polacino, P S; Agy, M B; Klaus, S J; Clark, E A

    1995-01-01

    We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.

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

    SciTech Connect

    Zhai, Q.; Fisher, R.D.; Chung, H.-Y.; Myszka, D.G.; Sundquist, W.I.; Hill, C.P.

    2009-05-28

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

  13. Cellular immune correlates analysis of an HIV-1 preexposure prophylaxis trial

    PubMed Central

    Kuebler, Peter J.; Mehrotra, Megha L.; McConnell, J. Jeff; Holditch, Sara J.; Shaw, Brian I.; Tarosso, Leandro F.; Leadabrand, Kaitlyn S.; Milush, Jeffrey M.; York, Vanessa A.; Raposo, Rui André Saraiva; Cheng, Rex G.; Eriksson, Emily M.; McMahan, Vanessa; Glidden, David V.; Shiboski, Stephen; Grant, Robert M.; Nixon, Douglas F.; Kallás, Esper G.

    2015-01-01

    HIV-1–specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case–control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1–negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19–0.66 and HR = 0.52, 95% CI = 0.28–0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1–specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes. PMID:26100867

  14. Molecular dynamics modeling the synthetic and biological polymers interactions pre-studied via docking: anchors modified polyanions interference with the HIV-1 fusion mediator.

    PubMed

    Tsvetkov, Vladimir B; Serbin, Alexander V

    2014-06-01

    In previous works we reported the design, synthesis and in vitro evaluations of synthetic anionic polymers modified by alicyclic pendant groups (hydrophobic anchors), as a novel class of inhibitors of the human immunodeficiency virus type 1 (HIV-1) entry into human cells. Recently, these synthetic polymers interactions with key mediator of HIV-1 entry-fusion, the tri-helix core of the first heptad repeat regions [HR1]3 of viral envelope protein gp41, were pre-studied via docking in terms of newly formulated algorithm for stepwise approximation from fragments of polymeric backbone and side-group models toward real polymeric chains. In the present article the docking results were verified under molecular dynamics (MD) modeling. In contrast with limited capabilities of the docking, the MD allowed of using much more large models of the polymeric ligands, considering flexibility of both ligand and target simultaneously. Among the synthesized polymers the dinorbornen anchors containing alternating copolymers of maleic acid were selected as the most representative ligands (possessing the top anti-HIV activity in vitro in correlation with the highest binding energy in the docking). To verify the probability of binding of the polymers with the [HR1]3 in the sites defined via docking, various starting positions of polymer chains were tried. The MD simulations confirmed the main docking-predicted priority for binding sites, and possibilities for axial and belting modes of the ligands-target interactions. Some newly MD-discovered aspects of the ligand's backbone and anchor units dynamic cooperation in binding the viral target clarify mechanisms of the synthetic polymers anti-HIV activity and drug resistance prevention.

  15. Rapid screening and identification of active ingredients in licorice extract interacting with V3 loop region of HIV-1 gp120 using ACE and CE-MS.

    PubMed

    Li, Zhongjie; Zhao, Yiran; Lin, Weiwei; Ye, Min; Ling, Xiaomei

    2015-01-01

    The binding of envelope protein gp120 to glycosphingolipids is very important during the human immunodeficiency virus entering into the host cell. This step occurs in the V3 loop region in particularly. The conserved core sequence of V3 loop in gp120 was named R15K. Anti-HIV drug targeting to R15K would avoid the drug-resistance caused by HIV-1 genetic diversity. Here, for the first time, affinity capillary electrophoresis (ACE) and capillary electrophoresis-mass spectrometry (CE-MS) were used for establishing a simple, rapid and effective method of screening the licorice extract for biological activity (anti-HIV), which avoided the complicated isolation and purification process. R15K, 3'-sialyllactose (the positive control), and d-galactose (the negative control) were used for the development and validation of ACE method. After the interaction between licorice extract and R15K was confirmed by ACE, the relative active ingredients were isolated by SPE and their structures were determined by CE-ESI-MS online. In this research, two mixtures from licorice extract were found to be active. Furthermore, glycyrrhizin and licorice saponin G2 were verified as the main ingredients that significantly interacted with R15K via CE-MS and LC-MS. The results of quantitative assays showed that the active mixture contained glycyrrhizin of 74.23% and licorice saponin G2 of 9.52%. Calculated by Scatchard analysis method, glycyrrhizin/R15K complex had the highest binding constant (1.69 ± 0.08) × 10(7)L/mol among 27 compounds isolated from licorice extract. The anti-HIV activity of glycyrrhizin was further confirmed by bioactive experiment of cellular level. This strategy might provide a high throughput screening and identifying platform for seeking HIV-1 inhibitors in natural products.

  16. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

    PubMed

    Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  17. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

    PubMed Central

    Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  18. Large-scale expression and purification of the major HIV-1 coreceptor CCR5 and characterization of its interaction with RANTES.

    PubMed

    Nisius, Lydia; Rogowski, Marco; Vangelista, Luca; Grzesiek, Stephan

    2008-10-01

    The G protein-coupled receptor CCR5 is a human chemokine receptor involved in the activation and migration of leukocytes. CCR5 is also the major HIV-1 coreceptor that, together with human CD4 and the viral glycoprotein gp120, promotes virus entry into host cells. Thus inhibition of the CCR5-gp120 interaction presents a promising route to prevent HIV infections. Atomic structural details of the interaction between CCR5 and its cognate chemokines or gp120 are presently unknown due to the general difficulties of membrane protein structure determination. Here, we report the high-yield expression of human CCR5 in baculovirus-infected Sf9 insect cells. Highly purified (>90%) CCR5 is obtained in detergent-solubilized form at yields of about 1mg/l cell culture. The conformational integrity of recombinant CCR5 after purification is shown by immunoprecipitation with the conformation-dependent monoclonal antibody 2D7, CD and NMR spectroscopy. The detergent micelles contain CCR5 in monomeric and dimeric forms, which can be separated by size exclusion chromatography and characterized individually. Further functional characterization by isothermal titration calorimetry indicates that the recombinant receptor interacts with its cognate chemokine RANTES. This interaction is strongly suppressed when sulfation of CCR5 is inhibited in the insect cells.

  19. Slow binding-tight binding interaction between benzimidazol-2-one inhibitors and HIV-1 reverse transcriptase containing the lysine 103 to asparagine mutation.

    PubMed

    Samuele, Alberta; Crespan, Emmanuele; Vitellaro, Samanta; Monforte, Anna-Maria; Logoteta, Patrizia; Chimirri, Alba; Maga, Giovanni

    2010-06-01

    Novel benzimidazol-2-one non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been recently identified, through rational structure-based molecular modeling and docking approaches, as highly effective inhibitors of the wild type and drug-resistant HIV-1 reverse transcriptase (RT). These compounds also showed potent anti-HIV activities against viral strains, superior to the clinically approved NNRTI efavirenz. However, they were still of limited efficacy towards the K103N mutant. Here we report a detailed enzymatic analysis elucidating the molecular mechanism of interaction between benzimidazol-2-one derivatives and the K103N mutant RT. The loss of potency of these molecules towards the K103N RT was specifically due to a reduction of their association rate to the enzyme. Unexpectedly, these compounds showed a strongly reduced dissociation rate from the K103N mutant, as compared to the wild type enzyme, suggesting that, once occupied by the drug, the mutated binding site could achieve a more stable interaction with these molecules. The characterization of this slow binding-tight binding mutant-specific mechanism of interaction may pave the way to the design of more effective new generation benzimidazol-2-one NNRTIs with promising drug resistant profile and minimal toxicity. PMID:20307579

  20. 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.

  1. 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.

  2. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

    PubMed Central

    Racine, Pierre-Jean; Chamontin, Célia; de Rocquigny, Hugues; Bernacchi, Serena; Paillart, Jean-Christophe; Mougel, Marylène

    2016-01-01

    HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT. PMID:27273064

  3. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

    PubMed

    Racine, Pierre-Jean; Chamontin, Célia; de Rocquigny, Hugues; Bernacchi, Serena; Paillart, Jean-Christophe; Mougel, Marylène

    2016-01-01

    HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT. PMID:27273064

  4. Platelets and HIV-1 infection: old and new aspects.

    PubMed

    Torre, Donato; Pugliese, Agostino

    2008-09-01

    In this review we summarize the data on interaction of platelets with HIV-1 infection. Thrombocytopenia is a common finding among HIV-1 infected patients; several combined factors contribute to low peripheral platelet counts, which are present during all the stages of the disease. In addition, a relationship between platelet count, plasma viral load and disease progression has been reported, and this shows the potential influence platelets may have on the natural history of HIV-1 disease. Several lines of evidence have shown that platelets are an integral part of inflammation, and can be also potent effector cells of innate immune response as well as of adaptive immunity. Thus, we rewieved the role of inflammatory cytokines, and chemokines as activators of platelets during HIV-1 infection. Moreover, platelets show a direct interaction with HIV-1 itself, through different pathogenic mechanisms as binding, engulfment, internalisation of HIV-1, playing a role in host defence during HIV-1 infection, by limiting viral spread and probably by inactivating viral particles. Platelets may also play an intriguing role on endothelial dysfunction present in HIV-1 infection, and this topic begins to receive systematic study, inasmuch as interaction between platelets and endothelial cells is important in the pathogenesis of atherosclerosis in HIV-1 infected patients, especially in those patients treated with antiretroviral drugs. Finally, this review attempts to better define the state of this emerging issue, to focus areas of potential clinical relevance, and to suggest several directions for future research.

  5. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    NASA Astrophysics Data System (ADS)

    Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-02-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

  6. Characterization of the HIV-1 TAR RNA-Tat peptide and drug interactions by on-line acoustic wave sensor

    NASA Astrophysics Data System (ADS)

    Tassew, Nardos Gobena

    This thesis presents the application of the thickness shear-mode (TSM) acoustic wave sensor to the study of RNA-protein and RNA-drug interactions at the solid-liquid interface. The binding of the human immunodeficiency virus-type 1 Tat protein to the trans-activation responsive RNA element (TAR) has been studied using this sensor. Data from such measurements show that the sensor is able to discriminate between different Tat peptides derived from the parent protein based on size. The effects of mutations introduced at specific sites in the protein and RNA on the TAR-Tat binding have also been examined in detail. Reduced level of response in acoustic parameters due to mutations was observed indicating that the decrease in binding in response to site specific mutations can be acoustically detected. Data from acoustic wave sensor measurements indicate that the TAR-Tat binding is also affected by ionic strength. Both the frequency and motional resistance signals show periodic responses when varying concentrations of salt are introduced on a TAR-modified surface. The binding of the two molecules seems to be a function of the response of the nucleic acid to salt concentrations. The kinetics of binding of Tat peptides to TAR RNA and to a bulge mutant analogue (MTAR) is also examined from the rate of change of the series resonant frequency. Results from such analysis illustrate longer Tat peptides formed more stable complexes with TAR RNA and exhibited increased discrimination between mutant and wild type TAR. The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to TAR RNA and their effectiveness in preventing TAR-Tat complex formation has been studied in detail. Binding affinity is directly correlated with the inhibitory potency of these molecules and the TSM sensor shows that neomycin exhibits at least a ten fold greater affinity to TAR and that it is also a more potent inhibitor than streptomycin. The results from this research involving TAR-Tat and

  7. A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

    PubMed Central

    Eckhardt, Manon; Anders, Maria; Muranyi, Walter; Heilemann, Mike; Krijnse-Locker, Jacomine; Müller, Barbara

    2011-01-01

    Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIVSNAP, which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. PMID:21799764

  8. Sialic acid associated with αvβ3 integrin mediates HIV-1 Tat protein interaction and endothelial cell proangiogenic activation.

    PubMed

    Chiodelli, Paola; Urbinati, Chiara; Mitola, Stefania; Tanghetti, Elena; Rusnati, Marco

    2012-06-01

    Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies. PMID:22528484

  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. 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.

  11. Opiate addiction therapies and HIV-1 Tat: interactive effects on glial [Ca²⁺]i, oxyradical and neuroinflammatory chemokine production and correlative neurotoxicity.

    PubMed

    Fitting, Sylvia; Zou, Shiping; El-Hage, Nazira; Suzuki, Masami; Paris, Jason J; Schier, Christina J; Rodríguez, José W; Rodriguez, Myosotys; Knapp, Pamela E; Hauser, Kurt F

    2014-01-01

    Few preclinical studies have compared the relative therapeutic efficacy of medications used to treat opiate addiction in relation to neuroAIDS. Here we compare the ability of methadone and buprenorphine, and the prototypic opiate morphine, to potentiate the neurotoxic and proinflammatory ([Ca²⁺]i, ROS, H₂O₂, chemokines) effects of HIV-1 Tat in neuronal and/or mixed-glial co-cultures. Repeated observations of neurons during 48 h exposure to combinations of Tat, equimolar concentrations (500 nM) of morphine, methadone, or buprenorphine exacerbated neurotoxicity significantly above levels seen with Tat alone. Buprenorphine alone displayed marked neurotoxicity at 500 nM, prompting additional studies of its neurotoxic effects at 5 nM and 50 nM concentrations ± Tat. In combination with Tat, buprenorphine displayed paradoxical, concentration-dependent, neurotoxic and neuroprotective actions. Buprenorphine neurotoxicity coincided with marked elevations in [Ca²⁺]i, but not increases in glial ROS or chemokine release. Tat by itself elevated the production of CCL5/RANTES, CCL4/MIP-1β, and CCL2/MCP-1. Methadone and buprenorphine alone had no effect, but methadone interacted with Tat to further increase production of CCL5/RANTES. In combination with Tat, all drugs significantly increased glial [Ca²⁺]i, but ROS was only significantly increased by co-exposure with morphine. Taken together, the increases in glial [Ca²⁺]i, ROS, and neuroinflammatory chemokines were not especially accurate predictors of neurotoxicity. Despite similarities, opiates displayed differences in their neurotoxic and neuroinflammatory interactions with Tat. Buprenorphine, in particular, was partially neuroprotective at a low concentration, which may result from its unique pharmacological profile at multiple opioid receptors. Overall, the results reveal differences among addiction medications that may impact neuroAIDS.

  12. Escherichia coli LysU is a potential surrogate for human lysyl tRNA synthetase in interactions with the C-terminal domain of HIV-1 capsid protein.

    PubMed

    Boonyalai, Nonlawat; Pullen, James R; Abdul Wahab, Mohd Firdaus; Wright, Michael; Miller, Andrew D

    2013-01-28

    Human lysyl-tRNA synthetase (hLysRS) is known to interact directly with human immunodeficiency virus type-1 (HIV-1) GagPol polyproteins, and both hLysRS with tRNA(Lys3) are selectively packaged into emerging HIV-1 viral particles. This packaging process appears to be mediated by contact between the motif 1 helix h7 of hLysRS and the C-terminal dimerization domain of the HIV-1 capsid protein (CA) segment of Gag or GagPol. Given similarities between hLysRS and Escherichia coli (E. coli) heat shock protein LysU, we investigate if LysU might be an hLysRS surrogate for interactions with Gag or GagPol proteins. We report on a series of studies involving three CA C-domains: CA(146) (intact domain), CA(151) (truncated domain), and CA(146)-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA(146) are dimeric whilst CA(151) and M185A remain monomeric, we use glutathione S-transferase (GST) pull-down assays to demonstrate the existence of specific interactions between LysU and all three CA-C domains. By means of (1)H-NMR titration experiments, we estimate K(d) values of 50 μM for the interaction between LysU and CA(146) or >500 μM for interactions between LysU and CA(151) or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA(146) take place through dimer-dimer interactions resulting in a α(2)β(2) heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA(146) proteins, and hLysRS/Gag binding interactions have also been suggested to involve only αβ heterodimer formation. Nevertheless, we propose that LysU could act as a surrogate for hLysRS with respect to Gag and GagPol polyprotein interactions although arguably not sufficiently for LysU to act as an inhibitor of the HIV-1 life cycle without further adaptation or mutation. Potentially, LysU and/or LysU mutants could represent a new class of anti-HIV-1 therapeutic agent.

  13. The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+ Smokers

    PubMed Central

    Barjaktarevic, Igor Z.; Crystal, Ronald G.

    2016-01-01

    Rationale. Matrix metalloproteinase-9 (MMP-9) expression is upregulated in alveolar macrophages (AM) of HIV1+ smokers who develop emphysema. Knowing that lung epithelial lining fluid (ELF) of HIV1+ smokers contains increased levels of inflammatory cytokines compared to HIV1− smokers, we hypothesized that upregulation of lung cytokines in HIV1+ smokers may be functionally related to increased MMP-9 expression. Methods. Cytokine arrays evaluated cytokine protein levels in ELF obtained from 5 groups of individuals: HIV1− healthy nonsmokers, HIV1− healthy smokers, HIV1− smokers with low diffusing capacity (DLCO), HIV1+ nonsmokers, and HIV1+ smokers with low DLCO. Results. Increased levels of the Th17 related cytokine IL-23 were found in HIV1− smokers with low DLCO and HIV1+ smokers and nonsmokers. Relative IL-23 gene expression was increased in AM of HIV1+ individuals, with greater expression in AM of HIV1+ smokers with low DLCO. Infection with HIV1 in vitro induced IL-23 expression in normal AM. IL-23 stimulation of AM/lymphocyte cocultures in vitro induced upregulation of MMP-9. Lung T lymphocytes express receptor IL-23R and interact with AM in order to upregulate MMP-9. Conclusion. This mechanism may contribute to the increased tissue destruction in the lungs of HIV1+ smokers and suggests that Th17 related inflammation may play a role. PMID:27446965

  14. The Role of Interleukin-23 in the Early Development of Emphysema in HIV1(+) Smokers.

    PubMed

    Barjaktarevic, Igor Z; Crystal, Ronald G; Kaner, Robert J

    2016-01-01

    Rationale. Matrix metalloproteinase-9 (MMP-9) expression is upregulated in alveolar macrophages (AM) of HIV1(+) smokers who develop emphysema. Knowing that lung epithelial lining fluid (ELF) of HIV1(+) smokers contains increased levels of inflammatory cytokines compared to HIV1(-) smokers, we hypothesized that upregulation of lung cytokines in HIV1(+) smokers may be functionally related to increased MMP-9 expression. Methods. Cytokine arrays evaluated cytokine protein levels in ELF obtained from 5 groups of individuals: HIV1(-) healthy nonsmokers, HIV1(-) healthy smokers, HIV1(-) smokers with low diffusing capacity (DLCO), HIV1(+) nonsmokers, and HIV1(+) smokers with low DLCO. Results. Increased levels of the Th17 related cytokine IL-23 were found in HIV1(-) smokers with low DLCO and HIV1(+) smokers and nonsmokers. Relative IL-23 gene expression was increased in AM of HIV1(+) individuals, with greater expression in AM of HIV1(+) smokers with low DLCO. Infection with HIV1 in vitro induced IL-23 expression in normal AM. IL-23 stimulation of AM/lymphocyte cocultures in vitro induced upregulation of MMP-9. Lung T lymphocytes express receptor IL-23R and interact with AM in order to upregulate MMP-9. Conclusion. This mechanism may contribute to the increased tissue destruction in the lungs of HIV1(+) smokers and suggests that Th17 related inflammation may play a role. PMID:27446965

  15. Discovery of novel low-molecular-weight HIV-1 inhibitors interacting with cyclophilin A using in silico screening and biological evaluations.

