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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. HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants

    PubMed Central

    Smith, Jessica L.; Izumi, Taisuke; Borbet, Timothy C.; Hagedorn, Ariel N.

    2014-01-01

    ABSTRACT Human APOBEC3 (A3) restriction factors provide intrinsic immunity against zoonotic transmission of pathogenic viruses. A3D, A3F, A3G, and A3H haplotype II (A3H-hapII) can be packaged into virion infectivity factor (Vif)-deficient HIVs to inhibit viral replication. To overcome these restriction factors, Vif binds to the A3 proteins in viral producer cells to target them for ubiquitination and proteasomal degradation, thus preventing their packaging into assembling virions. Therefore, the Vif-A3 interactions are attractive targets for novel drug development. HIV-1 and HIV-2 arose via distinct zoonotic transmission events of simian immunodeficiency viruses from chimpanzees and sooty mangabeys, respectively, and Vifs from these viruses have limited homology. To gain insights into the evolution of virus-host interactions that led to successful cross-species transmission of lentiviruses, we characterized the determinants of the interaction between HIV-2 Vif (Vif2) with human A3 proteins and compared them to the previously identified HIV-1 Vif (Vif1) interactions with the A3 proteins. We found that A3G, A3F, and A3H-hapII, but not A3D, were susceptible to Vif2-induced degradation. Alanine-scanning mutational analysis of the first 62 amino acids of Vif2 indicated that Vif2 determinants important for degradation of A3G and A3F are completely distinct from these regions in Vif1, as are the determinants in A3G and A3F that are critical for Vif2-induced degradation. These observations suggest that distinct Vif-A3 interactions evolved independently in different SIVs and their nonhuman primate hosts and conservation of the A3 determinants targeted by the SIV Vif proteins resulted in successful zoonotic transmission into humans. IMPORTANCE Primate APOBEC3 proteins provide innate immunity against invading pathogens, and Vif proteins of primate lentiviruses have evolved to overcome these host defenses by interacting with them and inducing their proteasomal degradation. HIV

  3. MULTIPLE WAYS OF TARGETING APOBEC3/VIF INTERACTIONS FOR ANTI-HIV-1 DRUG DEVELOPMENT

    PubMed Central

    Smith, Jessica L.; Bu, Wei; Burdick, Ryan C.; Pathak, Vinay K.

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1) infections and the resulting acquired immunodeficiency syndrome (AIDS) pandemic remain a global challenge in the absence of a protective vaccine and because of rapid selection of drug-resistant viral variants in response to all currently available antiviral therapies. Development of new and highly active antiviral agents would greatly facilitate effective clinical management of HIV-1 infections and delay the onset of AIDS. Recent advances in our understanding of intracellular immunity conferred by host cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F), and the mechanism by which the virally encoded Virion Infectivity Factor (Vif) protein induces their proteasomal degradation, provide fresh opportunities for the development of novel antiviral treatments. Interestingly, the interactions between Vif-A3G and Vif-A3F that overcome this host defense mechanism are structurally distinct, and provide two potential targets for antiviral drug development. This review provides an overview of the current knowledge of APOBEC3/Vif interactions and recent efforts to target these interactions for antiviral drug development. PMID:19837465

  4. APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

    PubMed

    Batisse, Julien; Guerrero, Santiago Xavier; Bernacchi, Serena; Richert, Ludovic; Godet, Julien; Goldschmidt, Valérie; Mély, Yves; Marquet, Roland; de Rocquigny, Hugues; Paillart, Jean-Christophe

    2013-06-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell. PMID:23576497

  5. APOBEC3G Impairs the Multimerization of the HIV-1 Vif Protein in Living Cells

    PubMed Central

    Batisse, Julien; Guerrero, Santiago Xavier; Bernacchi, Serena; Richert, Ludovic; Godet, Julien; Goldschmidt, Valérie; Mély, Yves; Marquet, Roland; de Rocquigny, Hugues

    2013-01-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55Gag. Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain (161PPLP164) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55Gag, Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell. PMID:23576497

  6. 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. PMID:26529466

  7. RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

    PubMed

    Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

    2007-09-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

  8. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    PubMed Central

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  9. Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

    PubMed

    Henriet, Simon; Richer, Delphine; Bernacchi, Serena; Decroly, Etienne; Vigne, Robert; Ehresmann, Bernard; Ehresmann, Chantal; Paillart, Jean-Christophe; Marquet, Roland

    2005-11-18

    The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F. PMID:16236319

  10. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  11. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif

    PubMed Central

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-01-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain 161PPLP164 regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the 161PPLP164 domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs. PMID:21076154

  12. Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F

    PubMed Central

    Siu, Karen K.; Sultana, Azmiri; Azimi, Farshad C.; Lee, Jeffrey E.