    PubMed

    Tian, Yu-Shi; Verathamjamras, Chris; Kawashita, Norihito; Okamoto, Kousuke; Yasunaga, Teruo; Ikuta, Kazuyoshi; Kameoka, Masanori; Takagi, Tatsuya

    2013-01-01

    Cyclophilin A has attracted attention recently as a new target of anti-human immunodeficiency virus type 1 (HIV-1) drugs. However, so far no drug against HIV-1 infection exhibiting this mechanism of action has been approved. To identify new potent candidates for inhibitors, we performed in silico screening of a commercial database of more than 1,300 drug-like compounds by using receptor-based docking studies. The candidates selected from docking studies were subsequently tested using biological assays to assess anti-HIV activities. As a result, two compounds were identified as the most active. Specifically, both exhibited anti-HIV activity against viral replication at a low concentration and relatively low cytotoxicity at the effective concentration inhibiting viral growth by 50%. Further modification of these molecules may lead to the elucidation of potent inhibitors of HIV-1.

  16. The Effect of N-acetylation and N-methylation of Lysine Residue of Tat Peptide on its Interaction with HIV-1 TAR RNA

    PubMed Central

    Kumar, Santosh; Maiti, Souvik

    2013-01-01

    Post-translational modification (PTM) of RNA binding proteins (RBPs) play a very important role in determining their binding to cognate RNAs and therefore regulate the downstream effects. Lysine can undergo various PTMs and thereby contribute to the regulation of different cellular processes. It can be reversibly acetylated and methylated using a pool of respective enzymes, to act as a switch for controlling the binding efficiency of RBPs. Here we have delineated the thermodynamic and kinetic effects of N-acetylation and N-monomethylation of lysine on interaction between HIV-1 TAR RNA and its cognate binder Tat peptide ( a model system). Our results indicate that acetylation of lysine 50 (K50), leads to eight- fold reduction in binding affinity, originating exclusively from entropy changes whereas, lysine 51 (K51) acetylation resulted only in three fold decrease with large enthalpy-entropy compensation. The measurement of kinetic parameters indicated major change (4.5 fold) in dissociation rate in case of K50 acetylation however, K51 acetylation showed similar effect on both association and dissociation rates. In contrast, lysine methylation did not affect the binding affinity of Tat peptide to TAR RNA at K50, nonetheless three fold enhancement in binding affinity was observed at K51 position. In spite of large enthalpy-entropy compensation, lysine methylation seems to have more pronounced position specific effect on the kinetic parameters. In case of K50 methylation, simultaneous increase was observed in the rate of association and dissociation leaving binding affinity unaffected. The increased binding affinity for methylated Tat at K51 stems from faster association rate with slightly slower dissociation rate. PMID:24147034

  17. 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.

  18. Pharmacokinetic Interactions between BMS-626529, the Active Moiety of the HIV-1 Attachment Inhibitor Prodrug BMS-663068, and Ritonavir or Ritonavir-Boosted Atazanavir in Healthy Subjects

    PubMed Central

    Hruska, Matthew; Hwang, Carey; Shah, Vaishali; Furlong, Michael; Hanna, George J.; Bertz, Richard; Savant Landry, Ishani

    2015-01-01

    BMS-663068 is a prodrug of BMS-626529, a first-in-class attachment inhibitor that binds directly to HIV-1 gp120, preventing initial viral attachment and entry into host CD4+ T cells. This open-label, multiple-dose, four-sequence, crossover study addressed potential two-way drug-drug interactions following coadministration of BMS-663068 (BMS-626529 is a CYP3A4 substrate), atazanavir (ATV), and ritonavir (RTV) (ATV and RTV are CYP3A4 inhibitors). Thirty-six healthy subjects were randomized 1:1:1:1 to receive one of four treatment sequences with three consecutive treatments: BMS-663068 at 600 mg twice daily (BID), BMS-663068 at 600 mg BID plus RTV at 100 mg once daily (QD), ATV at 300 mg QD plus RTV at 100 mg QD (RTV-boosted ATV [ATV/r]), or BMS-663068 at 600 mg BID plus ATV at 300 mg QD plus RTV at 100 mg QD. Compared with the results obtained by administration of BMS-663068 alone, coadministration of BMS-663068 with ATV/r increased the BMS-626529 maximum concentration in plasma (Cmax) and the area under the concentration-time curve in one dosing interval (AUCtau) by 68% and 54%, respectively. Similarly, coadministration of BMS-663068 with RTV increased the BMS-626529 Cmax and AUCtau by 53% and 45%, respectively. Compared with the results obtained by administration of ATV/r alone, ATV and RTV systemic exposures remained similar following coadministration of BMS-663068 with ATV/r. BMS-663068 was generally well tolerated, and there were no adverse events (AEs) leading to discontinuation, serious AEs, or deaths. Moderate increases in BMS-626529 systemic exposure were observed following coadministration of BMS-663068 with ATV/r or RTV. However, the addition of ATV to BMS-663068 plus RTV did not further increase BMS-626529 systemic exposure. ATV and RTV exposures remained similar following coadministration of BMS-663068 with either ATV/r or RTV. BMS-663068 was generally well tolerated alone or in combination with either RTV or ATV/r. PMID:25870057

  19. Interaction analysis of HIV-1 antibody 2G12 and Man9GlcNAc2 ligand: Theoretical calculations by fragment molecular orbital and MD methods

    NASA Astrophysics Data System (ADS)

    Koyama, Yuka; Ueno-Noto, Kaori; Takano, Keiko

    2013-07-01

    In HIV-1 infection, human antibody 2G12 is capable of recognizing the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. To investigate the ligand binding mechanisms of antibody 2G12 with glycans aiming for the contribution to the medications, we carried out classical molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the antibody 2G12 complex with its high-mannose ligand. We found that Mannose D1 of the ligand had the largest binding affinity with the antibody, which was well consistent with experimental reports. Furthermore, significant roles of Mannose 4 and 4‧ in the ligand binding were theoretically indicated.

  20. 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

  1. Target Cell APOBEC3C Can Induce Limited G-to-A Mutation in HIV-1

    PubMed Central

    Bourara, Khaoula; Liegler, Teri J; Grant, Robert M

    2007-01-01

    The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition. PMID:17967058

  2. Evidence of at Least Two Introductions of HIV-1 in the Amerindian Warao Population from Venezuela

    PubMed Central

    Rangel, Héctor R.; Maes, Mailis; Villalba, Julian; Sulbarán, Yoneira; de Waard, Jacobus H.; Bello, Gonzalo; Pujol, Flor H.

    2012-01-01

    Background The Venezuelan Amerindians were, until recently, free of human immunodeficiency virus (HIV) infection. However, in 2007, HIV-1 infection was detected for the first time in the Warao Amerindian population living in the Eastern part of Venezuela, in the delta of the Orinoco river. The aim of this study was to analyze the genetic diversity of the HIV-1 circulating in this population. Methodology/Principal Findings The pol genomic region was sequenced for 16 HIV-1 isolates and for some of them, sequences from env, vif and nef genomic regions were obtained. All HIV-1 isolates were classified as subtype B, with exception of one that was classified as subtype C. The 15 subtype B isolates exhibited a high degree of genetic similarity and formed a highly supported monophyletic cluster in each genomic region analyzed. Evolutionary analyses of the pol genomic region indicated that the date of the most recent common ancestor of the Waraos subtype B clade dates back to the late 1990s. Conclusions/Significance At least two independent introductions of HIV-1 have occurred in the Warao Amerindians from Venezuela. The HIV-1 subtype B was successfully established and got disseminated in the community, while no evidence of local dissemination of the HIV-1 subtype C was detected in this study. These results warrant further surveys to evaluate the burden of this disease, which can be particularly devastating in this Amerindian population, with a high prevalence of tuberculosis, hepatitis B, among other infectious diseases, and with limited access to primary health care. PMID:22808212

  3. Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups.

    PubMed

    Fan, Xing; Zhang, Feng-Hua; Al-Safi, Rasha I; Zeng, Li-Fan; Shabaik, Yumna; Debnath, Bikash; Sanchez, Tino W; Odde, Srinivas; Neamati, Nouri; Long, Ya-Qiu

    2011-08-15

    HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

  4. Design of HIV-1 Integrase Inhibitors Targeting the Catalytic Domain as Well as Its Interaction with LEDGF/p75: A Scaffold Hopping Approach Using Salicylate and Catechol Groups

    PubMed Central

    Fan, Xing; Zhang, Feng-Hua; Al-Safi, Rasha I.; Zeng, Li-Fan; Shabaik, Yumna; Debnath, Bikash; Sanchez, Tino W.; Odde, Srinivas; Neamati, Nouri; Long, Ya-Qiu

    2011-01-01

    HIV-1 integrase (IN) is a validated therapeutic target for antiviral agents. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands new structure and new mechanism IN inhibitors. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC50 = 5 μM) with more than 40-fold selectivity for the strand transfer over the 3′-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC50 value of 8 μM. Based on the molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. And the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site in IN protein. This work provided a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors. PMID:21778063

  5. 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

  6. APOBEC3G restricts early HIV-1 replication in the cytoplasm of target cells

    SciTech Connect

    Anderson, Jenny L.; Hope, Thomas J.

    2008-05-25

    Cellular APOBEC3G (A3G) protein is packaged into human immunodeficiency virus type 1 (HIV-1) virions in producer cells yet restricts viral replication in target cells. To characterize this restriction in target cells, the effect of A3G on generating various HIV-1 cDNA products was measured by quantitative real-time PCR. A3G decreased cDNA products from Vif-deficient HIV-1, with minor effects on early reverse transcripts and larger declines in late reverse transcripts. However, the greatest decline was typically observed in nuclear 2-LTR circles. Moreover, the magnitude of these declines varied with A3G dose. Adding integration inhibitor did not stop the A3G-mediated loss in 2-LTR circles. Moreover, obstructing HIV-1 nuclear entry using vesicular stomatitis virus matrix protein did not stop the A3G-mediated decline in late reverse transcripts. Collectively, these data suggest that A3G has important restriction activity in the cytoplasm and progressively diminishes viral cytoplasmic and nuclear cDNA forms with increasing magnitude during restriction.

  7. Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions.

    PubMed

    Xu, Hongzhan; Chertova, Elena; Chen, Jianbo; Ott, David E; Roser, James D; Hu, Wei-Shau; Pathak, Vinay K

    2007-04-10

    A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication. PMID:17126871

  8. The visiting internet Fiancé/ée (VIF): an emerging group of international travelers.

    PubMed

    Sofarelli, Theresa A; Birich, Holly K; Hale, DeVon C

    2014-01-01

    Here we describe an emerging category of travelers called the Visiting Internet Fiancé/ée (VIF), characterized by their travel to pursue a romantic relationship with an individual they have only encountered online. The VIF is not well identified in travel medicine literature despite having a higher risk for several travel-related issues including sexually transmitted infections, monetary fraud, and international scams. We also propose specific counseling interventions designed to minimize the adverse outcomes faced by the VIF traveler.

  9. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  10. Phylogenetic analysis provides evidence of interactions between Italian heterosexual and South American homosexual males as the main source of national HIV-1 subtype C epidemics.

    PubMed

    Lai, Alessia; Bozzi, Giorgio; Franzetti, Marco; Binda, Francesca; Simonetti, Francesco R; Micheli, Valeria; Meraviglia, Paola; Corsi, Paola; Bagnarelli, Patrizia; De Luca, Andrea; Ciccozzi, Massimo; Zehender, Gianguglielmo; Zazzi, Maurizio; Balotta, Claudia

    2014-05-01

    The HIV-1 clade C is prevalent worldwide and spread from Africa to South East Asia and South America early in the course of the epidemic. As a consequence of migration waves about 13% of the Italian HIV-1 epidemic is sustained by this clade. Two hundred fifty-four C pol sequences from the Italian ARCA database collected during 1997-2011 were analyzed. Epidemiological networks and geographical fluxes were identified through phylogeny using Bayesian approaches. Patients' country of origin was Italy, Africa, South America, and South East Asia for 44.9%, 23.6%, 4.7%, and 1.6%, respectively. Heterosexuals and men having sex with men accounted for 83.2% and 16.8%, respectively. Modality of infection was distributed differently: heterosexuals were largely prevalent among Italians (84.1%) and Africans (95.3%), while men having sex with men predominated among South Americans (66.7%). Eight significant clusters encompassing 111 patients (43.7%) were identified. Comparison between clustering and non-clustering patients indicated significant differences in country of origin, modality of infection and gender. Men having sex with men were associated to a higher probability to be included in networks (70% for men having sex with men vs. 30.3% for heterosexuals). Phylogeography highlighted two significant groups. One contained Indian strains and the second encompassed South Americans and almost all Italian strains. Phylogeography indicated that the spread of C subtype among Italians is related to South American variant. Although Italian patients mainly reported themselves as heterosexuals, homo-bisexual contacts were likely their source of infection. Phylogenetic monitoring is warranted to guide public health interventions aimed at controlling HIV infection.

  11. 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

  12. 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.

  13. HIV-1 antiretroviral drug therapy.

    PubMed

    Arts, Eric J; Hazuda, Daria J

    2012-04-01

    The most significant advance in the medical management of HIV-1 infection has been the treatment of patients with antiviral drugs, which can suppress HIV-1 replication to undetectable levels. The discovery of HIV-1 as the causative agent of AIDS together with an ever-increasing understanding of the virus replication cycle have been instrumental in this effort by providing researchers with the knowledge and tools required to prosecute drug discovery efforts focused on targeted inhibition with specific pharmacological agents. To date, an arsenal of 24 Food and Drug Administration (FDA)-approved drugs are available for treatment of HIV-1 infections. These drugs are distributed into six distinct classes based on their molecular mechanism and resistance profiles: (1) nucleoside-analog reverse transcriptase inhibitors (NNRTIs), (2) non-nucleoside reverse transcriptase inhibitors (NNRTIs), (3) integrase inhibitors, (4) protease inhibitors (PIs), (5) fusion inhibitors, and (6) coreceptor antagonists. In this article, we will review the basic principles of antiretroviral drug therapy, the mode of drug action, and the factors leading to treatment failure (i.e., drug resistance).

  14. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    PubMed

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells.

  15. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    PubMed Central

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  16. 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

  17. Genotype dependent QSAR for HIV-1 protease inhibition.

    PubMed

    Boutton, Carlo W; De Bondt, Hendrik L; De Jonge, Marc R

    2005-03-24

    The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs. PMID:15771454

  18. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  19. COMMD1/Murr1 Reinforces HIV-1 Latent Infection through IκB-α Stabilization

    PubMed Central

    Taura, Manabu; Kudo, Eriko; Kariya, Ryusho; Goto, Hiroki; Matsuda, Kouki; Hattori, Shinichiro; Vaeteewoottacharn, Kulthida; McDonald, Fiona; Suico, Mary Ann; Shuto, Tsuyoshi; Kai, Hirofumi

    2014-01-01

    ABSTRACT The transcription factor NF-κB is important for HIV-1 transcription initiation in primary HIV-1 infection and reactivation in latently HIV-1-infected cells. However, comparative analysis of the regulation and function of NF-κB in latently HIV-1-infected cells has not been done. Here we show that the expression of IκB-α, an endogenous inhibitor of NF-κB, is enhanced by latent HIV-1 infection via induction of the host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells by using four sets of latently HIV-1-infected cells and the respective parental cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during Toll-like receptor ligand and tumor necrosis factor alpha treatment and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the phosphoinositol 3-kinase (PI3K)–JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Our findings indicate that COMMD1 induction is the NF-κB inhibition mechanism in latently HIV-1-infected cells that contributes to innate immune deficiency and reinforces HIV-1 latency. Thus, COMMD1 might be a double-edged sword that is beneficial in primary infection but not beneficial in latent infection when HIV-1 eradication is considered. IMPORTANCE HIV-1 latency is a major barrier to viral eradication in the era of combination antiretroviral therapy. In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IκB-α by increasing the interaction with IκB-α in latently HIV-1-infected myeloid cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during the innate immune response and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the PI3K-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Thus, the host-derived factor COMMD1 is beneficial in suppressing primary infection but enhances

  20. AKAP149 binds to HIV-1 reverse transcriptase and is involved in the reverse transcription.

    PubMed

    Lemay, Julie; Maidou-Peindara, Priscilla; Cancio, Reynel; Ennifar, Eric; Coadou, Gaël; Maga, Giovanni; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

    2008-11-21

    Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.

  1. 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.