    2016-01-01

    The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses. APOBEC3F and APOBEC3G are the most potent factors against HIV-1. As a countermeasure, HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation. Here, we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding. Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F. Electrostatic interactions likely mediate Vif binding. Moreover, structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site, and a novel ‘aromatic switch’ is proposed to explain DNA substrate specificities across the APOBEC3 family. This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis. PMID:24185281

  13. Codon pairs of the HIV-1 vif gene correlate with CD4+ T cell count

    PubMed Central

    2013-01-01

    Background The human APOBEC3G (A3G) protein activity is associated with innate immunity against HIV-1 by inducing high rates of guanosines to adenosines (G-to-A) mutations (viz., hypermutation) in the viral DNA. If hypermutation is not enough to disrupt the reading frames of viral genes, it may likely increase the HIV-1 diversity. To counteract host innate immunity HIV-1 encodes the Vif protein that binds A3G protein and form complexes to be degraded by cellular proteolysis. Methods Here we studied the pattern of substitutions in the vif gene and its association with clinical status of HIV-1 infected individuals. To perform the study, unique vif gene sequences were generated from 400 antiretroviral-naïve individuals. Results The codon pairs: 78–154, 85–154, 101–157, 105–157, and 105–176 of vif gene were associated with CD4+ T cell count lower than 500 cells per mm3. Some of these codons were located in the 81LGQGVSIEW89 region and within the BC-Box. We also identified codons under positive selection clustered in the N-terminal region of Vif protein, between 21WKSLVK26 and 40YRHHY44 regions (i.e., 31, 33, 37, 39), within the BC-Box (i.e., 155, 159) and the Cullin5-Box (i.e., 168) of vif gene. All these regions are involved in the Vif-induced degradation of A3G/F complexes and the N-terminal of Vif protein binds to viral and cellular RNA. Conclusions Adaptive evolution of vif gene was mostly to optimize viral RNA binding and A3G/F recognition. Additionally, since there is not a fully resolved structure of the Vif protein, codon pairs associated with CD4+ T cell count may elucidate key regions that interact with host cell factors. Here we identified and discriminated codons under positive selection and codons under functional constraint in the vif gene of HIV-1. PMID:23578255

  14. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    PubMed

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  15. Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif

    PubMed Central

    Britan-Rosich, Elena; Nowarski, Roni; Kotler, Moshe

    2011-01-01

    In the absence of HIV-1 Vif protein, the host antiviral deaminase APOBEC3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative ssDNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity, and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here we show that: (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif virus associated A3G; (ii) Encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) Purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes (PICs) following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G anti-viral activity. PMID:21763507

  16. Productive Replication of vif-Chimeric HIV-1 in Feline Cells▿

    PubMed Central

    Stern, Melissa A.; Hu, Chunling; Saenz, Dyana T.; Fadel, Hind J.; Sims, Olivia; Peretz, Mary; Poeschla, Eric M.

    2010-01-01

    Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G→A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1VF and HIV-1VS) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection. PMID:20463079

  17. Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein*

    PubMed Central

    Pery, Erez; Sheehy, Ann; Nebane, N. Miranda; Brazier, Andrew Jay; Misra, Vikas; Rajendran, Kottampatty S.; Buhrlage, Sara J.; Mankowski, Marie K.; Rasmussen, Lynn; White, E. Lucile; Ptak, Roger G.; Gabuzda, Dana

    2015-01-01

    APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC50 as low as 8.4 μm and a TC50 of >100 μm when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity. PMID:25724652

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

    PubMed Central

    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 Kd = 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. PMID:19910370

  19. 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. PMID:19910370

  20. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    PubMed

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. PMID:22728817

  1. 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.

  2. 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. PMID:23545135

  3. Ring finger protein ZIN interacts with human immunodeficiency virus type 1 Vif.

    PubMed

    Feng, Feng; Davis, Adam; Lake, Julie-Anne; Carr, Jill; Xia, Wei; Burrell, Christopher; Li, Peng

    2004-10-01

    Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-kappaB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed. PMID:15367624

  4. 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

  5. ASK1 restores the antiviral activity of APOBEC3G by disrupting HIV-1 Vif-mediated counteraction

    PubMed Central

    Miyakawa, Kei; Matsunaga, Satoko; Kanou, Kazuhiko; Matsuzawa, Atsushi; Morishita, Ryo; Kudoh, Ayumi; Shindo, Keisuke; Yokoyama, Masaru; Sato, Hironori; Kimura, Hirokazu; Tamura, Tomohiko; Yamamoto, Naoki; Ichijo, Hidenori; Takaori-Kondo, Akifumi; Ryo, Akihide

    2015-01-01

    APOBEC3G (A3G) is an innate antiviral restriction factor that strongly inhibits the replication of human immunodeficiency virus type 1 (HIV-1). An HIV-1 accessory protein, Vif, hijacks the host ubiquitin–proteasome system to execute A3G degradation. Identification of the host pathways that obstruct the action of Vif could provide a new strategy for blocking viral replication. We demonstrate here that the host protein ASK1 (apoptosis signal-regulating kinase 1) interferes with the counteraction by Vif and revitalizes A3G-mediated viral restriction. ASK1 binds the BC-box of Vif, thereby disrupting the assembly of the Vif–ubiquitin ligase complex. Consequently, ASK1 stabilizes A3G and promotes its incorporation into viral particles, ultimately reducing viral infectivity. Furthermore, treatment with the antiretroviral drug AZT (zidovudine) induces ASK1 expression and restores the antiviral activity of A3G in HIV-1-infected cells. This study thus demonstrates a distinct function of ASK1 in restoring the host antiviral system that can be enhanced by AZT treatment. PMID:25901786

  6. 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

  7. 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

  8. 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

  9. Both Rbx1 and Rbx2 exhibit a functional role in the HIV-1 Vif-Cullin5 E3 ligase complex in vitro.

    PubMed

    Wang, Xiaodan; Wang, Xiaoying; Wang, Weiran; Zhang, Jingyao; Wang, Jiawen; Wang, Chu; Lv, Mingyu; Zuo, Tao; Liu, Donglai; Zhang, Haihong; Wu, Jiaxin; Yu, Bin; Kong, Wei; Wu, Hui; Yu, Xianghui

    2015-06-12

    Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo. PMID:25912140

  10. 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

  11. Cellular Requirements for Bovine Immunodeficiency Virus Vif-Mediated Inactivation of Bovine APOBEC3 Proteins

    PubMed Central

    Zhang, Wenyan; Wang, Hong; Li, Zhaolong; Liu, Xin; Liu, Guanchen; Harris, Reuben S.

    2014-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) viral infectivity factor (Vif) form a CRL5 E3 ubiquitin ligase complex to suppress virus restriction by host APOBEC3 (A3) proteins. The primate lentiviral Vif complex is composed of the unique cofactor core binding factor β (CBF-β) and canonical ligase components Cullin 5 (CUL5), Elongin B/C (ELOB/C), and RBX2. However, the mechanism by which the Vif protein of the related lentivirus bovine immunodeficiency virus (BIV) overcomes its host A3 proteins is less clear. In this study, we show that BIV Vif interacts with Cullin 2 (CUL2), ELOB/C, and RBX1, but not with CBF-β or CUL5, to form a CRL2 E3 ubiquitin ligase and degrade the restrictive bovine A3 proteins (A3Z2Z3 and A3Z3). RNA interference-mediated knockdown of ELOB or CUL2 inhibited BIV Vif-mediated degradation of these A3 proteins, whereas knockdown of CUL5 or CBF-β did not. BIV Vif with mutations in the BC box (Vif SLQ-AAA) or putative VHL box (Vif YI-AA), which cannot interact with ELOB/C or CUL2, respectively, lost the ability to counteract bovine A3 proteins. Moreover, CUL2 and UBE2M dominant negative mutants competitively inhibited the BIV Vif-mediated degradation mechanism. Thus, although the general strategy for inhibiting A3 proteins is conserved between HIV-1/SIV and BIV, the precise mechanisms can differ substantially, with only the HIV-1/SIV Vif proteins requiring CBF-β as a cofactor, HIV-1/SIV Vif using CUL5-RBX2, and BIV Vif using CUL2-RBX1. IMPORTANCE Primate lentivirus HIV-1 and SIV Vif proteins form a ubiquitin ligase complex to target host antiviral APOBEC3 proteins for degradation. However, the mechanism by which the nonprimate lentivirus BIV Vif inhibits bovine APOBEC3 proteins is unclear. In the present study, we determined the mechanism for BIV Vif-mediated degradation of bovine APOBEC3 proteins and found that it differs from the mechanism of HIV-1/SIV Vif by being CBF-β independent and

  12. 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

  13. 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

  14. Toll-interacting protein inhibits HIV-1 infection and regulates viral latency.

    PubMed

    Li, Chuan; Kuang, Wen-Dong; Qu, Di; Wang, Jian-Hua

    2016-06-24

    HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4(+) T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4(+) T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4(+) T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency. PMID:27181351

  15. Proteomics in the investigation of HIV-1 interactions with host proteins

    PubMed Central

    Li, Ming

    2015-01-01

    Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, we focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. PMID:25523935

  16. 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.

  17. 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

  18. 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.

  19. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  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. HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway, Integration Targeting and Replication Efficiency

    PubMed Central

    Schaller, Torsten; Ocwieja, Karen E.; Rasaiyaah, Jane; Price, Amanda J.; Brady, Troy L.; Roth, Shoshannah L.; Hué, Stéphane; Fletcher, Adam J.; Lee, KyeongEun; KewalRamani, Vineet N.; Noursadeghi, Mahdad; Jenner, Richard G.; James, Leo C.; Bushman, Frederic D.; Towers, Greg J.

    2011-01-01

    Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment. PMID:22174692

  2. HIV-1 antibodies and vaccine antigen selectively interact with lipid domains.

    PubMed

    Hardy, Gregory J; Wong, Gene C; Nayak, Rahul; Anasti, Kara; Hirtz, Michael; Shapter, Joseph G; Alam, S Munir; Zauscher, Stefan