  2. Regional Differences in Prevalence of HIV-1 Discordance in Africa and Enrollment of HIV-1 Discordant Couples into an HIV-1 Prevention Trial

    PubMed Central

    Lingappa, Jairam R.; Lambdin, Barrot; Bukusi, Elizabeth Ann; Ngure, Kenneth; Kavuma, Linda; Inambao, Mubiana; Kanweka, William; Allen, Susan; Kiarie, James N.; Makhema, Joseph; Were, Edwin; Manongi, Rachel; Coetzee, David; de Bruyn, Guy; Delany-Moretlwe, Sinead; Magaret, Amalia; Mugo, Nelly; Mujugira, Andrew; Ndase, Patrick; Celum, Connie

    2008-01-01

    Background Most HIV-1 transmission in Africa occurs among HIV-1-discordant couples (one partner HIV-1 infected and one uninfected) who are unaware of their discordant HIV-1 serostatus. Given the high HIV-1 incidence among HIV-1 discordant couples and to assess efficacy of interventions for reducing HIV-1 transmission, HIV-1 discordant couples represent a critical target population for HIV-1 prevention interventions and prevention trials. Substantial regional differences exist in HIV-1 prevalence in Africa, but regional differences in HIV-1 discordance among African couples, has not previously been reported. Methodology/Principal Findings The Partners in Prevention HSV-2/HIV-1 Transmission Trial (“Partners HSV-2 Study”), the first large HIV-1 prevention trial in Africa involving HIV-1 discordant couples, completed enrollment in May 2007. Partners HSV-2 Study recruitment data from 12 sites from East and Southern Africa were used to assess HIV-1 discordance among couples accessing couples HIV-1 counseling and testing, and to correlate with enrollment of HIV-1 discordant couples. HIV-1 discordance at Partners HSV-2 Study sites ranged from 8–31% of couples tested from the community. Across all study sites and, among all couples with one HIV-1 infected partner, almost half (49%) of couples were HIV-1 discordant. Site-specific monthly enrollment of HIV-1 discordant couples into the clinical trial was not directly associated with prevalence of HIV-1 discordance, but was modestly correlated with national HIV-1 counseling and testing rates and access to palliative care/basic health care (r = 0.74, p = 0.09). Conclusions/Significance HIV-1 discordant couples are a critical target for HIV-1 prevention in Africa. In addition to community prevalence of HIV-1 discordance, national infrastructure for HIV-1 testing and healthcare delivery and effective community outreach strategies impact recruitment of HIV-1 discordant couples into HIV-1 prevention trials. PMID

  3. Translational regulation of HIV-1 replication by HIV-1 Rev cellular cofactors Sam68, eIF5A, hRIP, and DDX3.

    PubMed

    Liu, Jinfeng; Henao-Mejia, Jorge; Liu, Hao; Zhao, Yingren; He, Johnny J

    2011-06-01

    Nuclear export and translation of HIV-1 RNA are two important posttranscriptional events for HIV-1 gene expression and replication. HIV-1 Rev functions to export unspliced and incompletely spliced HIV-1 RNA from the nucleus to the cytoplasm; it requires interaction with several cellular cofactors such as Sam68, eIF5A, hRIP, and DDX3. Meanwhile, some studies have also implicated Rev and some of its cofactors such as Sam68 in HIV-1 RNA translation. Thus, in this study, we aimed to characterize the potential function of all these four Rev cofactors in HIV-1 RNA translation. Ectopic expression, siRNA knockdown, and trans-complementation assays confirmed that all these cofactors were very important for HIV-1 gene expression and production through Rev and, accordingly, Rev-dependent reporter gene expression. Importantly, these studies revealed for the first time that each of these cofactors also regulated Rev-independent reporter gene expression. To directly determine the roles of these cofactors in HIV-1 RNA translation, we designed and synthesized a full-length capped HIV-1 RNA in vitro, transfected it into cells to bypass the RNA nuclear export step, and determined HIV-1 Gag expression from the cytoplasmic RNA in the cells that had ectopically expressed or siRNA knocked down cofactors. Gag expression was found to closely correlate with the expression levels of all these cofactors. Furthermore, we took advantage of a HIV-1 internal ribosomal entry site (IRES)-based bicistronic reporter gene assay and determined the effects of these cofactors on cap-independent IRES-mediated HIV-1 translation. The results showed that DDX3, eIF5A, and hRIP enhanced HIV-1 IRES-mediated translation, whereas Sam68 did not. Taken together, these results show that HIV-1 Rev cofactors Sam68, eIF5A, hRIP, and DDX3 also function in the translation of HIV-1 RNA and suggest that the regulatory mechanisms of HIV-1 RNA translation are likely different among these cofactors.

  4. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding

    PubMed Central

    Lin, Pin-Fang; Blair, Wade; Wang, Tao; Spicer, Timothy; Guo, Qi; Zhou, Nannan; Gong, Yi-Fei; Wang, H.-G. Heidi; Rose, Ronald; Yamanaka, Gregory; Robinson, Brett; Li, Chang-Ben; Fridell, Robert; Deminie, Carol; Demers, Gwendeline; Yang, Zheng; Zadjura, Lisa; Meanwell, Nicholas; Colonno, Richard

    2003-01-01

    BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 μM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 μM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs. PMID:12930892

  5. 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

  6. 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

  7. M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner

    PubMed Central

    Wang, Qin; Zhang, Xiaolin; Han, Yuling; Wang, Xinlu; Gao, Guangxia

    2016-01-01

    M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production. PMID:27604950

  8. M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner.

    PubMed

    Wang, Qin; Zhang, Xiaolin; Han, Yuling; Wang, Xinlu; Gao, Guangxia

    2016-01-01

    M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production. PMID:27604950

  9. 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.

  10. Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

    PubMed Central

    Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

    2014-01-01

    HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

  11. Differentially-Expressed Pseudogenes in HIV-1 Infection.

    PubMed

    Gupta, Aditi; Brown, C Titus; Zheng, Yong-Hui; Adami, Christoph

    2015-10-01

    Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

  12. C-type lectin Mermaid inhibits dendritic cell mediated HIV-1 transmission to CD4+ T cells.

    PubMed

    Nabatov, Alexey A; de Jong, Marein A W P; de Witte, Lot; Bulgheresi, Silvia; Geijtenbeek, Teunis B H

    2008-09-01

    Dendritic cells (DCs) are important in HIV-1 transmission; DCs capture invading HIV-1 through the interaction of the gp120 oligosaccharides with the C-type lectin DC-SIGN and migrate to the lymphoid tissues where HIV-1 is transmitted to T cells. Thus, the HIV-1 envelope glycoprotein gp120 is an attractive target to prevent interactions with DCs and subsequent viral transmission. Here, we have investigated whether the structural homologue of DC-SIGN, the nematode C-type lectin Mermaid can be used to prevent HIV-1 transmission by DCs. Our data demonstrate that Mermaid interacts with high mannose structures present on HIV-1 gp120 and thereby inhibits HIV-1 binding to DC-SIGN on DCs. Moreover, Mermaid inhibits DC-SIGN-mediated HIV-1 transmission from DC to T cells. We have identified Mermaid as a non-cytotoxic agent that shares the glycan specificity with DC-SIGN and inhibits DC-SIGN-gp120 interaction. The results are important for the anti-HIV-1 microbicide development directed at preventing DC-HIV-1 interactions. PMID:18597806

  13. Mechanisms for macrophage-mediated HIV-1 induction.

    PubMed

    Devadas, Krishnakumar; Hardegen, Neil J; Wahl, Larry M; Hewlett, Indira K; Clouse, Kathleen A; Yamada, Kenneth M; Dhawan, Subhash

    2004-12-01

    Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.

  14. Antiretroviral drug levels and interactions affect lipid, lipoprotein and glucose metabolism in HIV-1 seronegative subjects: A pharmacokinetic-pharmacodynamic analysis

    PubMed Central

    Rosenkranz, Susan L.; Yarasheski, Kevin E.; Para, Michael F.; Reichman, Richard C.; Morse, Gene D.

    2007-01-01

    Background: HIV-infected patients treated with antiretroviral medications (ARVs) develop undesirable changes in lipid and glucose metabolism that mimic the metabolic syndrome and may be proatherogenic. Antiretroviral drug levels and their interactions may contribute to these metabolic alterations. Methods: Fifty-six HIV-seronegative adults were enrolled in an open-label, randomized, pharmacokinetic interaction study, and received a non-nucleoside reverse transcriptase inhibitor (efavirenz on days 1-21) plus a protease inhibitor (PI; amprenavir on days 11-21), with a second PI on days 15-21 (saquinavir, nelfinavir, indinavir, or ritonavir). Fasting triglycerides, total, LDL- and HDL-cholesterol, glucose, insulin and C-peptide levels were measured on days 0, 14, 21, and 2-3 weeks after discontinuing drugs. Regression models were used to estimate changes in these parameters and associations between these changes and circulating levels of study drugs. Results: Short-term efavirenz and amprenavir administration significantly increased cholesterol, triglycerides and glucose levels. Addition of a second protease inhibitor further increased triglycerides, total- and LDL-cholesterol levels. Higher amprenavir levels predicted larger increases in triglycerides, total and LDL-cholesterol. Two weeks after all study drugs were stopped, total, LDL- and HDL-cholesterol remained elevated above baseline. Conclusions: ARV regimens that include a non-nucleoside reverse transcriptase inhibitor plus single or boosted PIs are becoming more common, but the pharmacodynamic interactions associated with these regimens can result in persistent, undesirable alterations in serum lipid/lipoprotein levels. Additional pharmacodynamic studies are needed to examine the metabolic effects of ritonavir-boosted regimens, with and without efavirenz. PMID:18007962

  15. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    SciTech Connect

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

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTV and MDR HIV-1 protease.

  16. Different mutagenic potential of HIV-1 restriction factors APOBEC3G and APOBEC3F is determined by distinct single-stranded DNA scanning mechanisms.

    PubMed

    Ara, Anjuman; Love, Robin P; Chelico, Linda

    2014-03-01

    The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹² motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F ¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F ¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif

  17. Different Mutagenic Potential of HIV-1 Restriction Factors APOBEC3G and APOBEC3F Is Determined by Distinct Single-Stranded DNA Scanning Mechanisms

    PubMed Central

    Ara, Anjuman; Love, Robin P.; Chelico, Linda

    2014-01-01

    The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (−)HIV-1 DNA. Upon replication of the (−)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an 190NPM192 motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F 191Pro to 191Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F 190NGM192 could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5

  18. BioAfrica's HIV-1 proteomics resource: combining protein data with bioinformatics tools.

    PubMed

    Doherty, Ryan S; De Oliveira, Tulio; Seebregts, Chris; Danaviah, Sivapragashini; Gordon, Michelle; Cassol, Sharon

    2005-01-01

    Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource http://bioafrica.mrc.ac.za/proteomics/index.html is a website that contains detailed information about the HIV-1 proteome and protease cleavage sites, as well as data-mining tools that can be used to manipulate and query protein sequence data, a BLAST tool for initiating structural analyses of HIV-1 proteins, and a proteomics tools directory. The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: http

  19. Near full-length genome sequence of a novel HIV-1 recombinant form (CRF01_AE/B) detected among men who have sex with men in Jilin Province, China.

    PubMed

    Li, Xingguang; Feng, Yi; Yang, Yao; Chen, Yanli; Guo, Qi; Sun, Liuyan; Zang, Xihui; Xing, Hui; Shao, Yiming

    2014-07-01

    We report here a novel HIV-1 recombinant form (CRF01_AE/B) detected from a comprehensive HIV-1 molecular epidemiologic study among men who have sex with men (MSM) in Jilin province of northeastern China. The near full-length genome (NFLG) analyses showed that the novel HIV-1 recombinant isolate (JL.RF07) was composed of CRF01_AE cluster 5 (northeastern China origin) and subtype B (U.S. and European origin), with six recombinant breakpoints observed in the pol, vif, tat, rev, and env gene regions. To the best of our knowledge, this is the first detection of a novel HIV-1 recombinant form (CRF01_AE/B) in Jilin, which may indicate an active transmission network of HIV-1 infection among MSM in the region. Further studies of the molecular epidemiology of the HIV-1 epidemic among MSM in northeastern China are necessary to gain a fuller understanding of the transmission network and potential public health impact of HIV-1 among MSM in this region.

  20. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    DOE PAGESBeta

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

  1. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    SciTech Connect

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  2. The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

    PubMed Central

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP. PMID:25723178

  3. 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.

  4. Structural Insight into HIV-1 Restriction by MxB

    PubMed Central

    Alvarez, Frances Joan D.; Summers, Brady J.; Dewdney, Tamaria G.; Aiken, Christopher; Zhang, Peijun; Engelman, Alan; Xiong, Yong

    2014-01-01

    Summary The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended anti-parallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid and this interaction depends on dimerization and the N-terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1. PMID:25312384

  5. HIV-1 Vpr—a still “enigmatic multitasker”

    PubMed Central

    Guenzel, Carolin A.; Hérate, Cécile; Benichou, Serge

    2014-01-01

    Like other HIV-1 auxiliary proteins, Vpr is conserved within all the human (HIV-1, HIV-2) and simian (SIV) immunodeficiency viruses. However, Vpr and homologous HIV-2, and SIV Vpx are the only viral auxiliary proteins specifically incorporated into virus particles through direct interaction with the Gag precursor, indicating that this presence in the core of the mature virions is mainly required for optimal establishment of the early steps of the virus life cycle in the newly infected cell. In spite of its small size, a plethora of effects and functions have been attributed to Vpr, including induction of cell cycle arrest and apoptosis, modulation of the fidelity of reverse transcription, nuclear import of viral DNA in macrophages and other non-dividing cells, and transcriptional modulation of viral and host cell genes. Even if some more recent studies identified a few cellular targets that HIV-1 Vpr may utilize in order to perform its different tasks, the real role and functions of Vpr during the course of natural infection are still enigmatic. In this review, we will summarize the main reported functions of HIV-1 Vpr and their significance in the context of the viral life cycle. PMID:24744753

  6. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity

    PubMed Central

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao

    2015-01-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell–cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  7. Genetic determinants of pediatric HIV-1 infection: vertical transmission and disease progression among children.

    PubMed Central

    Matt, C.; Roger, M.

    2001-01-01

    It is very likely that perinatal human immunodeficiency virus type 1 (HIV-1) infection is influenced by a combination of virologic and host factors. A greater understanding of the role played by various risk factors for HIV-1 infection is crucial for the design of new preventive and therapeutic strategies. In recent years, a number of studies have suggested that host genetic factors are important determinants of both the susceptibility to perinatal HIV-1 infection and the subsequent pathogenesis of acquired immunodeficiency syndrome (AIDS). Control of HIV-1 infection involves the processing of specific viral peptides and their presentation to cells of the immune system by highly polymorphic human leukocyte antigen (HLA) alleles. The contribution of multiple HLA class I and II alleles in modulating pediatric HIV/AIDS outcomes has now been confirmed by several independent groups. Penetration of HIV-1 into cells is mediated by interaction between CD4 and chemokine receptors that serve as entry coreceptors. Genetic polymorphisms in chemokine ligand and chemokine receptor genes have recently been associated both with mother-to-child HIV-1 transmission and disease progression in children. These observations suggest a key role for genetic factors in pediatric HIV-1 infection. This article describes the current state of knowledge regarding host genetic influences on pediatric HIV-1 infection and discusses the role of these genes in HIV/AIDS pathogenesis. PMID:11778647

  8. Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

    PubMed

    Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

    2016-12-01

    Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.

  9. Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

    PubMed

    Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

    2016-12-01

    Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli. PMID:27590917

  10. Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

    SciTech Connect

    Sparger, Ellen E. Dubie, Robert A.; Shacklett, Barbara L.; Cole, Kelly S.; Chang, W.L.; Luciw, Paul A.

    2008-05-10

    Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-{gamma} enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.

  11. HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production.

    PubMed

    De Francesco, Maria A; Baronio, Manuela; Poiesi, Claudio

    2011-06-01

    HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.

  12. 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

  13. 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.

  14. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

    PubMed Central

    Chugh, Pauline; Bradel-Tretheway, Birgit; Monteiro-Filho, Carlos MR; Planelles, Vicente; Maggirwar, Sanjay B; Dewhurst, Stephen; Kim, Baek

    2008-01-01

    Background Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs. Results Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production. Conclusion Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs. PMID:18237430

  15. Substance abuse, HIV-1 and hepatitis

    PubMed Central

    Parikh, Nirzari; Nonnemacher, Michael R.; Pirrone, Vanessa; Block, Timothy; Mehta, Anand; Wigdahl, Brian

    2013-01-01

    During the course of human immunodeficiency virus type 1 (HIV-1) disease, the virus has been shown to effectively escape the immune response with the subsequent establishment of latent viral reservoirs in specific cell populations within the peripheral blood (PB) and associated lymphoid tissues, bone marrow (BM), brain, and potentially other end organs. HIV-1, along with hepatitis B and C viruses (HBV and HCV), are known to share similar routes of transmission, including intravenous drug use, blood transfusions, sexual intercourse, and perinatal exposure. Substance abuse, including the use of opioids and cocaine, is a significant risk factor for exposure to HIV-1 and the development of acquired immune deficiency syndrome, as well as HBV and HCV exposure, infection, and disease. Thus, coinfection with HIV-1 and HBV or HCV is common and may be impacted by chronic substance abuse during the course of disease. HIV-1 impacts the natural course of HBV and HCV infection by accelerating the progression of HBV/HCV-associated liver disease toward end-stage cirrhosis and quantitative depletion of the CD4+ T-cell compartment. HBV or HCV coinfection with HIV-1 is also associated with increased mortality when compared to either infection alone. This review focuses on the impact of substance abuse and coinfection with HBV and HCV in the PB, BM, and brain on the HIV-1 pathogenic process as it relates to viral pathogenesis, disease progression, and the associated immune response during the course of this complex interplay. The impact of HIV-1 and substance abuse on hepatitis virus-induced disease is also a focal point. PMID:22973853

  16. 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

  17. Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

    PubMed Central

    Ceballos, Ana; Remes Lenicov, Federico; Sabatté, Juan; Rodríguez Rodrígues, Christian; Cabrini, Mercedes; Jancic, Carolina; Raiden, Silvina; Donaldson, Mónica; Agustín Pasqualini, Rodolfo; Marin-Briggiler, Clara; Vazquez-Levin, Mónica; Capani, Francisco; Amigorena, Sebastián

    2009-01-01

    Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection. PMID:19858326

  18. Combination genetic therapy to inhibit HIV-1.

    PubMed

    Strayer, David S; Branco, Francisco; Landré, Julien; BouHamdan, Mohamad; Shaheen, Farida; Pomerantz, Roger J

    2002-01-01

    Compared with single agents, combination antilentiviral pharmacotherapy targets multiple HIV-1 functions simultaneously, maximizing efficacy and decreasing chances of escape mutations. Combination genetic therapy could theoretically enhance efficacy similarly, but delivery of even single genes to high percentages of hematopoietic cells or their derivatives has proven problematic. Because of their high efficiency of gene delivery, we tested recombinant SV40-derived vectors (rSV40s) for this purpose. We made six rSV40s, each carrying a different transgene that targeted a different lentiviral function. We tested the ability of these constructs, individually and in double and triple combinations, to protect SupT1 human T lymphoma cells from HIV-1 challenge. Single chain antibodies (SFv) against CXCR4 and against HIV-1 reverse transcriptase (RT) and integrase (IN) were used, as were polymeric TAR decoys (PolyTAR) and a dominant-negative mutant of HIV-1 Rev (RevM10). Immunostaining showed that virtually all doubly treated cells expressed both transgenes. All transgenes individually protected from HIV-1 but, except for anti-CXCR4 SFv, their effectiveness diminished as challenge doses increased from 40 through 2500 tissue culture infectious dose(50) (TCID(50))/10(6) cells. However, all combinations of transgenes protected target cells better than individual transgenes, even from the highest challenge doses. Thus, combination gene therapies may inhibit HIV-1 better than single agents, and rSV40s may facilitate delivery of multigene therapeutics.

  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. Exosomes: Implications in HIV-1 Pathogenesis.

    PubMed

    Madison, Marisa N; Okeoma, Chioma M

    2015-07-20

    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.

  1. 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

  2. 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

  3. Structural basis for germline antibody recognition of HIV-1 immunogens

    PubMed Central

    Scharf, Louise; West, Anthony P; Sievers, Stuart A; Chen, Courtney; Jiang, Siduo; Gao, Han; Gray, Matthew D; McGuire, Andrew T; Scheid, Johannes F; Nussenzweig, Michel C; Stamatatos, Leonidas; Bjorkman, Pamela J

    2016-01-01

    Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design. DOI: http://dx.doi.org/10.7554/eLife.13783.001 PMID:26997349

  4. Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

    PubMed

    Rivero-Buceta, Eva; Carrero, Paula; Casanova, Elena; Doyagüez, Elisa G; Madrona, Andrés; Quesada, Ernesto; Peréz-Pérez, María Jesús; Mateos, Raquel; Bravo, Laura; Mathys, Leen; Noppen, Sam; Kiselev, Evgeny; Marchand, Christophe; Pommier, Yves; Liekens, Sandra; Balzarini, Jan; Camarasa, María José; San-Félix, Ana

    2015-12-01

    The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1. PMID:26540494

  5. Roles of HIV-1 capsid in viral replication and immune evasion.

    PubMed

    Le Sage, Valerie; Mouland, Andrew J; Valiente-Echeverría, Fernando

    2014-11-26

    The primary roles of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein are to encapsidate and protect the viral RNA genome. It is becoming increasing apparent that HIV-1 CA is a multifunctional protein that acts early during infection to coordinate uncoating, reverse transcription, nuclear import of the pre-integration complex and integration of double stranded viral DNA into the host genome. Additionally, numerous recent studies indicate that CA is playing a crucial function in HIV-1 immune evasion. Here we summarize the current knowledge on HIV-1 CA and its interactions with the host cell to promote infection. The fact that CA engages in a number of different protein-protein interactions with the host makes it an interesting target for the development of new potent antiviral agents.

  6. Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress

    PubMed Central

    Rowson, Sydney A.; Harrell, Constance S.; Bekhbat, Mandakh; Gangavelli, Apoorva; Wu, Matthew J.; Kelly, Sean D.; Reddy, Renuka; Neigh, Gretchen N.

    2016-01-01

    Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was

  7. Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress.

    PubMed

    Rowson, Sydney A; Harrell, Constance S; Bekhbat, Mandakh; Gangavelli, Apoorva; Wu, Matthew J; Kelly, Sean D; Reddy, Renuka; Neigh, Gretchen N

    2016-01-01

    Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was

  8. Identification of a Novel HIV-1 Intra-CRF01_AE in China: A Descendant of the Previously Identified CRF01_AE transmission clusters 1 and 6

    PubMed Central

    Ning, Chuanyi; Li, Xingguang; Tang, Weiming; Zhou, Bo; Cai, Weiping; Tucker, Joseph D.

    2015-01-01

    We report here a novel HIV-1 intra-CRF01_AE recombinant form (CRF01-1AE/CRF01-6AE) composed of CRF01_AE transmission clusters 1 and 6, identified among heterosexuals in Fujian, with one breakpoint observed in vif gene. The CRF01_AE region I (HXB2: 868-5184) of the recombinant was clustered with the CRF01_AE transmission cluster 1, which is mainly circulating among IDUs and heterosexuals from the southern and southwestern China, with the support of 100% bootstrap value. The CRF01_AE region II (HXB2: 5185-9605) of the recombinant was clustered with the CRF01_AE transmission cluster 6, which is mainly prevailing among heterosexuals from southern China, with the support of 100% bootstrap value. To our best knowledge, this is the first detection of a novel HIV-1 intra-CRF01_AE recombinant form (CRF01-1_AE/CRF01-6_AE) in Fujian Province, which indicates ongoing active HIV-1 transmission among heterosexuals in this region. It may help illustrate CRF01_AE genetic diversity and contribute to our understanding of HIV-1 epidemiology, pathogenesis, and vaccine development. PMID:26100011

  9. HIV-1 infection in Juba, southern Sudan.

    PubMed

    McCarthy, M C; Khalid, I O; El Tigani, A

    1995-05-01

    Thirty years of civil war in the Sudan have resulted in the isolation of the southern provinces which border Central and East Africa. Consequently, little is known about the epidemiology of HIV-1 infection in this region. To estimate the prevalence of HIV-1 infection in southern Sudan and the risk factors associated with disease transmission, a seroepidemiologic survey was conducted in the township of Juba. Study subjects invited to participate in this study included medical outpatients, inpatients hospitalized for active tuberculosis, and female prostitutes. A total of 401 subjects participated in the study. HIV-1 infection was confirmed in 25 subjects. The prevalence of HIV-1 infection was 19% (8/42) among tuberculosis patients, 16% (8/50) among prostitutes, and 3% (9/309) among outpatients. A significantly higher prevalence of HIV-1 infection was found among female prostitutes when compared to female outpatients: 16% (8/50) vs. 2% (4/178), P < 0.001. Correspondingly, the prevalence of seropositives was significantly higher among male outpatients reporting a history of sexual relations with prostitutes during the prior 10 years compared to male outpatients denying relations with prostitutes: 14% (5/37) vs. 0% (0/94), P = 0.0011. A history of a sexually transmitted disease (STD) was also associated with HIV-1 infection among male outpatients. The findings of this study indicate that HIV-1 infection is highly prevalent in southern Sudan and that prostitutes and their sexual partners represent a major reservoir of HIV infection in this population. This epidemiologic pattern resembles that seen in the African nations neighboring southern Sudan.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. 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

  11. The Role of Cationic Polypeptides in Modulating HIV-1 Infection of the Cervicovaginal Mucosa

    PubMed Central

    Cole, Amy Liese; Cole, Alexander M.

    2014-01-01

    The mucosa and overlying fluid of the female reproductive tract (FRT) are portals for the heterosexual transmission of HIV-1. Toward the ongoing development of topically applied microbicides and mucosal vaccines against HIV-1, it is evermore important to understand how the dynamic FRT mucosa is involved in controlling transmission and infection of HIV-1. Cationic peptides and proteins are the principal innate immune effector molecules of mucosal surfaces, and interact in a combinatorial fashion to modulate HIV-1 infection of the cervix and vagina. While cationic peptides and proteins have historically been categorized as antimicrobial or have other host-benefitting roles, an increasing number of these molecules have been found to augment HIV-1 infection and potentially antagonize host defense. Complex environmental factors such as hormonal fluctuations and/or bacterial and viral co-infections provide additional challenges to both experimentation and interpretation of results. In the context of heterosexual transmission of HIV-1, this review explores how various cationic peptides and proteins participate in modulating host defense against HIV-1 of the cervicovaginal mucosa. PMID:27025760

  12. Epigenetics of μ-Opioid receptors: Intersection with HIV-1 infection of the Central Nervous System

    PubMed Central

    Regan, Patrick M.; Dave, Rajnish S.; Datta, Prasun K.; Khalili, Kamel

    2014-01-01

    The abuse of intravenous drugs, such as heroin, has become a major public health concern due to the increased risk of HIV-1 infection. Opioids such as heroin were originally identified and subsequently abused for their analgesic effects. However, many investigations have found additional effects of opioids, including regulation of the immune system. As such, chronic opioid abuse has been shown to promote HIV-1 pathogenesis and facilitate HIV-1-associated neurocognitive dysfunction. Clinical opioids, such as morphine and methadone, as well as illicit opioids, such as heroin, exert their effects primarily through interactions with the μ-opioid receptor (MOR). However, the mechanisms by which opioids enhance neurocognitive dysfunction through MOR-mediated signaling pathways are not completely understood. New findings in the regulation of MOR expression, particularly epigenetic and transcriptional regulation as well as alternative splicing, sheds new insights into possible mechanisms of HIV-1 and opiate synergy. In this review, we identify mechanisms regulating MOR expression and propose novel mechanisms by which opioids and HIV-1 may modulate this regulation. Additionally, we suggest that differential regulation of newly identified MOR isoforms by opioids and HIV-1 has functional consequence in enhancing HIV-1 neurocognitive dysfunction. PMID:22034138

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

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

    2014-09-01

    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.

  19. 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

  20. 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.

  1. Restriction Factors in HIV-1 Disease Progression.

    PubMed

    Merindol, Natacha; Berthoux, Lionel

    2015-01-01

    About 35 million people worldwide were living with HIV-1 at the end of 2013 and over 25 million have already died of AIDS. AIDS patients show high variability in the speed of disease progression in the absence of treatment. While certain immunological traits have been shown to correlate with accelerated or slowed progression in some subjects, including slow progressors, factors controlling HIV-1 replication and disease kinetics remain largely enigmatic. The importance of T lymphocytes and of protective HLA-alleles is undeniable, but not sufficient to explain every attenuated phenotype. A thorough understanding of HIV-1 infection control in these patient subsets may help the development of novel strategies for treatment and prevention. Restriction factors are type I interferon-induced specialized cellular proteins that block viruses at different steps of their life cycle. TRIM5α, Mx2/MxB, TRIM22/Staf50, SAMHD1, p21/CDKN1, tetherin/BST2/CD137, APOBEC3G and APOBEC3F have all been proposed to inhibit HIV-1, often with gene variant- or cellular context-specificity. Recent evidence highlights their possible implication in AIDS disease progression. In this review, we depict their restrictive activity against HIV-1 and recapitulate the latest data on their potential role in vivo, in both normal and slow progressors.

  2. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    PubMed Central

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  3. Vif determines the requirement for CBF-β in APOBEC3 degradation

    PubMed Central

    Yoshikawa, Rokusuke; Takeuchi, Junko S.; Yamada, Eri; Nakano, Yusuke; Ren, Fengrong; Tanaka, Hiroshi; Münk, Carsten; Harris, Reuben S.; Miyazawa, Takayuki; Koyanagi, Yoshio

    2015-01-01

    APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by Vif (viral infectivity factor) of lentiviruses, which binds APOBEC3s and promotes their degradation. CBF-β (core binding factor subunit β) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-β in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here, we determined the sequence of feline CBFB and performed phylogenetic analyses. These analyses revealed that mammalian CBFB is under purifying selection. Moreover, we demonstrated that CBF-β is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggested that primate lentiviruses have adapted to use CBF-β, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses. PMID:25516542

  4. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    PubMed Central

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-01-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. PMID:26940118

  5. A Computational Model of Inhibition of HIV-1 by Interferon-Alpha.

    PubMed

    Browne, Edward P; Letham, Benjamin; Rudin, Cynthia

    2016-01-01

    Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses. PMID:27010978

  6. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-03-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

  7. A Computational Model of Inhibition of HIV-1 by Interferon-Alpha

    PubMed Central

    Browne, Edward P.; Letham, Benjamin; Rudin, Cynthia

    2016-01-01

    Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses. PMID:27010978

  8. The Presence and Anti-HIV-1 Function of Tenascin C in Breast Milk and Genital Fluids.

    PubMed

    Mansour, Robin G; Stamper, Lisa; Jaeger, Frederick; McGuire, Erin; Fouda, Genevieve; Amos, Joshua; Barbas, Kimberly; Ohashi, Tomoo; Alam, S Munir; Erickson, Harold; Permar, Sallie R

    2016-01-01

    Tenascin-C (TNC) is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.

  9. 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

  10. Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells

    PubMed Central

    Braaten, Douglas; Luban, Jeremy

    2001-01-01

    The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4+ T cells by homologous recombination. HIV-1 replication in PPIA–/– cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag’s interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA+/+ and PPIA–/– cells. Stable re-expression of CypA in PPIA–/– cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA–/– cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions. PMID:11250896

  11. Blocking of integrins inhibits HIV-1 infection of human cervical mucosa immune cells with free and complement-opsonized virions

    PubMed Central

    Tjomsland, Veronica; Ellegård, Rada; Kjölhede, Preben; Wodlin, Ninni Borendal; Hinkula, Jorma; Lifson, Jeffrey D; Larsson, Marie

    2013-01-01

    The initial interaction between HIV-1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV-1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV-1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV-1 infection of dendritic cells (DCs) compared with that by free HIV-1, but this increased infection was not observed with CD4+ T cells. Blockage of the α4-, β7-, and β1-integrins significantly inhibited HIV-1 infection of both DCs and CD4+ T cells. We found a greater impairment of HIV-1 infection in DCs for complement-opsonized virions compared with that of free virions when αM/β2- and α4-integrins were blocked. Blocking the C-type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4+ T-cell infection. We show that blocking of integrins decreases the HIV-1 infection of both mucosal DCs and CD4+ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV-1 infection. PMID:23686382

  12. Assessment of mucosal immunity to HIV-1.

    PubMed

    Jespers, Vicky; Harandi, Ali M; Hinkula, Jorma; Medaglini, Donata; Le Grand, Roger; Stahl-Hennig, Christiane; Bogers, Willy; El Habib, Raphaelle; Wegmann, Frank; Fraser, Carol; Cranage, Martin; Shattock, Robin J; Spetz, Anna-Lena

    2010-04-01

    A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines. PMID:20370549

  13. 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.

  14. 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

  15. 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.

  16. Strategies to inhibit viral protein nuclear import: HIV-1 as a target.

    PubMed

    Levin, Aviad; Loyter, Abraham; Bukrinsky, Michael

    2011-09-01

    Nuclear import is a critical step in the life cycle of HIV-1. During the early (preintegration) stages of infection, HIV-1 has to transport its preintegration complex into the nucleus for integration into the host cell chromatin, while at the later (postintegration) stages viral regulatory proteins Tat and Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclear export of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1 life cycle, this step presents an attractive target for antiviral therapeutic intervention. In this review, we describe the current state of our understanding of the interactions regulating nuclear import of the HIV-1 preintegration complex and describe current approaches to inhibit it. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

  17. Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2009-07-01

    HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

  18. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    SciTech Connect

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D.

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  19. The impact of host genetic variation on infection with HIV-1.

    PubMed

    McLaren, Paul J; Carrington, Mary

    2015-06-01

    The outcome after infection with the human immunodeficiency virus type 1 (HIV-1) is a complex phenotype determined by interactions among the pathogen, the human host and the surrounding environment. An impact of host genetic variation on HIV-1 susceptibility was identified early in the pandemic, with a major role attributed to the genes encoding class I human leukocyte antigens (HLA) and the chemokine receptor CCR5. Studies using genome-wide data sets have underscored the strength of these associations relative to variants located throughout the rest of the genome. However, the extent to which additional polymorphisms influence HIV-1 disease progression, and how much of the variability in outcome can be attributed to host genetics, remain largely unclear. Here we discuss findings concerning the functional impact of associated variants, outline methods for quantifying the host genetic component and examine how available genome-wide data sets may be leveraged to discover gene variants that affect the outcome of HIV-1 infection.

  20. Promoter Targeting RNAs: Unexpected Contributors to the Control of HIV-1 Transcription

    PubMed Central

    Suzuki, Kazuo; Ahlenstiel, Chantelle; Marks, Katherine; Kelleher, Anthony D

    2015-01-01

    In spite of prolonged and intensive treatment with combined antiretroviral therapy (cART), which efficiently suppresses plasma viremia, the integrated provirus of HIV-1 persists in resting memory CD4+ T cells as latent infection. Treatment with cART does not substantially reduce the burden of latent infection. Once cART is ceased, HIV-1 replication recrudesces from these reservoirs in the overwhelming majority of patients. There is increasing evidence supporting a role for noncoding RNAs (ncRNA), including microRNAs (miRNAs), antisense (as)RNAs, and short interfering (si)RNA in the regulation of HIV-1 transcription. This appears to be mediated by interaction with the HIV-1 promoter region. Viral miRNAs have the potential to act as positive or negative regulators of HIV transcription. Moreover, inhibition of virally encoded long-asRNA can induce positive transcriptional regulation, while antisense strands of siRNA targeting the NF-κB region suppress viral transcription. An in-depth understanding of the interaction between ncRNAs and the HIV-1 U3 promoter region may lead to new approaches for the control of HIV reservoirs. This review focuses on promoter associated ncRNAs, with particular emphasis on their role in determining whether HIV-1 establishes active or latent infection. PMID:25625613

  1. The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors.

    PubMed

    Jiang, Fan; Chen, Wei; Yi, Kejia; Wu, Zhiqiang; Si, Yiling; Han, Weidong; Zhao, Yali

    2010-12-01

    Four catechins with the galloyl moiety, including catechin gallate (CG), epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), and epicatechin gallate (ECG), were found to inhibit HIV-1 integrase effectively as determined by our ELISA method. In our docking study, it is proposed that when the HIV-1 integrase does not combine with virus DNA, the four catechins may bind to Tyr143 and Gln148, thus altering the flexibility of the loop (Gly140-Gly149), which could lead to an inhibition of HIV-1 integrase activity. In addition, after combining HIV-1 integrase with virus DNA, the four catechins may bind between the integrase and virus DNA, consequently, disrupt this interaction. Thus, the four catechins may reduce the activity of HIV-1 integrase by disrupting its interaction with virus DNA. The four catechins have a highly cooperative inhibitory effect (IC₅₀=0.1 μmol/L). Our study suggests that catechins with the galloyl moiety could be a novel and effective class of HIV-1 integrase inhibitors.