    2014-10-01

    The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies. PMID:25019685

  3. Evolutionary interactions between N-linked glycosylation sites in the HIV-1 envelope.

    PubMed

    Poon, Art F Y; Lewis, Fraser I; Pond, Sergei L Kosakovsky; Frost, Simon D W

    2007-01-19

    The addition of asparagine (N)-linked polysaccharide chains (i.e., glycans) to the gp120 and gp41 glycoproteins of human immunodeficiency virus type 1 (HIV-1) envelope is not only required for correct protein folding, but also may provide protection against neutralizing antibodies as a "glycan shield." As a result, strong host-specific selection is frequently associated with codon positions where nonsynonymous substitutions can create or disrupt potential N-linked glycosylation sites (PNGSs). Moreover, empirical data suggest that the individual contribution of PNGSs to the neutralization sensitivity or infectivity of HIV-1 may be critically dependent on the presence or absence of other PNGSs in the envelope sequence. Here we evaluate how glycan-glycan interactions have shaped the evolution of HIV-1 envelope sequences by analyzing the distribution of PNGSs in a large-sequence alignment. Using a "covarion"-type phylogenetic model, we find that the rates at which individual PNGSs are gained or lost vary significantly over time, suggesting that the selective advantage of having a PNGS may depend on the presence or absence of other PNGSs in the sequence. Consequently, we identify specific interactions between PNGSs in the alignment using a new paired-character phylogenetic model of evolution, and a Bayesian graphical model. Despite the fundamental differences between these two methods, several interactions are jointly identified by both. Mapping these interactions onto a structural model of HIV-1 gp120 reveals that negative (exclusive) interactions occur significantly more often between colocalized glycans, while positive (inclusive) interactions are restricted to more distant glycans. Our results imply that the adaptive repertoire of alternative configurations in the HIV-1 glycan shield is limited by functional interactions between the N-linked glycans. This represents a potential vulnerability of rapidly evolving HIV-1 populations that may provide useful glycan

  4. Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

    PubMed Central

    Tekeste, Shewit S.; Wilkinson, Thomas A.; Weiner, Ethan M.; Xu, Xiaowen; Miller, Jennifer T.; Le Grice, Stuart F. J.; Clubb, Robert T.

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance (NMR) spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT prebound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were noninfectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a cosedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle. IMPORTANCE To establish a productive infection, human immunodeficiency virus type 1 (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by the HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN

  5. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  6. Computational Analysis of HIV-1 Resistance Based on Gene Expression Profiles and the Virus-Host Interaction Network

    PubMed Central

    Huang, Tao; Xu, Zhongping; Chen, Lei; Cai, Yu-Dong; Kong, Xiangyin

    2011-01-01

    A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS) therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR) and Incremental Feature Selection (IFS) methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection. PMID:21394196

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

    PubMed

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

    2005-01-21

    APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif. PMID:15581898

  8. 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

  9. Highly Accurate Structure-Based Prediction of HIV-1 Coreceptor Usage Suggests Intermolecular Interactions Driving Tropism

    PubMed Central

    Kieslich, Chris A.; Tamamis, Phanourios; Guzman, Yannis A.; Onel, Melis; Floudas, Christodoulos A.

    2016-01-01

    HIV-1 entry into host cells is mediated by interactions between the V3-loop of viral glycoprotein gp120 and chemokine receptor CCR5 or CXCR4, collectively known as HIV-1 coreceptors. Accurate genotypic prediction of coreceptor usage is of significant clinical interest and determination of the factors driving tropism has been the focus of extensive study. We have developed a method based on nonlinear support vector machines to elucidate the interacting residue pairs driving coreceptor usage and provide highly accurate coreceptor usage predictions. Our models utilize centroid-centroid interaction energies from computationally derived structures of the V3-loop:coreceptor complexes as primary features, while additional features based on established rules regarding V3-loop sequences are also investigated. We tested our method on 2455 V3-loop sequences of various lengths and subtypes, and produce a median area under the receiver operator curve of 0.977 based on 500 runs of 10-fold cross validation. Our study is the first to elucidate a small set of specific interacting residue pairs between the V3-loop and coreceptors capable of predicting coreceptor usage with high accuracy across major HIV-1 subtypes. The developed method has been implemented as a web tool named CRUSH, CoReceptor USage prediction for HIV-1, which is available at http://ares.tamu.edu/CRUSH/. PMID:26859389

  10. 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

  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. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    PubMed

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients. PMID:25332688

  13. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination

    PubMed Central

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤4 µg/ml) and stimulates CSR at high concentrations (≥8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients. PMID:25332688

  14. 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

  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. 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

  17. 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

  18. Predicting drug resistance of the HIV-1 protease using molecular interaction energy components.

    PubMed

    Hou, Tingjun; Zhang, Wei; Wang, Jian; Wang, Wei

    2009-03-01

    Drug resistance significantly impairs the efficacy of AIDS therapy. Therefore, precise prediction of resistant viral mutants is particularly useful for developing effective drugs and designing therapeutic regimen. In this study, we applied a structure-based computational approach to predict mutants of the HIV-1 protease resistant to the seven FDA approved drugs. We analyzed the energetic pattern of the protease-drug interaction by calculating the molecular interaction energy components (MIECs) between the drug and the protease residues. Support vector machines (SVMs) were trained on MIECs to classify protease mutants into resistant and nonresistant categories. The high prediction accuracies for the test sets of cross-validations suggested that the MIECs successfully characterized the interaction interface between drugs and the HIV-1 protease. We conducted a proof-of-concept study on a newly approved drug, darunavir (TMC114), on which no drug resistance data were available in the public domain. Compared with amprenavir, our analysis suggested that darunavir might be more potent to combat drug resistance. To quantitatively estimate binding affinities of drugs and study the contributions of protease residues to causing resistance, linear regression models were trained on MIECs using partial least squares (PLS). The MIEC-PLS models also achieved satisfactory prediction accuracy. Analysis of the fitting coefficients of MIECs in the regression model revealed the important resistance mutations and shed light into understanding the mechanisms of these mutations to cause resistance. Our study demonstrated the advantages of characterizing the protease-drug interaction using MIECs. We believe that MIEC-SVM and MIEC-PLS can help design new agents or combination of therapeutic regimens to counter HIV-1 protease resistant strains. PMID:18704937

  19. Cyclic Peptide Inhibitors of HIV-1 Capsid-Human Lysyl-tRNA Synthetase Interaction

    PubMed Central

    2012-01-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNALys,3, which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents. PMID:22276994

  20. Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

    PubMed

    Kanevsky, Igor; Chaminade, Françoise; Ficheux, Damien; Moumen, Abdeladim; Gorelick, Robert; Negroni, Matteo; Darlix, Jean-Luc; Fossé, Philippe

    2005-05-20

    During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer. PMID:15854644

  1. 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).

  2. 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

  3. Molecular Structure and Biochemical Properties of the HCCH-Zn2+ Site in HIV-1 Vif†

    PubMed Central

    Giri, Kalyan; Scott, Robert A.; Maynard, Ernest L.

    2009-01-01

    Virion infectivity factor (Vif) is an HIV accessory protein that is essential for the infection of CD4+ T cells. Vif recruits a Cullin-5 (Cul5)-based ubiquitin ligase that targets a host cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G), for proteasomal degradation. The Vif N-terminus binds APOBEC3G and the C-terminus interacts with the Cul5-based ubiquitin ligase machinery. Within the C-terminus, a highly conserved H108-X5-C114-X17–18-C133-X3–5-H139 (HCCH) motif binds zinc and is implicated in the Vif-Cul5 interaction. We have employed the biomimetic peptide HCCHp (HIV-1 Vif amino acids 101–142) in order to determine the zinc ligands and investigate the role of zinc binding in Cul5 recognition. Using CD spectroscopy, a competitive zinc binding assay, and a light scattering assay, we found that mutation of the conserved His and Cys residues in HCCHp had little effect on secondary structure, but reduced zinc-binding affinity and altered the aggregation properties of the peptides. X-ray absorption spectroscopy was used to study zinc coordination in wild type HCCHp. The data are consistent with S2N(imid)2 coordination and strongly suggest that His-108, Cys-114, Cys-133, and His-139 are zinc ligands. Mutation of one or both conserved Cys residues in HCCHp led to a decrease in Cys ligation, and an increase in the number of (N,O) ligands, with noninteger coordination numbers suggesting zinc site heterogeneity. A purified fragment of human Cul5 was found to inhibit zinc-induced aggregation of HCCHp, and pull-down experiments revealed that zinc binding to HCCHp increases the strength of the HCCHp-Cul5 interaction by eight-fold. PMID:19588889

  4. A review of in silico approaches for analysis and prediction of HIV-1-human protein-protein interactions.

    PubMed

    Bandyopadhyay, Sanghamitra; Ray, Sumanta; Mukhopadhyay, Anirban; Maulik, Ujjwal

    2015-09-01

    The computational or in silico approaches for analysing the HIV-1-human protein-protein interaction (PPI) network, predicting different host cellular factors and PPIs and discovering several pathways are gaining popularity in the field of HIV research. Although there exist quite a few studies in this regard, no previous effort has been made to review these works in a comprehensive manner. Here we review the computational approaches that are devoted to the analysis and prediction of HIV-1-human PPIs. We have broadly categorized these studies into two fields: computational analysis of HIV-1-human PPI network and prediction of novel PPIs. We have also presented a comparative assessment of these studies and proposed some methodologies for discussing the implication of their results. We have also reviewed different computational techniques for predicting HIV-1-human PPIs and provided a comparative study of their applicability. We believe that our effort will provide helpful insights to the HIV research community. PMID:25479794

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

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

    PubMed

    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

  11. 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

  12. Mining quasi-bicliques from HIV-1-human protein interaction network: a multiobjective biclustering approach.

    PubMed

    Maulik, Ujjwal; Mukhopadhyay, Anirban; Bhattacharyya, Malay; Kaderali, Lars; Brors, Benedikt; Bandyopadhyay, Sanghamitra; Eils, Roland

    2013-01-01

    In this work, we model the problem of mining quasi-bicliques from weighted viral-host protein-protein interaction network as a biclustering problem for identifying strong interaction modules. In this regard, a multiobjective genetic algorithm-based biclustering technique is proposed that simultaneously optimizes three objective functions to obtain dense biclusters having high mean interaction strengths. The performance of the proposed technique has been compared with that of other existing biclustering methods on an artificial data. Subsequently, the proposed biclustering method is applied on the records of biologically validated and predicted interactions between a set of HIV-1 proteins and a set of human proteins to identify strong interaction modules. For this, the entire interaction information is realized as a bipartite graph. We have further investigated the biological significance of the obtained biclusters. The human proteins involved in the strong interaction module have been found to share common biological properties and they are identified as the gateways of viral infection leading to various diseases. These human proteins can be potential drug targets for developing anti-HIV drugs. PMID:23929866