  2. Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor

    PubMed Central

    Hagiwara, Kyoji; Ishii, Hideki; Murakami, Tomoyuki; Takeshima, Shin-nosuke; Chutiwitoonchai, Nopporn; Kodama, Eiichi N.; Kawaji, Kumi; Kondoh, Yasumitsu; Honda, Kaori; Osada, Hiroyuki; Tsunetsugu-Yokota, Yasuko; Suzuki, Masaaki; Aida, Yoko

    2015-01-01

    The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54–74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection. PMID:26701275

  3. The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions

    PubMed Central

    2014-01-01

    Background Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. Results A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. Conclusion Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection. PMID:24447338

  4. Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

    PubMed

    Weinberg, J B; Shugars, D C; Sherman, P A; Sauls, D L; Fyfe, J A

    1998-05-19

    Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase. PMID:9610370

  5. 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

  6. A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1

    PubMed Central

    Langer, Simon M.; Hopfensperger, Kristina; Iyer, Shilpa S.; Kreider, Edward F.; Learn, Gerald H.; Lee, Lan-Hui; Hahn, Beatrice H.; Sauter, Daniel

    2015-01-01

    Background Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown. Results Examining infectious molecular clones (IMCs) of HIV-1 that encode the rev1-vpu polymorphism, we show that a fusion protein is expressed in infected cells. Due to the splicing pattern of viral mRNA, however, these same IMCs also express a regular Vpu protein, which is produced at much higher levels. To investigate the function of the fusion gene, we characterized isogenic IMC pairs differing only in their ability to express a Rev1-Vpu protein. Analysis in transfected HEK293T and infected CD4+ T cells showed that all of these viruses were equally active in known Vpu functions, such as down-modulation of CD4 or counteraction of tetherin. Furthermore, the polymorphism did not affect Vpu-mediated inhibition of NF-кB activation or Rev-dependent nuclear export of incompletely spliced viral mRNAs. There was also no evidence for enhanced replication of Rev1-Vpu expressing viruses in primary PBMCs or ex vivo infected human lymphoid tissues. Finally, the frequency of HIV-1 quasispecies members that encoded a rev1-vpu fusion gene did not change in HIV-1 infected individuals over time. Conclusions Expression of a rev1-vpu fusion gene does not affect regular Rev and Vpu functions or alter HIV-1 replication in primary target cells. Since there is no evidence for increased replication fitness of rev1-vpu encoding viruses, this polymorphism likely emerged in the context of other mutations within and/or outside the rev1-vpu intergenic region, and may have a neutral phenotype. PMID:26554585

  7. Pentosan polysulfate as an inhibitor of extracellular HIV-1 Tat.

    PubMed

    Rusnati, M; Urbinati, C; Caputo, A; Possati, L; Lortat-Jacob, H; Giacca, M; Ribatti, D; Presta, M

    2001-06-22

    HIV-1 Tat protein, released from HIV-infected cells, may act as a pleiotropic heparin-binding growth factor. From this observation, extracellular Tat has been implicated in the pathogenesis of AIDS and of AIDS-associated pathologies. Here we demonstrate that the heparin analog pentosan polysulfate (PPS) inhibits the interaction of glutathione S-transferase (GST)-Tat protein with heparin immobilized to a BIAcore sensor chip. Competition experiments showed that Tat-PPS interaction occurs with high affinity (K(d) = 9.0 nm). Also, GST.Tat prevents the binding of [(3)H]heparin to GST.Tat immobilized to glutathione-agarose beads. In vitro, PPS inhibits GST.Tat internalization and, consequently, HIV-1 long terminal repeat transactivation in HL3T1 cells. Also, PPS inhibits cell surface interaction and mitogenic activity of GST.Tat in murine adenocarcinoma T53 Tat-less cells. In all assays, PPS exerts its Tat antagonist activity with an ID(50) equal to approximately 1.0 nm. In vivo, PPS inhibits the neovascularization induced by GST.Tat or by Tat-overexpressing T53 cells in the chick embryo chorioallantoic membrane. In conclusion, PPS binds Tat protein and inhibits its cell surface interaction, internalization, and biological activity in vitro and in vivo. PPS may represent a prototypic molecule for the development of novel Tat antagonists with therapeutic implications in AIDS and AIDS-associated pathologies, including Kaposi's sarcoma.

  8. 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

  9. 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

  10. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

    PubMed Central

    Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S.; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J.

    2016-01-01

    The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280 in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. PMID:26810656

  11. Two subtypes of HIV-1 among injection-drug users in southern China.

    PubMed

    Yu, X F; Chen, J; Shao, Y; Beyrer, C; Lai, S

    1998-04-25

    The rate of HIV-1 infection has increased steadily in China by about 80% annually and by the end of September 1997, 8277 HIV-1 cases had been reported, of whom more than 75% were IV drug users (IVDUs). UNAIDS, however, has estimated that up to 200,000 people could actually be infected with HIV-1 in China. Guangxi Province borders Yunnan province in the west and Vietnam to the south, and is a major transit area for heroin trafficking from the opium-growing region of Laos and Myanmar. Phylogenetic analyses of HIV-1 env sequences (C2-V3) obtained from 14 IVDUs found that 9 subjects from Pingxiang City were infected with subtype E and 5 from Baise City with subtype C. The 9 subtype E and 5 subtype C HIV-1 sequences were clustered together within each group, with significant bootstrap values of 100% and 95%, respectively. The subtype E sequences were more closely related to HIV-1 subtype E from Thailand than to those from Africa, and the subtype C sequences were clustered more closely to those from India than to those from Africa. Study results suggest 2 epidemiologically unrelated epidemics and 2 different sources; subtype C probably transmitted from Yunnan to Baise City through drug trafficking and IVDU interaction, and subtype E coming into China from Vietnam. PMID:9643749

  12. Dynamic correlation between intrahost HIV-1 quasispecies evolution and disease progression.

    PubMed

    Lee, Ha Youn; Perelson, Alan S; Park, Su-Chan; Leitner, Thomas

    2008-12-01

    Quantifying the dynamics of intrahost HIV-1 sequence evolution is one means of uncovering information about the interaction between HIV-1 and the host immune system. In the chronic phase of infection, common dynamics of sequence divergence and diversity have been reported. We developed an HIV-1 sequence evolution model that simulated the effects of mutation and fitness of sequence variants. The amount of evolution was described by the distance from the founder strain, and fitness was described by the number of offspring a parent sequence produces. Analysis of the model suggested that the previously observed saturation of divergence and decrease of diversity in later stages of infection can be explained by a decrease in the proportion of offspring that are mutants as the distance from the founder strain increases rather than due to an increase of viral fitness. The prediction of the model was examined by performing phylogenetic analysis to estimate the change in the rate of evolution during infection. In agreement with our modeling, in 13 out of 15 patients (followed for 3-12 years) we found that the rate of intrahost HIV-1 evolution was not constant but rather slowed down at a rate correlated with the rate of CD4+ T-cell decline. The correlation between the dynamics of the evolutionary rate and the rate of CD4+ T-cell decline, coupled with our HIV-1 sequence evolution model, explains previously conflicting observations of the relationships between the rate of HIV-1 quasispecies evolution and disease progression.

  13. Dendritic Cells in HIV-1 and HCV Infection: Can They Help Win the Battle?

    PubMed Central

    Sehgal, Mohit; Khan, Zafar K; Talal, Andrew H; Jain, Pooja

    2013-01-01

    Persistent infections with human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are a major cause of morbidity and mortality worldwide. As sentinels of our immune system, dendritic cells (DCs) play a central role in initiating and regulating a potent antiviral immune response. Recent advances in our understanding of the role of DCs during HIV-1 and HCV infection have provided crucial insights into the mechanisms employed by these viruses to impair DC functions in order to evade an effective immune response against them. Modulation of the immunological synapse between DC and T-cell, as well as dysregulation of the crosstalk between DCs and natural killer (NK) cells, are emerging as two crucial mechanisms. This review focuses on understanding the interaction of HIV-1 and HCV with DCs not only to understand the immunopathogenesis of chronic HIV-1 and HCV infection, but also to explore the possibilities of DC-based immunotherapeutic approaches against them. Host genetic makeup is known to play major roles in infection outcome and rate of disease progression, as well as response to anti-viral therapy in both HIV-1 and HCV-infected individuals. Therefore, we highlight the genetic variations that can potentially affect DC functions, especially in the setting of chronic viral infection. Altogether, we address if DCs’ potential as critical effectors of antiviral immune response could indeed be utilized to combat chronic infection with HIV-1 and HCV. PMID:25512691

  14. The HIV-1 Protein Vpr Targets the Endoribonuclease Dicer for Proteasomal Degradation to Boost Macrophage Infection

    PubMed Central

    Klockow, Laurieann Casey; Sharifi, Hamayun J.; Wen, Xiaoyun; Flagg, Meg; Furuya, Andrea K. M.; Nekorchuk, Michael; de Noronha, Carlos M.

    2013-01-01

    The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4DCAF1 ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4DCAF1. Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4DCAF1. We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4DCAF1 ubiquitin ligase complex and its subsequent degradation. PMID:23849790

  15. Dendritic Cells in HIV-1 and HCV Infection: Can They Help Win the Battle?

    PubMed

    Sehgal, Mohit; Khan, Zafar K; Talal, Andrew H; Jain, Pooja

    2013-01-01

    Persistent infections with human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are a major cause of morbidity and mortality worldwide. As sentinels of our immune system, dendritic cells (DCs) play a central role in initiating and regulating a potent antiviral immune response. Recent advances in our understanding of the role of DCs during HIV-1 and HCV infection have provided crucial insights into the mechanisms employed by these viruses to impair DC functions in order to evade an effective immune response against them. Modulation of the immunological synapse between DC and T-cell, as well as dysregulation of the crosstalk between DCs and natural killer (NK) cells, are emerging as two crucial mechanisms. This review focuses on understanding the interaction of HIV-1 and HCV with DCs not only to understand the immunopathogenesis of chronic HIV-1 and HCV infection, but also to explore the possibilities of DC-based immunotherapeutic approaches against them. Host genetic makeup is known to play major roles in infection outcome and rate of disease progression, as well as response to anti-viral therapy in both HIV-1 and HCV-infected individuals. Therefore, we highlight the genetic variations that can potentially affect DC functions, especially in the setting of chronic viral infection. Altogether, we address if DCs' potential as critical effectors of antiviral immune response could indeed be utilized to combat chronic infection with HIV-1 and HCV. PMID:25512691

  16. Functional roles of HIV-1 Tat protein in the nucleus.

    PubMed

    Musinova, Yana R; Sheval, Eugene V; Dib, Carla; Germini, Diego; Vassetzky, Yegor S

    2016-02-01

    Human immunodeficiency virus-1 (HIV-1) Tat protein is one of the most important regulatory proteins for viral gene expression in the host cell and can modulate different cellular processes. In addition, Tat is secreted by the infected cell and can be internalized by neighboring cells; therefore, it affects both infected and uninfected cells. Tat can modulate cellular processes by interacting with different cellular structures and signaling pathways. In the nucleus, Tat might be localized either in the nucleoplasm or the nucleolus depending on its concentration. Here we review the distinct functions of Tat in the nucleoplasm and the nucleolus in connection with viral infection and HIV-induced oncogenesis. PMID:26507246

  17. Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration.

    PubMed

    Chin, Christopher R; Perreira, Jill M; Savidis, George; Portmann, Jocelyn M; Aker, Aaron M; Feeley, Eric M; Smith, Miles C; Brass, Abraham L

    2015-11-24

    Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA's interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1's depth of nuclear invasion, potentially aiding the virus's integration into gene-dense regions.

  18. 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

  19. Progesterone augments cell susceptibility to HIV-1 and HIV-1/HSV-2 co-infections.

    PubMed

    Ragupathy, Viswanath; Xue, Wang; Tan, Ji; Devadas, Krishnakumar; Gao, Yamei; Hewlett, Indira

    2016-10-01

    In human immunodeficiency virus type 1 (HIV-1)-infected women, oral or injectable progesterone containing contraceptive pills may enhance HIV-1 acquisition in vivo, and the mechanism by which this occurs is not fully understood. In developing countries, Herpes simplex virus type-2 (HSV-2) co-infection has been shown to be a risk for increase of HIV-1 acquisition and, if co-infected women use progesterone pills, infections may increase several fold. In this study, we used an in vitro cell culture system to study the effects of progesterone on HIV-1 replication and to explore the molecular mechanism of progesterone effects on infected cells. In our in vitro model, CEMss cells (lymphoblastoid cell line) were infected with either HIV-1 alone or co-infected with HSV-2. HIV-1 viral load was measured with and without sex hormone treatment. Progesterone-treated cells showed an increase in HIV-1 viral load (1411.2 pg/mL) compared with cells without progesterone treatment (993.1 pg/mL). Increased cell death was noted with HSV-2 co-infection and in progesterone-treated cells. Similar observations were noted in peripheral blood mononuclear cells (PBMC) cells derived from three female donors. Progesterone-treated cells also showed reduced antiviral efficacy. Inflammatory cytokines and associations with biomarkers of disease progression were explored. Progesterone upregulated inflammatory cytokines and chemokines conversely and downregulated anti-apoptotic Bcl-2 expression. Nuclear protein analysis by electrophoretic mobility shift assay showed the association of progesterone with progesterone response element (PRE), which may lead to downregulation of Bcl-2. These data indicate that progesterone treatment enhances HIV-1 replication in infected cells and co-infection with HSV-2 may further fuel this process. PMID:27538988

  20. HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

    PubMed

    Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A; Grunwald, David; Sardo, Luca; Burdick, Ryan C; Plisov, Sergey; Liang, Edward; Tai, Sheldon; Pathak, Vinay K; Hu, Wei-Shau

    2016-01-12

    Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

  1. HIV-1 gp120 as a therapeutic target: Navigating a moving labyrinth

    PubMed Central

    Acharya, Priyamvada; Lusvarghi, Sabrina; Bewley, Carole A.; Kwong, Peter D.

    2015-01-01

    Introduction The HIV-1 gp120 envelope (Env) glycoprotein mediates attachment of virus to human target cells that display requisite receptors, CD4 and co-receptor, generally CCR5. Despite high affinity interactions with host receptors and proof-of-principle by the drug maraviroc that interference with CCR5 provides therapeutic benefit, no licensed drug currently targets gp120. Areas covered An overview of the role of gp120 in HIV-1 entry and of sites of potential gp120 vulnerability to therapeutic inhibition is presented. Viral defenses that protect these sites and turn gp120 into a moving labyrinth are discussed together with strategies for circumventing these defenses to allow therapeutic targeting of gp120 sites of vulnerability. Expert opinion The gp120 envelope glycoprotein interacts with host proteins through multiple interfaces and has conserved structural features at these interaction sites. In spite of this, targeting gp120 for therapeutic purposes is challenging. Env mechanisms evolved to evade the humoral immune response also shield it from potential therapeutics. Nevertheless, substantial progress has been made in understanding HIV-1 gp120 structure and its interactions with host receptors, and in developing therapeutic leads that potently neutralize diverse HIV-1 strains. Synergies between advances in understanding, needs for therapeutics against novel viral targets, and characteristics of breadth and potency for a number of gp120-targetting lead molecules bodes well for gp120 as a HIV-1 therapeutic target. PMID:25724219

  2. Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

    PubMed

    Métifiot, Mathieu; Johnson, Barry C; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves

    2016-08-19

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed. PMID:27369381

  3. Inhibition of HIV-1 reactivation by a telomerase-derived peptide in a HSP90-dependent manner

    PubMed Central

    Kim, Hong; Choi, Myung-Soo; Inn, Kyung-Soo; Kim, Bum-Joon

    2016-01-01

    A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells. PMID:27363520

  4. New and old complex recombinant HIV-1 strains among patients with primary infection in 1996–2006 in France: The French ANRS CO06 primo cohort study

    PubMed Central

    Frange, Pierre; Galimand, Julie; Vidal, Nicole; Goujard, Cécile; Deveau, Christiane; Souala, Faouzi; Peeters, Martine; Meyer, Laurence; Rouzioux, Christine; Chaix, Marie-Laure

    2008-01-01

    Background Prevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs). Methods Among 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures. Results Among V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006–2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France. Conclusion This study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity. PMID:18673538

  5. MxB Is Not Responsible for the Blocking of HIV-1 Infection Observed in Alpha Interferon-Treated Cells

    PubMed Central

    Opp, Silvana; Vieira, Daniel A. S. A.; Schulte, Bianca; Chanda, Sumit K.

    2015-01-01

    ABSTRACT MxB restricts HIV-1 infection by directly interacting with the HIV-1 core, which is made of viral capsid; however, the contribution of MxB to the HIV-1 restriction observed in alpha interferon (IFN-α)-treated human cells is unknown. To understand this contribution, we used HIV-1 bearing the G208R capsid mutant (HIV-1-G208R), which overcomes the restriction imposed by cells expressing MxB. Here we showed that the reason why MxB does not block HIV-1-G208R is that MxB does not interact with HIV-1 cores bearing the mutation G208R. To understand whether MxB contributes to the HIV-1 restriction imposed by IFN-α-treated human cells, we challenged IFN-α-treated cells with HIV-G208R and found that MxB does not contribute to the restriction imposed by IFN-α-treated cells. To more directly test the contribution of MxB, we challenged IFN-α-treated human cells that are knocked out for the expression of MxB with HIV-1. These experiments suggested that MxB does not contribute to the HIV-1 restriction observed in IFN-α-treated human cells. IMPORTANCE MxB is a restriction factor that blocks HIV-1 infection in human cells. Although it has been postulated that MxB is the factor that blocks HIV-1 infection in IFN-α-treated cells, this is a hard concept to grasp due to the great number of genes that are induced by IFN-α in cells from the immune system. The work presented here elegantly demonstrates that MxB has minimal or no contribution to the ability of IFN-α-treated human cells to block HIV-1 infection. Furthermore, this work suggests the presence of novel restriction factors in IFN-α-treated human cells that block HIV-1 infection. PMID:26719253

  6. [Analysis of genetic recombination between human immunodeficiency virus type 1 (HIV-1) and HIV-2].

    PubMed

    Motomura, Kazushi

    2009-03-01

    It is estimated that one million people are dually infected with Human Immunodeficiency Virus type-I (HIV-1) and type-II (HIV-2) in West Africa and parts of India. HIV-1 and HIV-2 use the same receptor and coreceptors for entry into cells, and thus target the same cell populations in the host. Additionally, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. Therefore these properties suggest that in the dually infected population, it is likely that some cells can be infected by both HIV-1 and HIV-2, thereby providing opportunities for these two viruses to interact with each other. We constructed recombination assay system for measurement recombination frequencies and analyzed recombination rate between HIV-1 and HIV-2. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect copackaging of HIV-1 and HIV-2 RNAs. In this study, approximately 0.2% of infection events generated the GFP phenotype. Therefore, the appearance of the GFP+ phenotype in the current system is approximately 35-fold lower than that between two homologous HIV-1 or HIV-2 viruses. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns including those only had crossovers in gfp. The most common hybrid genomes had heterologous LTRs. Although infrequent, crossovers were also identified in the viral sequences. Such chimeric HIV-1 and HIV-2 viruses have yet to be observed in the infected population. It is unclear whether the lack of observed chimeras is due to the divergence between HIV-1 and HIV-2 being too great for such an event to occur, or whether such events could occur but have not yet been observed. Given the number of coinfected people, the potential for

  7. Tyrosine-sulfated V2 peptides inhibit HIV-1 infection via coreceptor mimicry.

    PubMed

    Cimbro, Raffaello; Peterson, Francis C; Liu, Qingbo; Guzzo, Christina; Zhang, Peng; Miao, Huiyi; Van Ryk, Donald; Ambroggio, Xavier; Hurt, Darrell E; De Gioia, Luca; Volkman, Brian F; Dolan, Michael A; Lusso, Paolo

    2016-08-01

    Tyrosine sulfation is a post-translational modification that facilitates protein-protein interaction. Two sulfated tyrosines (Tys173 and Tys177) were recently identified within the second variable (V2) loop of the major HIV-1 envelope glycoprotein, gp120, and shown to contribute to stabilizing the intramolecular interaction between V2 and the third variable (V3) loop. Here, we report that tyrosine-sulfated peptides derived from V2 act as structural and functional mimics of the CCR5 N-terminus and potently block HIV-1 infection. Nuclear magnetic and surface plasmon resonance analyses indicate that a tyrosine-sulfated V2 peptide (pV2α-Tys) adopts a CCR5-like helical conformation and directly interacts with gp120 in a CD4-dependent fashion, competing with a CCR5 N-terminal peptide. Sulfated V2 mimics, but not their non-sulfated counterparts, inhibit HIV-1 entry and fusion by preventing coreceptor utilization, with the highly conserved C-terminal sulfotyrosine, Tys177, playing a dominant role. Unlike CCR5 N-terminal peptides, V2 mimics inhibit a broad range of HIV-1 strains irrespective of their coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site in gp120. These results document the use of receptor mimicry by a retrovirus to occlude a key neutralization target site and provide leads for the design of therapeutic strategies against HIV-1. PMID:27389109

  8. The Global Transmission Network of HIV-1

    PubMed Central

    Wertheim, Joel O.; Leigh Brown, Andrew J.; Hepler, N. Lance; Mehta, Sanjay R.; Richman, Douglas D.; Smith, Davey M.; Kosakovsky Pond, Sergei L.

    2014-01-01

    Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression. PMID:24151309

  9. Latency: the hidden HIV-1 challenge

    PubMed Central

    Marcello, Alessandro

    2006-01-01

    Eradication of HIV-1 from an infected individual cannot be achieved by current regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy for a long time and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptionally silent provirus. Since the molecular mechanisms that permit long term transcriptional control of proviral gene expression in these cells are still obscure, this review aims at summarizing the various aspects of the problem that need to be considered. In particular, this review will focus the attention on the control of transcription imposed by chromatin through various epigenetic mechanisms. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. PMID:16412247

  10. APOBEC3 Has Not Left an Evolutionary Footprint on the HIV-1 Genome▿†

    PubMed Central

    Ebrahimi, Diako; Anwar, Firoz; Davenport, Miles P.

    2011-01-01

    It is known that the human immune proteins APOBEC3G and -F (hA3G/F) can inhibit Vif-deficient HIV by G-to-A mutation; however, the roles of these enzymes in the evolution of HIV are debated. We argue that if evolutionary pressure from hA3G/F exists there should be evidence of their imprint on the HIV genome in the form of (i) underrepresentation of hA3G/F target motifs (e.g., TGGG [targeted position is underlined]) and overrepresentation of product motifs (e.g., TAGG) and/or (ii) an increase in the ratio of nonsynonymous to synonymous (NS/S) G-to-A changes among hA3G/F target motifs and a decrease of NS/S A-to-G changes among hA3G/F product motifs. To test the first hypothesis, we studied the representation of hA3G/F target and product motifs in 1,932 complete HIV-1 genomes using Markov models. We found that the highly targeted motifs are not underrepresented and their product motifs are not overrepresented. To test the second hypothesis, we determined the NS/S G↔A changes among the hA3G/F target and product motifs in 1,540 complete sets of nine HIV-1 genes. The NS/S changes did not show an increasing/decreasing trend within the target/product motifs, but the NS/S changes within the motif AG was exceptionally low. We observed the same pattern by analyzing 740 human genes. Given that hA3G/F do not act on the human genome, this suggests a small NS/S change within AG has arisen by other mechanisms. We therefore find no evidence of an evolutionary footprint of hA3G/F. We postulate several mechanisms to explain why the HIV-1 genome does not contain the hA3G/F footprint. PMID:21697498

  11. Nanochemistry-based immunotherapy for HIV-1.

    PubMed

    Lori, F; Calarota, S A; Lisziewicz, J

    2007-01-01

    Highly active antiretroviral treatment (HAART), i.e. the combination of three or more drugs against human immunodeficiency virus type 1 (HIV-1), has greatly improved the clinical outcome of HIV-1-infected individuals. However, HAART is unable to reconstitute HIV-specific immunity and eradicate the virus. Several observations in primate models and in humans support the notion that cell-mediated immunity can control viral replication and slow disease progression. Thus, besides drugs, an immunotherapy that induces long-lasting HIV-specific T-cell responses could play a role in the treatment of HIV/AIDS. To induce such immune responses, DermaVir Patch has been developed. DermaVir consists of an HIV-1 antigen-encoding plasmid DNA that is chemically formulated in a nanoparticle. DermaVir is administered under a patch after a skin preparation that supports the delivery of the nanoparticle to Langerhans cells (LC). Epidermal LC trap and transport the nanomedicine to draining lymph nodes. While in transit, LC mature into dendritic cells (DC), which can efficiently present the DNA-encoded antigens to naïve T-cells for the induction of cellular immunity. Pre-clinical studies and Phase I clinical testing of DermaVir in HIV-1-infected individuals have demonstrated the safety and tolerability of DermaVir Patch. To further modulate cellular immunity, molecular adjuvants might be added into the nanoparticle. DermaVir Patch represents a new nanomedicine platform for immunotherapy of HIV/AIDS. In this review, the antiviral activity of DermaVir-induced cellular immunity is discussed. Furthermore, the action of some cytokines currently being tested as adjuvants are highlighted and the adjuvant effect of cytokine plasmid DNA included in the DermaVir nanoparticle is reviewed.

  12. HIV-1 infection kinetics in tissue cultures.

    PubMed

    Spouge, J I; Shrager, R I; Dimitrov, D S

    1996-11-01

    Despite intensive experimental work on HIV-1, very little theoretical work has focused on HIV-1 spread in tissue culture. This article uses two systems of ordinary differential equations to model two modes of viral spread, cell-free virus and cell-to-cell contact. The two models produce remarkably similar qualitative results. Simulations using realistic parameter regimes showed that starting with a small fraction of cells infected, both cell-free viral spread and direct cell-to-cell transmission give an initial exponential phase of viral growth, followed by either a crash or a gradual decline, extinguishing the culture. Under some conditions, an oscillatory phase may precede the extinction. Some previous models of in vivo HIV-1 infection oscillate, but only in unrealistic parameter regimes. Experimental tissue infections sometimes display several sequential cycles of oscillation, however, so our models can at least mimic them qualitatively. Significantly, the models show that infective oscillations can be explained by infection dynamics; biological heterogeneity is not required. The models also display proportionality between infected cells and cell-free virus, which is reassuringly consistent with assumptions about the equivalence of several measures of viral load, except that the proportionality requires a relatively constant total cell concentration. Tissue culture parameter values can be determined from accurate, controlled experiments. Therefore, if verified, our models should make interpreting experimental data and extrapolating it to in vivo conditions sharper and more reliable.

  13. Progress in HIV-1 Vaccine Development

    PubMed Central

    Haynes, Barton F.; McElrath, M. Juliana

    2014-01-01

    Purpose of the Review In this review, examples of recent progress in HIV-1 vaccine research are discussed. Recent Findings New insights from the immune correlates analyses of the RV144 efficacy trial have accelerated vaccine development with leads to follow in non-human primate studies and improved vaccine designs. Several new vaccine vector approaches offer promise in exquisite control of acute infection and in improving the breadth of T cell responses. New targets of broadly neutralizing antibodies (BnAbs) have been elucidated, and improved understanding of how the human host controls BnAb development have emerged from BnAb knockin mice and from analyses of BnAb maturation and virus evolution in subjects followed from the time of HIV-1 transmission to BnAb induction. Summary Based on these observations, it is clear that development of a successful HIV-1 vaccine will require new vaccine approaches and iterative testing of immunogens in well-designed animal and human trials. PMID:23743722

  14. Virus Maturation as a Novel HIV-1 Therapeutic Target

    PubMed Central

    Adamson, Catherine S.; Salzwedel, Karl; Freed, Eric O.

    2009-01-01

    Development of novel therapeutic targets against HIV-1 is a high research priority due to the serious clinical consequences associated with acquisition of resistance to current antiretroviral drugs. The HIV-1 structural protein Gag represents a potential novel therapeutic target as it plays a central role in virus particle production, yet is not targeted by any of the currently approved antiretroviral drugs. The Gag polyprotein precursor multimerizes to form immature particles that bud from the infected cell. Concomitant with virus release, the Gag precursor undergoes proteolytic processing by the viral protease to generate the mature Gag proteins, which include capsid (CA). Once liberated from the Gag polyprotein precursor, CA molecules interact to reassemble into a condensed conical core, which organizes the viral RNA genome and several viral proteins to facilitate virus replication in the next round of infection. Correct Gag proteolytic processing and core assembly are therefore essential for virus infectivity. In this review, we discuss novel strategies to inhibit maturation by targeting proteolytic cleavage sites in Gag or CA-CA interactions required for core formation. The identification and development of lead maturation inhibitors are highlighted. PMID:19534569

  15. 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

  16. 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.

  17. Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

    PubMed

    Flausino, O A; Dufau, L; Regasini, L O; Petrônio, M S; Silva, D H S; Rose, T; Bolzani, V S; Reboud-Ravaux, M

    2012-01-01

    The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the β-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular β-sheets involved in protein-protein interactions.

  18. Neutrophils Turn Plasma Proteins into Weapons against HIV-1

    PubMed Central

    Hagleitner, Magdalena; Rambach, Günter; Van Aken, Hugo; Dierich, Manfred; Kehrel, Beate E.

    2013-01-01

    As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms

  19. 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

  20. 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.

  1. HIV-1 prevalence in selected Tijuana sub-populations.

    PubMed Central

    Güereña-Burgueño, F; Benenson, A S; Sepúlveda-Amor, J

    1991-01-01

    To assess the prevalence of HIV-1 (human immunodeficiency virus) infection among high-risk populations in Tijuana, Mexico, HIV-1 antibody status was determined and information on risk behavior was obtained from 1,069 individuals in three high-risk groups. The prevalence of HIV-1 among 415 prostitutes was 0.5 percent; 410 prisoners, 1.2 percent; 233 homosexual/bisexual men, 11.6 percent; and 106 intravenous drug abusers, 1.9 percent. The potential for spread of HIV-1 exists in Tijuana despite the current relatively low seroprevalence of HIV-1. PMID:2014864

  2. HIV-1 phylogenetic analysis shows HIV-1 transits through the meninges to brain and peripheral tissues.

    PubMed

    Lamers, Susanna L; Gray, Rebecca R; Salemi, Marco; Huysentruyt, Leanne C; McGrath, Michael S

    2011-01-01

    Brain infection by the human immunodeficiency virus type 1 (HIV-1) has been investigated in many reports with a variety of conclusions concerning the time of entry and degree of viral compartmentalization. To address these diverse findings, we sequenced HIV-1 gp120 clones from a wide range of brain, peripheral and meningeal tissues from five patients who died from several HIV-1 associated disease pathologies. High-resolution phylogenetic analysis confirmed previous studies that showed a significant degree of compartmentalization in brain and peripheral tissue subpopulations. Some intermixing between the HIV-1 subpopulations was evident, especially in patients that died from pathologies other than HIV-associated dementia. Interestingly, the major tissue harboring virus from both the brain and peripheral tissues was the meninges. These results show that (1) HIV-1 is clearly capable of migrating out of the brain, (2) the meninges are the most likely primary transport tissues, and (3) infected brain macrophages comprise an important HIV reservoir during highly active antiretroviral therapy.

  3. 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

  4. Roles played by acidic lipids in HIV-1 Gag membrane binding.

    PubMed

    Olety, Balaji; Ono, Akira

    2014-11-26

    The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids.

  5. 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.

  6. An Alix fragment potently inhibits HIV-1 budding: characterization of binding to retroviral YPXL late domains.

    PubMed

    Munshi, Utpal M; Kim, Jaewon; Nagashima, Kunio; Hurley, James H; Freed, Eric O

    2007-02-01

    The retroviral structural protein, Gag, contains small peptide motifs known as late domains that promote efficient virus release from the infected cell. In addition to the well characterized PTAP late domain, the p6 region of HIV-1 Gag contains a binding site for the host cell protein Alix. To better understand the functional role of the Gag/Alix interaction, we overexpressed an Alix fragment composed of residues 364-716 (Alix 364-716) and examined the effect on release of wild type (WT) and Alix binding site mutant HIV-1. We observed that Alix 364-716 expression significantly inhibited WT virus release and Gag processing and that mutation of the Alix binding site largely relieved this inhibition. Furthermore, Alix 364-716 expression induced a severe defect on WT but not mutant particle morphology. Intriguingly, the impact of Alix 364-716 expression on HIV-1 release and Gag processing was markedly different from that induced by mutation of the Alix binding site in p6. The association of Alix 364-716 with HIV-1 and equine infectious anemia virus late domains was quantitatively evaluated by isothermal titration calorimetry and surface plasmon resonance techniques, and the effects of mutations in these viral sequences on Alix 364-716 binding was determined. This study identifies a novel Alix-derived dominant negative inhibitor of HIV-1 release and Gag processing and provides quantitative information on the interaction between Alix and viral late domains.

  7. The QSAR and docking calculations of fullerene derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Saleh, Noha A.

    2015-02-01

    The inhibition of HIV-1 protease is considered as one of the most important targets for drug design and the deactivation of HIV-1. In the present work, the fullerene surface (C60) is modified by adding oxygen atoms as well as hydroxymethylcarbonyl (HMC) groups to form 6 investigated fullerene derivative compounds. These compounds have one, two, three, four or five O atoms + HMC groups at different positions on phenyl ring. The effect of the repeating of these groups on the ability of suggested compounds to inhibit the HIV protease is studied by calculating both Quantitative Structure Activity Relationship (QSAR) properties and docking simulation. Based on the QSAR descriptors, the solubility and the hydrophilicity of studied fullerene derivatives increased with increasing the number of oxygen atoms + HMC groups in the compound. While docking calculations indicate that, the compound with two oxygen atoms + HMC groups could interact and binds with HIV-1 protease active site. This is could be attributed to the active site residues of HIV-1 protease are hydrophobic except the two aspartic acids. So that, the increase in the hydrophilicity and polarity of the compound is preventing and/or decreasing the hydrophobic interaction between the compound and HIV-1 protease active site.

  8. Effects of drug-resistant mutations on the dynamic properties of HIV-1 protease and inhibition by Amprenavir and Darunavir.

    PubMed

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2015-01-01

    Molecular dynamics simulations are performed to investigate the dynamic properties of wild-type HIV-1 protease and its two multi-drug-resistant variants (Flap + (L10I/G48V/I54V/V82A) and Act (V82T/I84V)) as well as their binding with APV and DRV inhibitors. The hydrophobic interactions between flap and 80 s (80's) loop residues (mainly I50-I84' and I50'-I84) play an important role in maintaining the closed conformation of HIV-1 protease. The double mutation in Act variant weakens the hydrophobic interactions, leading to the transition from closed to semi-open conformation of apo Act. APV or DRV binds with HIV-1 protease via both hydrophobic and hydrogen bonding interactions. The hydrophobic interactions from the inhibitor is aimed to the residues of I50 (I50'), I84 (I84'), and V82 (V82') which create hydrophobic core clusters to further stabilize the closed conformation of flaps, and the hydrogen bonding interactions are mainly focused with the active site of HIV-1 protease. The combined change in the two kinds of protease-inhibitor interactions is correlated with the observed resistance mutations. The present study sheds light on the microscopic mechanism underlying the mutation effects on the dynamics of HIV-1 protease and the inhibition by APV and DRV, providing useful information to the design of more potent and effective HIV-1 protease inhibitors. PMID:26012849

  9. Immune Compromise in HIV-1/HTLV-1 Coinfection With Paradoxical Resolution of CD4 Lymphocytosis During Antiretroviral Therapy

    PubMed Central

    Rockwood, N.; Cook, L.; Kagdi, H.; Basnayake, S.; Bangham, C.R.M.; Pozniak, A.L.; Taylor, G.P.