  13. Mining Quasi-Bicliques from HIV-1--Human Protein Interaction Network: A Multiobjective Biclustering Approach.

    PubMed

    Maulik, Ujjwal; Mukhopadhyay, Anirban; Bhattacharyya, Malay; Kaderali, Lars; Brors, Benedikt; Bandyopadhyay, Sanghamitra; Eils, Roland

    2012-11-28

    In this work, we model the problem of mining quasi-bicliques from weighted viral-host protein-protein interaction network as a biclustering problem for identifying strong interaction modules. In this regard, a multiobjective genetic algorithm based biclustering technique is proposed that simultaneously optimizes three objective functions to obtain dense biclusters having high mean interaction strengths. The performance of the proposed technique has been compared with that of other existing biclustering methods on an artificial data. Subsequently, the proposed biclustering method is applied on the records of biologically validated and predicted interactions between a set of HIV-1 proteins and a set of human proteins to identify strong interaction modules. For this, the entire interaction information is realized as a bipartite graph. We have further investigated the biological significance of the obtained biclusters. The human proteins involved in the strong interaction module have been found to share common biological properties and they are identified as the gateways of viral infection leading to various diseases. These human proteins can be potential drug targets for developing anti-HIV drugs. PMID:23209057

  14. An in silico approach for identification of novel inhibitors as a potential therapeutics targeting HIV-1 viral infectivity factor.

    PubMed

    Sinha, Chanda; Nischal, Anuradha; Bandaru, Srinivas; Kasera, Priyadarshani; Rajput, Ashish; Nayarisseri, Anuraj; Khattri, Sanjay

    2015-01-01

    Currently available antiviral drugs target the pol-encoded retroviral enzymes or integrases, in addition, inhibitors that target HIV-1 envelope-receptor interactions have also been recently approved. Recent understanding of the interactions between HIV-1 and host restriction factors has provided fresh avenues for development of novel antiviral drugs. For example, viral infectivity factor (Vif) now surfaced as an important therapeutic target in treatment of HIV infection. Vif suppresses A3G antiviral activity by targeting these proteins for polyubiquitination and proteasomal degradation. In the present study we analyzed the inhibitory potential of VEC5 and RN18 to inhibit the Vif-A3G interaction through protein- protein docking studies. Perusal of the study showed that, VEC5 and RN18 though inhibits the interaction however showed sub optimal potential. To overcome this set back, we identified 35 structural analogues of VEC5 and 18 analogues of RN18 through virtual screening approach. Analogue with PubCID 71624757 and 55358204 (AKOS006479723) -structurally akin to VEC5 and RN18 respectively showed much appreciable interaction than their respective parent compound. Evident from Vif-A3G; protein - protein docking studies, analogue PubCID 71624757 demonstrated 1.08 folds better inhibitory potential than its parent compound VEC5 while analogue PubCID 55358204 was 1.15 folds better than RN18. Further these analogues passed drug likeness filters and predicted to be non- toxic. We expect these analogues can be put to pharmacodynamic studies that can pave way the breakthrough in HIV therapeutics. PMID:25579575

  15. Probing interactions of Vpu from HIV-1 with amiloride-based compounds.

    PubMed

    Rosenberg, Matthew R; Weaver, Llara M; Casarotto, Marco G

    2016-04-01

    Viral ion channels or viroporins are short membrane proteins that participate in wide-ranging functions including virus replication and entry, assembly, and virus release. One such viroporin is the 81 amino acid residue Vpu protein derived from HIV-1. This protein consists of one transmembrane (TM) and two cytoplasmic helical domains, the former of which oligomerises to form cation-selective ion channels. In this study, we investigate the binding properties of amiloride compounds to Vpu embedded into liposomes using surface plasmon resonance (SPR). We explore the Vpu ion channel inhibitor, hexamethylene amiloride (HMA), as a molecular tool to examine the potential interactive role of key TM residues, Trp23, Ser24, and Glu29, in terms of positioning of these residues on the channel pore and the orientation of its constituent helices. The study provides experimental support that a direct interaction between Ser24 and HMA occurs and that this residue is most likely located in the channel pore. Mutation of Trp23 does not impact HMA affinity suggesting no direct involvement in binding and that this residue is lipid facing. These findings indicate that small molecules such as amilorides are capable of specifically interacting with Vpu ion channels. Although a correlation between ion channel and functional activity cannot be dismissed, alternative mechanisms involving protein-protein interactions may play an important role in the efficacy of these compounds. PMID:26724207

  16. COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

    PubMed Central

    Marban, Céline; Redel, Laetitia; Suzanne, Stella; Van Lint, Carine; Lecestre, Dominique; Chasserot-Golaz, Sylvette; Leid, Mark; Aunis, Dominique; Schaeffer, Evelyne; Rohr, Olivier

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region. PMID:15849318

  17. 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

  18. 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

  19. Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

    PubMed Central

    Nanda, Hirsh; Datta, Siddhartha A.K.; Heinrich, Frank; Lösche, Mathias; Rein, Alan; Krueger, Susan; Curtis, Joseph E.

    2010-01-01

    The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions. PMID:20959092

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

    PubMed

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

    2016-02-26

    HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection. PMID:26733199

  1. Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

    PubMed Central

    Kyrychenko, Alexander; Freites, J. Alfredo; He, Jing; Tobias, Douglas J.; Wimley, William C.; Ladokhin, Alexey S.