    2015-01-01

    Abstract Human immunodeficiency virus type-1 (HIV-1) and human T lymphotropic virus type-1 (HTLV-1) infections have complex effects on adaptive immunity, with specific tropism for, but contrasting effects on, CD4 T lymphocytes: depletion with HIV-1, proliferation with HTLV-1. Impaired T lymphocyte function occurs early in HIV-1 infection but opportunistic infections (OIs) rarely occur in the absence of CD4 lymphopenia. In the unusual case where a HIV-1 infected individual with a high CD4 count presents with recurrent OIs, a clinician is faced with the possibility of a second underlying comorbidity. We present a case of pseudo-adult T cell leukemia/lymphoma (ATLL) in HIV-1/HTLV-1 coinfection where the individual fulfilled Shimoyama criteria for chronic ATLL and had pulmonary Mycobacterium kansasii, despite a high CD4 lymphocyte count. However, there was no evidence of clonal T-cell proliferation by T-cell receptor gene rearrangement studies nor of monoclonal HTLV-1 integration by high-throughput sequencing. Mutually beneficial interplay between HIV-1 and HTLV-1, maintaining high level HIV-1 and HTLV-1 viremia and proliferation of poorly functional CD4 cells despite chronicity of infection is a postulated mechanism. Despite good microbiological response to antimycobacterial therapy, the patient remained systemically unwell with refractory anemia. Subsequent initiation of combined antiretroviral therapy led to paradoxical resolution of CD4 T lymphocytosis as well as HIV-1 viral suppression and decreased HTLV-1 proviral load. This is proposed to be the result of attenuation of immune activation post-HIV virological control. This case illustrates the importance of screening for HTLV-1 in HIV-1 patients with appropriate clinical presentation and epidemiological risk factors and explores mechanisms for the complex interactions on HIV-1/HTLV-1 adaptive immunity. PMID:26683952

  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. G3BP1 restricts HIV-1 replication in macrophages and T-cells by sequestering viral RNA.

    PubMed

    Cobos Jiménez, Viviana; Martinez, Fernando O; Booiman, Thijs; van Dort, Karel A; van de Klundert, Maarten A A; Gordon, Siamon; Geijtenbeek, Teunis B H; Kootstra, Neeltje A

    2015-12-01

    HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

  12. 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

  13. Raman spectroscopy of HIV-1 antigen and antibody

    NASA Astrophysics Data System (ADS)

    Zinin, Pavel V.; Hu, Ningjie; Kamemoto, Lori E.; Yu, Qigui; Misra, Anupam K.; Sharma, Shiv K.

    2011-05-01

    Raman spectra of anti-HIV-1 antibody, HIV-1 antigen (p24), and HIV-1 antibody-antigen complex have been measured in near-infrared and UV regions: 785 nm; 830 nm; and 244 nm laser excitations. The spectrum of the HIV-1 antigen was excited with an infrared laser and contains numerous Raman peaks. The most prominent peaks are broad bands at 1343, 1449, 1609 and 1655 cm-1, which are characteristic of the Raman spectra of biological cells. The UV Raman spectrum of the HIV-1 antigen has a completely different structure. It has two strong peaks at 1613 cm-1 and 1173 cm-1. The peak at 1613 cm-1 is associated with vibrations of the aromatic amino acids tyrosine (Tyr) and tryptophan (Try). The second strongest peak at 1173 cm-1 is associated with the vibration of Tyr. The Raman peak pattern of the HIV-1 antigen-antibody complex is very similar to that of the HIV-1 antigen. The only difference is that the peak at 1007 cm-1 of the Raman spectrum of the HIV-1 antigen-antibody complex is slightly enhanced compared to that of the HIV-1 antigen. This indicates that the peaks of the HIV-1 antigen dominate the Raman spectrum of the HIV-1 antigen-antibody complex.

  14. Short Communication: Neutralizing Antibodies in HIV-1-Infected Brazilian Individuals

    PubMed Central

    Morgado, Mariza Gonçalvez; Côrtes, Fernanda Heloise; Guimarães, Monick Lindermeyer; Mendonça-Lima, Leila; Pilotto, Jose Henrique; Grinsztejn, Beatriz; Veloso, Valdiléa Gonçalves; Bongertz, Vera

    2013-01-01

    Abstract Tests for the detection of the humoral immune response to HIV-1 have to be standardized and established, demanding regional efforts. For this purpose the neutralizing antibody (NAb) assay for HIV-1 in TZM-bl cells was introduced in Brazil. Twenty plasma samples from HIV-1-infected individuals were assayed: 10 progressors and 10 long-term nonprogressors. These were tested against eight env-pseudotyped viruses (psVs) in the TZM-bl NAb assay and against HIV-1 strain HTLV/IIIB (HIV-1 IIIB) in primary lymphocytes. Forty-four percent of the samples showed neutralizing titers for psVs and 55% for HIV-1 IIIB. Plasma from progressors showed a broader neutralization and a higher potency. The introduction of these reference reagents encourages the participation of Brazil in future comparative assessments of anti-HIV-1 antibodies. PMID:23145941

  15. Significant impact of non-B HIV-1 variants genetic diversity in Gabon on plasma HIV-1 RNA quantitation.

    PubMed

    Mouinga-Ondémé, Augustin; Mabika-Mabika, Arsène; Alalade, Patrick; Mongo, Arnaud Delis; Sica, Jeanne; Liégeois, Florian; Rouet, François

    2014-01-01

    Evaluations of HIV-1 RNA viral load assays are lacking in Central Africa. The main objective of our study was to assess the reliability of HIV-1 RNA results obtained with three different assays for samples collected in Gabon. A total of 137 plasma specimens were assessed for HIV-1 RNA using the Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ® version 2.0 assays. It included HIV-1 non-B samples (n = 113) representing six subtypes, 10 CRFs and 18 URFs from patients infected with HIV-1 and treated with antiretrovirals that were found HIV-1 RNA positive (≥300 copies/ml) with the Generic HIV viral load® assay; and samples (n = 24) from untreated individuals infected with HIV-1 but showing undetectable (<300 copies/ml) results with the Biocentric kit. For samples found positive with the Generic HIV viral load® test, correlation coefficients obtained between the three techniques were relatively low (R = 0.65 between Generic HIV viral load® and Abbott RealTime HIV-1®, 0.50 between Generic HIV viral load® and Nuclisens HIV-1 EasyQ®, and 0.66 between Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ®). Discrepancies by at least one log10 were obtained for 19.6%, 33.7%, and 20% of samples, respectively, irrespective of genotype. Most of samples (22/24) from untreated study patients, found negative with the Biocentric kit, were also found negative with the two other techniques. In Central Africa, HIV-1 genetic diversity remains challenging for viral load testing. Further studies are required in the same area to confirm the presence of HIV-1 strains that are not amplified with at least two different viral load assays.

  16. Mechanism of Multivalent Nanoparticle Encounter with HIV-1 for Potency Enhancement of Peptide Triazole Virus Inactivation*

    PubMed Central

    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

  17. Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets

    PubMed Central

    Phogat, S.; Wyatt, R. T.; Hedestam, G. B. Karlsson

    2008-01-01

    Phogat S, Wyatt RT, Karlsson Hedestam GB (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; Department of Microbiology Tumor and Cell Biology, Karolinska Institutet, Stockholm; and the Swedish Institute for Infectious Disease Control, Solna, Sweden). Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets (Review). Vaccine-induced antibodies that interfere with viral entry are the protective correlate of most existing prophylactic vaccines. However, for highly variable viruses such as HIV-1, the ability to elicit broadly neutralizing antibody responses through vaccination has proven to be extremely difficult. The major targets for HIV-1 neutralizing antibodies are the viral envelope glycoprotein trimers on the surface of the virus that mediate receptor binding and entry. HIV-1 has evolved many mechanisms on the surface of envelope glyco-proteins to evade antibody-mediated neutralization, including the masking of conserved regions by glycan, quaternary protein interactions and the presence of immunodominant variable elements. The primary challenge in the development of an HIV-1 vaccine that elicits broadly neutralizing antibodies therefore lies in the design of suitable envelope glycoprotein immunogens that circumvent these barriers. Here, we describe neutralizing determinants on the viral envelope glyco-proteins that are defined by their function in receptor binding or by rare neutralizing antibodies isolated from HIV-infected individuals. We also describe the nonvariable cellular receptors involved in the HIV-1 entry process, or other cellular proteins, and ongoing studies to determine if antibodies against these proteins have efficacy as therapeutic reagents or, in some cases, as vaccine targets to interfere with HIV-1 entry. PMID:17598813

  18. Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors.

    PubMed Central

    Balzarini, J; Karlsson, A; Vandamme, A M; Pérez-Pérez, M J; Zhang, H; Vrang, L; Oberg, B; Bäckbro, K; Unge, T; San-Félix, A

    1993-01-01

    We recently reported that a newly discovered class of nucleoside analogues--[2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains. HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138-->Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT. PMID:7688467

  19. 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.

  20. Human cytosolic extracts stabilize the HIV-1 core.

    PubMed

    Fricke, Thomas; Brandariz-Nuñez, Alberto; Wang, Xiaozhao; Smith, Amos B; Diaz-Griffero, Felipe

    2013-10-01

    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 on HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure the stability of in vitro-assembled HIV-1 CA-NC complexes. The assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core. Interestingly, stabilization of in vitro-assembled HIV-1 CA-NC complexes is not due solely to macromolecular crowding, suggesting the presence of specific cellular factors that stabilize the HIV-1 core. By using our novel assay, we measured the abilities of different drugs, such as PF74, CAP-1, IXN-053, cyclosporine, Bi2 (also known as BI-2), and the peptide CAI, to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes. Interestingly, we found that PF74 and Bi2 strongly stabilized HIV-1 CA-NC complexes. On the other hand, the peptide CAI destabilized HIV-1 CA-NC complexes. We also found that purified cyclophilin A destabilizes in vitro-assembled HIV-1 CA-NC complexes in the presence of cellular extracts in a cyclosporine-sensitive manner. In agreement with previous observations using the fate-of-the-capsid assay, we also demonstrated the ability of recombinant CPSF6 to stabilize HIV-1 CA-NC complexes. Overall, our findings suggested that cellular extracts specifically stabilize the HIV-1 core. We believe that our assay can be a powerful tool to assess HIV-1 core stability in vitro.

  1. Developing a Dynamic Pharmacophore Model for HIV-1 Integrase

    SciTech Connect

    Carlson, Heather A.; Masukawa, Keven M.; Rubins, Kathleen; Bushman, Frederic; Jorgensen, William L.; Lins, Roberto; Briggs, James; Mccammon, Andy

    2000-05-11

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of ''dynamic'' pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a ''static'' pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.

  2. HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.

    PubMed

    Simonich, Cassandra A; Williams, Katherine L; Verkerke, Hans P; Williams, James A; Nduati, Ruth; Lee, Kelly K; Overbaugh, Julie

    2016-06-30

    HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM. PMID:27345369

  3. Developing a dynamic pharmacophore model for HIV-1 integrase.

    PubMed

    Carlson, H A; Masukawa, K M; Rubins, K; Bushman, F D; Jorgensen, W L; Lins, R D; Briggs, J M; McCammon, J A

    2000-06-01

    We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of "dynamic" pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a "static" pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors. PMID:10841789

  4. Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials

    PubMed Central

    Huang, Yunda; Follmann, Dean; Nason, Martha; Zhang, Lily; Huang, Ying; Mehrotra, Devan V.; Moodie, Zoe; Metch, Barbara; Janes, Holly; Keefer, Michael C.; Churchyard, Gavin; Robb, Merlin L.; Fast, Patricia E.; Duerr, Ann; McElrath, M. Juliana; Corey, Lawrence; Mascola, John R.; Graham, Barney S.; Sobieszczyk, Magdalena E.; Kublin, James G.; Robertson, Michael; Hammer, Scott M.; Gray, Glenda E.; Buchbinder, Susan P.; Gilbert, Peter B.

    2015-01-01

    Background Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow–up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines. Methods We included participant-level data from all three efficacy trials, and three Phase 1–2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests. Findings Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99–1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11–1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61–1.26, P = 0.48). Results were similar when including the Phase 1–2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5

  5. Molecular docking guided structure based design of symmetrical N,N'-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors.

    PubMed

    Sivan, Sree Kanth; Vangala, Radhika; Manga, Vijjulatha

    2013-08-01

    Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N'-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations.

  6. Vpu Protein: The Viroporin Encoded by HIV-1

    PubMed Central

    González, María Eugenia

    2015-01-01

    Viral protein U (Vpu) is a lentiviral viroporin encoded by human immunodeficiency virus type 1 (HIV-1) and some simian immunodeficiency virus (SIV) strains. This small protein of 81 amino acids contains a single transmembrane domain that allows for supramolecular organization via homoligomerization or interaction with other proteins. The topology and trafficking of Vpu through subcellular compartments result in pleiotropic effects in host cells. Notwithstanding the high variability of its amino acid sequence, the functionality of Vpu is well conserved in pandemic virus isolates. This review outlines our current knowledge on the interactions of Vpu with the host cell. The regulation of cellular physiology by Vpu and the validity of this viroporin as a therapeutic target are also discussed. PMID:26247957

  7. Crystallization studies on HIV-1 reverse transcriptase

    NASA Astrophysics Data System (ADS)

    Lloyd, Lesley F.; Brick, Peter; Blow, David M.; Mei-Zhen, Lou

    1992-08-01

    HIV-1 reverse transcriptase has been crystallized in a variety of forms. Various ligands used for co-crystallization are described and the results presented. All of these crystals showed disorder when examined in the X-ray beam. The best diffraction currently achieved has been approximately 7A˚. The possible reasons for crystal disorder are discussed. An example of another protein, car☐ypeptidase G 2, which initially yielded non-diffracting crystals, is used to illustrate the value of applying random or incomplete factorial screens to sample wider parameter space for conditions to grow well-ordered crystals.

  8. Rigidity analysis of HIV-1 protease

    NASA Astrophysics Data System (ADS)

    Heal, J. W.; Wells, S. A.; Jimenez-Roldan, E.; Freedman, R. F.; Römer, R. A.

    2011-03-01

    We present a rigidity analysis on a large number of X-ray crystal structures of the enzyme HIV-1 protease using the 'pebble game' algorithm of the software FIRST. We find that although the rigidity profile remains similar across a comprehensive set of high resolution structures, the profile changes significantly in the presence of an inhibitor. Our study shows that the action of the inhibitors is to restrict the flexibility of the β-hairpin flaps which allow access to the active site. The results are discussed in the context of full molecular dynamics simulations as well as data from NMR experiments.

  9. 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

  10. The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid.

    PubMed

    York, Ashley; Kutluay, Sebla B; Errando, Manel; Bieniasz, Paul D

    2016-08-01

    The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions. PMID:27541140

  11. Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography

    PubMed Central

    Jun, Sangmi; Ke, Danxia; Debiec, Karl; Zhao, Gongpu; Meng, Xin; Ambrose, Zandrea; Gibson, Gregory A.; Watkins, Simon C.; Zhang, Peijun

    2011-01-01

    SUMMARY Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state, and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, direct observation of structural details of infecting HIV-1 has not been realized due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by developing a correlative high-speed 3D live-cell imaging and cryoET method. Using this methodology, we showed, for the first time under near-native conditions, that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host-cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantage of our correlative live-cell and cryoET approach to image dynamic processes, such as viral infection. PMID:22078557

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

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

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

  13. The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid

    PubMed Central

    Errando, Manel; Bieniasz, Paul D.

    2016-01-01

    The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions. PMID:27541140

  14. Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

    SciTech Connect

    Boggiano, Cesar; Jiang Shibo; Lu Hong; Zhao Qian; Liu Shuwen; Binley, James; Blondelle, Sylvie E. . E-mail: sylvieb@burnham.org

    2006-09-08

    Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide DC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While DC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1{alpha} to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of DC13 implies additional mode(s) of action. These results suggest that DC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.

  15. Modest Attenuation of HIV-1 Vpu Alleles Derived from Elite Controller Plasma

    PubMed Central

    Chen, Jingyan; Tibroni, Nadine; Sauter, Daniel; Galaski, Johanna; Miura, Toshiyuki; Alter, Galit; Mueller, Birthe; Haller, Claudia; Walker, Bruce D.; Kirchhoff, Frank; Brumme, Zabrina L.; Ueno, Takamasa; Fackler, Oliver T.

    2015-01-01

    In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection. PMID:25793728

  16. Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

    PubMed

    Schachter, Julieta; Delgado, Kelly Valcárcel; Barreto-de-Souza, Victor; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis; Meyer-Fernandes, José Roberto

    2015-05-01

    Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages.

  17. Identification of a small molecule HIV-1 inhibitor that targets the capsid hexamer.

    PubMed

    Xu, Jimmy P; Branson, Jeffrey D; Lawrence, Rae; Cocklin, Simon

    2016-02-01

    The HIV-1 CA protein is an attractive therapeutic target for the development of new antivirals. An inter-protomer pocket within the hexamer configuration of the CA, which is a binding site for key host dependency factors, is an especially appealing region for small molecule targeting. Using a field-based pharmacophore derived from an inhibitor known to interact with this region, coupled to biochemical and biological assessment, we have identified a new compound that inhibits HIV-1 infection and that targets the assembled CA hexamer. PMID:26747394

  18. HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling

    SciTech Connect

    András, Ibolya E. Toborek, Michal

    2014-04-15

    Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood–brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity of dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level. - Highlights: • HIV-1 induces nuclear accumulation of amyloid beta (Aβ) in brain endothelial cells. • EEA-1 and TGF-Β/Smad act in concert to regulate nuclear entry of Aβ. • Dynamin appropriates the EEA-1 and TGF-Β/Smad signaling. • Dynamin serves as a master regulator of HIV-1-induced nuclear accumulation of Aβ.

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

    PubMed

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

    2016-07-29

    Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1. PMID:27417497

  20. The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics*

    PubMed Central

    Huang, Xi; Britto, Manuel D.; Kear-Scott, Jamie L.; Boone, Christopher D.; Rocca, James R.; Simmerling, Carlos; Mckenna, Robert; Bieri, Michael; Gooley, Paul R.; Dunn, Ben M.; Fanucci, Gail E.

    2014-01-01

    HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR 15N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors. PMID:24742668

  1. Noninvasive micromanipulation of live HIV-1 infected cells via laser light

    SciTech Connect

    Mthunzi, Patience

    2015-12-31

    Live mammalian cells from various tissues of origin can be aseptically and noninvasively micromanipulated via lasers of different regimes. Laser-driven techniques are therefore paving a path toward the advancement of human immuno-deficiency virus (HIV-1) investigations. Studies aimed at the interaction of laser light, nanomaterials, and biological materials can also lead to an understanding of a wealth of disease conditions and result in photonics-based therapies and diagnostic tools. Thus, in our research, both continuous wave and pulsed lasers operated at varying wavelengths are employed, as they possess special properties that allow classical biomedical applications. This paper discusses photo-translocation of antiretroviral drugs into HIV-1 permissive cells and preliminary results of low-level laser therapy (LLLT) in HIV-1 infected cells.