    2014-01-01

    We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning. PMID:24507601

  2. 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

  3. HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction.

    PubMed

    Rom, Slava; Pacifici, Marco; Passiatore, Giovanni; Aprea, Susanna; Waligorska, Agnieszka; Del Valle, Luis; Peruzzi, Francesca

    2011-10-01

    The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia. PMID:21745501

  4. Two mutations in the vif gene of maedi-visna virus have different phenotypes, indicating more than one function of Vif.

    PubMed

    Franzdóttir, Sigrídur R; Ólafsdóttir, Katrín; Jónsson, Stefán R; Strobel, Hannah; Andrésson, Ólafur S; Andrésdóttir, Valgerdur

    2016-01-15

    Like most other lentiviruses, maedi-visna virus (MVV) requires Vif for replication in natural target cells and in vivo. Here, we show that Vif-deficient MVV accumulates G-A mutations in the sequence context characteristic of ovine APOBEC3, consistent with a role of MVV Vif in neutralizing APOBEC3. We studied two point mutations in the vif gene of MVV. One was a tryptophan to arginine mutation that affects the interaction with APOBEC3 and caused G-A hypermutation. The other mutation was a proline to serine mutation that together with a mutation in the capsid protein caused attenuated replication in fetal ovine synovial (FOS) cells but not in sheep choroid plexus (SCP) cells. There was no hypermutation associated with this mutation. These results suggest that MVV Vif exerts more than one function and that there may be interaction between Vif and the capsid. The results also suggest the involvement of an unknown host factor in MVV Vif function. PMID:26590796

  5. 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

  6. 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.

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

    SciTech Connect

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

    2013-08-05

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

  8. Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity

    PubMed Central

    Wang, Wei; Naiyer, Nada; Mitra, Mithun; Li, Jialin; Williams, Mark C.; Rouzina, Ioulia; Gorelick, Robert J.; Wu, Zhengrong; Musier-Forsyth, Karin

    2014-01-01

    During human immunodeficiency virus type 1 (HIV-1) maturation, three different forms of nucleocapsid (NC) protein—NCp15 (p9 + p6), NCp9 (p7 + SP2) and NCp7—appear successively. A mutant virus expressing NCp15 shows greatly reduced infectivity. Mature NCp7 is a chaperone protein that facilitates remodeling of nucleic acids (NAs) during reverse transcription. To understand the strict requirement for NCp15 processing, we compared the chaperone function of the three forms of NC. NCp15 anneals tRNA to the primer-binding site at a similar rate as NCp7, whereas NCp9 is the most efficient annealing protein. Assays to measure NA destabilization show a similar trend. Dynamic light scattering studies reveal that NCp15 forms much smaller aggregates relative to those formed by NCp7 and NCp9. Nuclear magnetic resonance studies suggest that the acidic p6 domain of HIV-1 NCp15 folds back and interacts with the basic zinc fingers. Neutralizing the acidic residues in p6 improves the annealing and aggregation activity of NCp15 to the level of NCp9 and increases the protein–NA aggregate size. Slower NCp15 dissociation kinetics is observed by single-molecule DNA stretching, consistent with the formation of electrostatic inter-protein contacts, which likely contribute to the distinct aggregate morphology, irregular HIV-1 core formation and non-infectious virus. PMID:24813443

  9. 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

  10. 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

  11. 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.

  12. Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription.

    PubMed

    Boudier, Christian; Humbert, Nicolas; Chaminade, Françoise; Chen, Yingying; de Rocquigny, Hugues; Godet, Julien; Mauffret, Olivier; Fossé, Philippe; Mély, Yves

    2014-01-01

    The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure-activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44-61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44-61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion. PMID:24153111

  13. Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription

    PubMed Central

    Boudier, Christian; Humbert, Nicolas; Chaminade, Françoise; Chen, Yingying; de Rocquigny, Hugues; Godet, Julien; Mauffret, Olivier; Fossé, Philippe; Mély, Yves

    2014-01-01

    The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44–61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44–61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure–activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44–61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44–61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion. PMID:24153111

  14. Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5

    PubMed Central

    Schatz, Octavian; Oft, Martin; Dascher, Christiane; Schebesta, Michael; Rosorius, Olaf; Jaksche, Herbert; Dobrovnik, Marika; Bevec, Dorian; Hauber, Joachim

    1998-01-01

    It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts. PMID:9465063

  15. HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter.

    PubMed

    Deng, Longwen; Ammosova, Tatyana; Pumfery, Anne; Kashanchi, Fatah; Nekhai, Sergei

    2002-09-13

    Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1 transcription in vitro. Tat-mediated CTD phosphorylation by CDK2 required cysteine 22 in the activation domain of Tat and amino acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by forming dynamic contacts with amino acids 15-24 and 36-49 of Tat. Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2 associated with RNAPII and was found in elongation complexes assembled on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in HeLa nuclear extract blocked Tat-dependent transcription. We suggest that CDK2 is part of a transcription complex that is required for Tat-dependent transcription and that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD phosphorylation by CDK2. PMID:12114499

  16. 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

  17. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    1993-01-01

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

  20. 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.