  2. Blocking of HIV-1 Infectivity by a Soluble, Secreted Form of the CD4 Antigen

    NASA Astrophysics Data System (ADS)

    Smith, Douglas H.; Byrn, Randal A.; Marsters, Scot A.; Gregory, Timothy; Groopman, Jerome E.; Capon, Daniel J.

    1987-12-01

    The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

  3. Noninvasive micromanipulation of live HIV-1 infected cells via laser light

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience

    2015-12-01

    Live mammalian cells from various tissues of origin can be aseptically and noninvasively micromanipulated via lasers of different regimes. Laser-driven techniques are therefore paving a path toward the advancement of human immuno-deficiency virus (HIV-1) investigations. Studies aimed at the interaction of laser light, nanomaterials, and biological materials can also lead to an understanding of a wealth of disease conditions and result in photonics-based therapies and diagnostic tools. Thus, in our research, both continuous wave and pulsed lasers operated at varying wavelengths are employed, as they possess special properties that allow classical biomedical applications. This paper discusses photo-translocation of antiretroviral drugs into HIV-1 permissive cells and preliminary results of low-level laser therapy (LLLT) in HIV-1 infected cells.

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

    PubMed

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

    2016-07-29

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

  5. Stapled HIV-1 Peptides Recapitulate Antigenic Structures and Engage Broadly Neutralizing Antibodies

    PubMed Central

    Bird, Gregory H.; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D.; Wilson, Ian A.; Walensky, Loren D.

    2014-01-01

    Hydrocarbon stapling can restore bioactive, α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here, we explore the capacity of peptide stapling to generate high fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease-resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically-stabilized antigens for vaccination. PMID:25420104

  6. 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 Central

    2015-01-01

    ABSTRACT 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

  7. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    PubMed Central

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  8. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

    PubMed

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  9. Identification of RNA helicases in human immunodeficiency virus 1 (HIV-1) replication - a targeted small interfering RNA library screen using pseudotyped and WT HIV-1.

    PubMed

    Williams, Claire A; Abbink, Truus E M; Jeang, Kuan-Teh; Lever, Andrew M L

    2015-06-01

    Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.

  10. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

    PubMed

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1.

  11. Fucoidans as Potential Inhibitors of HIV-1

    PubMed Central

    Prokofjeva, Maria M.; Imbs, Tatyana I.; Shevchenko, Natalya M.; Spirin, Pavel V.; Horn, Stefan; Fehse, Boris; Zvyagintseva, Tatyana N.; Prassolov, Vladimir S.

    2013-01-01

    The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001–100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001–0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors. PMID:23966033

  12. Fucoidans as potential inhibitors of HIV-1.

    PubMed

    Prokofjeva, Maria M; Imbs, Tatyana I; Shevchenko, Natalya M; Spirin, Pavel V; Horn, Stefan; Fehse, Boris; Zvyagintseva, Tatyana N; Prassolov, Vladimir S

    2013-08-19

    The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001-100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001-0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors.

  13. Species‐specific differences in the ability of feline lentiviral Vif to degrade feline APOBEC3 proteins

    PubMed Central

    Yoshikawa, Rokusuke; Nakano, Yusuke; Yamada, Eri; Izumi, Taisuke; Misawa, Naoko; Koyanagi, Yoshio

    2016-01-01

    ABSTRACT How host–virus co‐evolutionary relationships manifest is one of the most intriguing issues in virology. To address this topic, the mammal–lentivirus relationship can be considered as an interplay of cellular and viral proteins, particularly apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3 (APOBEC3) and viral infectivity factor (Vif). APOBEC3s enzymatically restrict lentivirus replication, whereas Vif antagonizes the host anti‐viral action mediated by APOBEC3. In this study, the focus was on the interplay between feline APOBEC3 proteins and two feline immunodeficiency viruses in cats and pumas. To our knowledge, this study provides the first evidence of non‐primate lentiviral Vif being incapable of counteracting a natural host's anti‐viral activity mediated via APOBEC3 protein. PMID:26935128

  14. 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

  15. [Inhibitory effect of human saliva on HIV-1 infectivity].

    PubMed

    Etsuko, K; Wei, S

    2001-08-01

    Human saliva is known to decrease human immunodeficiency virus type 1 (HIV-1) infectivity in vitro. The purpose of this study was to confirm these findings and to explore the mechanism of action of saliva. Whole saliva from seronegative donors was incubated with HIV-1IIIB chronically infected MOLT 4 cells (MOLT 4/HIV-1IIIB cells) or cell-free HIV-1IIIB or KMT strains. We monitored viral infectivity by using MAGI/CCR5 cells. Whole saliva with Na levels less than 20 mEq/l rapidly damaged MOLT 4/HIV-1IIIB cells, thereby HIV infection to MAGI/CCR5 cells by MOLT 4/HIV-1IIIB cells was nearly abolished. On the contrary, in the cace of whole saliva with Na levels more than 23 mEq/l which damaged few cells, cell-to-cell transmission of HIV-1IIIB was prevented by more than 50%. The infectivity of cell-free HIV-1IIIB to MAGI/CCR5 cells was abolished after incubating and filtering the HIV with whole saliva. Depletion of secretory leukocyte protease inhibitor (SLPI) from whole saliva resulted in a 11-28% decrease in the anti HIV-1KMT activity of saliva. Preincubation of host cells with whole saliva led to an enhancement of the HIV infection rather than inhibition. Whole saliva had no effect on the expression level of the cellular receptors (CD4, CXCR4 and CCR5). These results suggest that the inhibitory effect of whole saliva on HIV-1 infectivity is directly linked to the virus itself rather than on the host cell. Moreover, the physical entrapment of cell-free HIV-1 by whole saliva seems to have major salivaly defence mechanisms against HIV-1 infection through the oral cavity. PMID:16578966

  16. Raltegravir, an HIV-1 integrase inhibitor for HIV infection.

    PubMed

    Cabrera, Cecilia

    2008-08-01

    Merck & Co has developed and launched raltegravir, an HIV-1 integrase inhibitor for the treatment of HIV-1 infection in treatment-experienced adult patients who have evidence of viral replication and HIV-1 strains resistant to multiple antiretroviral agents. This drug is the lead from a series of integrase strand transfer inhibitors and, by April 2008, it had been launched in Canada, the US, the UK, France, Germany and Spain, and had been filed for approval in Japan.

  17. Disseminated human immunodeficiency virus 1 (HIV-1) infection in SCID- hu mice after peripheral inoculation with HIV-1

    PubMed Central

    1994-01-01

    A small animal model that could be infected with human immunodeficiency virus 1 (HIV-1) after peripheral inoculation would greatly facilitate the study of the pathophysiology of acute HIV-1 infection. The utility of SCID mice implanted with human fetal thymus and liver (SCID-hu mice) for studying peripheral HIV-1 infection in vivo has been hampered by the requirement for direct intraimplant injection of HIV-1 and the continued restriction of the resultant HIV-1 infection to the human thymus and liver (hu-thy/liv) implant. This may have been due to the very low numbers of human T cells present in the SCID-hu mouse peripheral lymphoid compartment. Since the degree of the peripheral reconstitution of SCID-hu mice with human T cells may be a function of the hu-thy/liv implant size, we increased the quantity of hu-thy/liv tissue implanted under the renal capsule and implanted hu-thy/liv tissue under the capsules of both kidneys. This resulted in SCID-hu mice in which significant numbers of human T cells were detected in the peripheral blood, spleens, and lymph nodes. After intraimplant injection of HIV-1 into these modified SCID-hu mice, significant HIV-1 infection was detected by quantitative coculture not only in the hu- thy/liv implant, but also in the spleen and peripheral blood. This indicated that HIV-1 infection can spread from the thymus to the peripheral lymphoid compartment. More importantly, a similar degree of infection of the hu-thy/liv implant and peripheral lymphoid compartment occurred after peripheral intraperitoneal inoculation with HIV-1. Active viral replication was indicated by the detection of HIV-1 gag DNA, HIV-1 gag RNA, and spliced tat/rev RNA in the hu-thy/liv implants, peripheral blood mononuclear cells (PBMC), spleens, and lymph nodes of these HIV-1-infected SCID-hu mice. As a first step in using our modified SCID-hu mouse model to investigate the pathophysiological consequences of HIV-1 infection, the effect of HIV-1 infection on the

  18. Structural Basis for the Recognition Between HIV-1 Integrase and Transcriptional Coactivator p75

    SciTech Connect

    Cherepanov,P.; Ambrosio, A.; Rahman, S.; Ellenberger, T.; Engelman, A.

    2005-01-01

    Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of {alpha}-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

  19. Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

    PubMed Central

    Yu, Xinwei; Feizpour, Amin; Ramirez, Nora-Guadalupe P.; Wu, Linxi; Akiyama, Hisashi; Xu, Fangda

    2014-01-01

    Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of HIV-1 viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions. PMID:24947940

  20. Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

    NASA Astrophysics Data System (ADS)

    Yu, Xinwei; Feizpour, Amin; Ramirez, Nora-Guadalupe P.; Wu, Linxi; Akiyama, Hisashi; Xu, Fangda; Gummuluru, Suryaram; Reinhard, Björn M.

    2014-06-01

    Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

  1. Structured treatment interruptions to control HIV-1 infection.

    PubMed

    Lori, F; Maserati, R; Foli, A; Seminari, E; Timpone, J; Lisziewicz, J

    2000-01-22

    Structured treatment interruptions progressively lowered the rate of viral rebound in some HIV-1 infected patients. This approach should be explored as an alternative to continuous antiretroviral therapies.

  2. 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

  3. Impaired IL-2 expression in latent HIV-1 infection.

    PubMed

    Shin, YoungHyun; Yoon, Cheol-Hee; Lim, Hoyong; Park, Jihwan; Roh, Tae-Young; Kang, Chun; Choi, Byeong-Sun

    2015-08-01

    Regarding the T cell function in HIV-1 infection, activation of T cells is enhanced in acutely HIV-1-infected T cells upon stimuli. However, T cell immune responses underlying the activation of T cell receptor (TCR) signaling molecules and interleukin (IL)-2 production in latently HIV-1-infected cells are poorly understood. The expression and activation of TCR components and its downstream molecules in acutely and latently HIV-1-infected T cells were compared using quantitative reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression and enzyme-linked immunosorbent assay (ELISA) for levels of IL-2 in phytohemagglutinin M (PHA-M). The levels of T cell surface molecules and TCR signaling molecules in latently HIV-1-infected cells were greatly decreased without changes in their mRNA levels. In addition, downstream TCR-signaling molecules in latently HIV-1-infected cells were not activated even in the presence of PHA-M. The phosphorylation of mitogen-activated protein kinases (MAPKs) in the presence of PHA-M was weakly induced in latently HIV-1-infected cells but was greater in acutely HIVNL4-3-infected cells. Finally, the production of IL-2 was significantly decreased in latently HIV-1-infected cells compared with uninfected parent cells. Thus, IL-2-related immunological functions in latently HIV-1-infected T cells were markedly impaired even in the presence of stimuli.

  4. Broad activation of latent HIV-1 in vivo

    PubMed Central

    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

  5. Detection of diverse HIV-1 genetic subtypes in the USA.

    PubMed

    Brodine, S K; Mascola, J R; Weiss, P J; Ito, S I; Porter, K R; Artenstein, A W; Garland, F C; McCutchan, F E; Burke, D S

    1995-11-01

    Of the nine genetic subtypes of HIV-1 that exist world wide, subtype B predominates in North America and Europe. Thus, most knowledge about HIV-1 and most vaccine development efforts are based on subtype B viruses. We document here the detection of HIV-1 subtypes A, D, and E in five US servicemen who acquired these non-subtype-B infections during overseas deployments. The dispersal of diverse HIV-1 subtypes into regions of the world with previously restricted genetic diversity may have important implications for the epidemiology of the epidemic and for the design and implementation of vaccine trials. PMID:7475661

  6. [Effective components against HIV-1 replicative enzymes isolated from plants].

    PubMed

    Peng, Zong-gen; Xu, Li-jia; Ye, Wen-cai; Xiao, Pei-gen; Chen, Hong-shan

    2010-02-01

    Plant active components characterized of many different structures and activities on multiple targets, have made them to be the important sources of inhibitors on HIV-1. For finding leading compounds with new structure against HIV-1, three key HIV-1 replicative enzymes (reverse transcriptase, protease and integrase) were used as screening models. The in vitro activities of 45 plant derived components isolated from Schisandraceae, Rutaceae and Ranunculaceae were reported. Within twelve triterpene components isolated, eight compounds were found to inhibit HIV-1 protease, in these eight active compounds, kadsuranic acid A (7) and nigranoic acid (8), inhibited both HIV-1 protease and integrase; Among fifteen lignans, meso-dihydroguaiaretic acid (15) and kadsurarin (16) were active on HIV-1 reverse transcriptase, and 4, 4-di(4-hydroxy-3-methoxyphenly)-2, 3-dimethylbutanol (13) active on HIV-1 integrase. All of the six alkaloids, seven flavones, and five others compounds were not active or only with low activities against HIV-1 replicative enzymes. Further studies of the triterpene components showing strong inhibitory activities on HIV-1 were warranted.

  7. 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

  8. 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

    2016-09-08

    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.

  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. [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.

  11. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    SciTech Connect

    Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2006-08-01

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5{alpha} with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain.

  12. Stable Phenotypic Changes of the Host T Cells Are Essential to the Long-Term Stability of Latent HIV-1 Infection

    PubMed Central

    Seu, Lillian; Sabbaj, Steffanie; Duverger, Alexandra; Wagner, Frederic; Anderson, Joshua C.; Davies, Elizabeth; Wolschendorf, Frank; Willey, Christopher D.; Saag, Michael S.; Goepfert, Paul

    2015-01-01

    ABSTRACT The extreme stability of the latent HIV-1 reservoir in the CD4+ memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. IMPORTANCE The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4+ memory T cells that

  13. 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

  14. HIV-1 infection may be on the rise in Peru.

    PubMed

    The results of a national survey have indicated that "... HIV-1 infection is epidemic in Peru among groups at high risk of sexually and parenterally transmitted diseases," a multicenter group reported (AIDS 1996; 10: 1141-1145). Although the risk of infection appears to be very low in the general population, it may possibly be increasing, according to Dr. Michael C. McCarthy, US National Naval Medical Center in Bethesda, Maryland. McCarthy's group evaluated over 140,000 serum samples for antibodies to HIV-1 between January 1986 and December 1990 in Peru. HIV-1 antibody was detected in 26% of samples from homosexual men, 10% of samples from male sexually transmitted disease patients, and 13% of samples from drug users. 10% of the samples from hemophiliacs and unlicensed female prostitutes were positive for antibodies to HIV-1. In general, he concluded that the patterns of the HIV-1 epidemic in Peru are similar to those seen in Brazil and "... are also similar to initial transmission patterns of HIV-1 infection of North America." However, McCarthy also noted a substantial increase in the prevalence of HIV-1 infection between the beginning and the end of the survey period. Although there was a low prevalence of HIV-1 infection among military personnel and among women seen at prenatal clinics, a "low but rising prevalence of HIV-1 antibody" among military personnel points to a potential increase in the general population. "The fact that many HIV-1 antibody-positive men were married and reported bisexual behavior (28%) highlights the potential of this group to transmit HIV-1 to female partners," he added.

  15. Microtubule-associated proteins 1 (MAP1) promote human immunodeficiency virus type I (HIV-1) intracytoplasmic routing to the nucleus.

    PubMed

    Fernandez, Juliette; Portilho, Débora M; Danckaert, Anne; Munier, Sandie; Becker, Andreas; Roux, Pascal; Zambo, Anaba; Shorte, Spencer; Jacob, Yves; Vidalain, Pierre-Olivier; Charneau, Pierre; Clavel, François; Arhel, Nathalie J

    2015-02-20

    After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

  16. Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

    PubMed Central

    Fernandez, Juliette; Portilho, Débora M.; Danckaert, Anne; Munier, Sandie; Becker, Andreas; Roux, Pascal; Zambo, Anaba; Shorte, Spencer; Jacob, Yves; Vidalain, Pierre-Olivier; Charneau, Pierre; Clavel, François; Arhel, Nathalie J.

    2015-01-01

    After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules. PMID:25505242

  17. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

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

    SciTech Connect

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

    2012-05-09

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

  19. 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.

  20. 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

  1. Probing the Effect of Force on HIV-1 Receptor CD4

    PubMed Central

    2015-01-01

    Cell-surface proteins are central for the interaction of cells with their surroundings and are also associated with numerous diseases. These molecules are exposed to mechanical forces, but the exact relation between force and the functions and pathologies associated with cell-surface proteins is unclear. An important cell-surface protein is CD4, the primary receptor of HIV-1. Here we show that mechanical force activates conformational and chemical changes on CD4 that may be important during viral attachment. We have used single-molecule force spectroscopy and analysis on HIV-1 infectivity to demonstrate that the mechanical extension of CD4 occurs in a time-dependent manner and correlates with HIV-1 infectivity. We show that Ibalizumab, a monoclonal antibody that blocks HIV-1, prevents the mechanical extension of CD4 domains 1 and 2. Furthermore, we demonstrate that thiol/disulfide exchange in CD4 requires force for exposure of cryptic disulfide bonds. This mechanical perspective provides unprecedented information that can change our understanding on how viruses interact with their hosts. PMID:25299596

  2. Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites

    PubMed Central

    Prescher, Jens; Baumgärtel, Viola; Ivanchenko, Sergey; Torrano, Adriano A.; Bräuchle, Christoph; Müller, Barbara; Lamb, Don C.

    2015-01-01

    The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck

  3. 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. PMID:27617163

  4. 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.

  5. Quantitative HIV-1 proviral DNA detection: a multicentre analysis.

    PubMed

    De Rossi, Anita; Zanchetta, Marisa; Vitone, Francesca; Antonelli, Guido; Bagnarelli, Patrizia; Buonaguro, Luigi; Capobianchi, Maria Rosaria; Clementi, Massimo; Abbate, Isabella; Canducci, Filippo; Monachetti, Alessia; Riva, Elisabetta; Rozera, Gabriella; Scagnolari, Carolina; Tagliamonte, Maria; Re, Maria Carla

    2010-10-01

    Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients. PMID:21213587

  6. 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 cel