  1. 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.

  2. 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.

  3. Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

    PubMed

    Yoshikawa, Rokusuke; Izumi, Taisuke; Nakano, Yusuke; Yamada, Eri; Moriwaki, Miyu; Misawa, Naoko; Ren, Fengrong; Kobayashi, Tomoko; Koyanagi, Yoshio; Sato, Kei

    2016-06-01

    Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3. PMID:27193350

  4. 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.

  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. 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.

  7. INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo

    PubMed Central

    2013-01-01

    Background Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication. Results A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1NL4-3 and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro. Conclusions Our results demonstrate that a

  8. 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

  9. 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

  10. An extended CCR5-ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions

    PubMed Central

    Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-01-01

    The chemokine receptor CCR5 serves as a co-receptor for the Human Immunodefficiency Virus type-1, HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing the ECL1 and ECL3 were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study we determined that in aqueous buffer the segment Q188-Q194 in an elongated ECL2 peptide (R168 to K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two dimensional Saturation Transfer Difference NMR spectroscopy and dynamic filtering studies revealed the involvement of Y187, F189, W190 and F193 of the helical segment, in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule and therefore, could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. PMID:25703038

  11. 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

  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. 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.

  14. 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

  15. Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface promote dimer formation.

    PubMed

    Tsiang, Manuel; Jones, Gregg S; Hung, Magdeleine; Samuel, Dharmaraj; Novikov, Nikolai; Mukund, Susmith; Brendza, Katherine M; Niedziela-Majka, Anita; Jin, Debi; Liu, Xiaohong; Mitchell, Michael; Sakowicz, Roman; Geleziunas, Romas

    2011-03-15

    We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation. PMID:21222490

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

    PubMed Central

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

    2016-01-01

    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. PMID:26927806

  17. 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.

  18. Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil*S⃞

    PubMed Central

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

    2009-01-01

    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 (KD = 30 nm), close to its IC50 concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (KD = 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 (KD = 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 affinity T20

  19. Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin

    PubMed Central

    Bugatti, Antonella; Giagulli, Cinzia; Urbinati, Chiara; Caccuri, Francesca; Chiodelli, Paola; Oreste, Pasqua; Fiorentini, Simona; Orro, Alessandro; Milanesi, Luciano; D'Ursi, Pasqualina; Caruso, Arnaldo; Rusnati, Marco

    2013-01-01

    Once released by HIV+ cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists. PMID:23166320

  20. 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.

  1. Free energy calculation of single molecular interaction using Jarzynski's identity method: the case of HIV-1 protease inhibitor system

    NASA Astrophysics Data System (ADS)

    Li, De-Chang; Ji, Bao-Hua

    2012-06-01

    Jarzynski' identity (JI) method was suggested a promising tool for reconstructing free energy landscape of biomolecular interactions in numerical simulations and experiments. However, JI method has not yet been well tested in complex systems such as ligand-receptor molecular pairs. In this paper, we applied a huge number of steered molecular dynamics (SMD) simulations to dissociate the protease of human immunodeficiency type I virus (HIV-1 protease) and its inhibitors. We showed that because of intrinsic complexity of the ligand-receptor system, the energy barrier predicted by JI method at high pulling rates is much higher than experimental results. However, with a slower pulling rate and fewer switch times of simulations, the predictions of JI method can approach to the experiments. These results suggested that the JI method is more appropriate for reconstructing free energy landscape using the data taken from experiments, since the pulling rates used in experiments are often much slower than those in SMD simulations. Furthermore, we showed that a higher loading stiffness can produce higher precision of calculation of energy landscape because it yields a lower mean value and narrower bandwidth of work distribution in SMD simulations.

  2. Interaction between Artemether-Lumefantrine and Nevirapine-Based Antiretroviral Therapy in HIV-1-Infected Patients▿

    PubMed Central

    Kredo, T.; Mauff, K.; Van der Walt, J. S.; Wiesner, L.; Maartens, G.; Cohen, K.; Smith, P.; Barnes, K. I.

    2011-01-01

    Artemether-lumefantrine and nevirapine-based antiretroviral therapy (ART) are the most commonly recommended first-line treatments for malaria and HIV, respectively, in Africa. Artemether, lumefantrine, and nevirapine are metabolized by the cytochrome P450 3A4 enzyme system, which nevirapine induces, creating potential for important drug interactions. In a parallel-design pharmacokinetic study, concentration-time profiles were obtained in two groups of HIV-infected patients: ART-naïve patients and those stable on nevirapine-based therapy. Both groups received the recommended artemether-lumefantrine dose. Patients were admitted for intense pharmacokinetic sampling (0 to 72 h) with outpatient sampling until 21 days. Concentrations of lumefantrine, artemether, dihydroartemisinin, and nevirapine were determined by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The primary outcome was observed day 7 lumefantrine concentrations, as these are associated with therapeutic response in malaria. We enrolled 36 patients (32 females). Median (range) day 7 lumefantrine concentrations were 622 ng/ml (185 to 2,040 ng/ml) and 336 ng/ml (29 to 934 ng/ml) in the nevirapine and ART-naïve groups, respectively (P = 0.0002). The median artemether area under the plasma concentration-time curve from 0 to 8 h [AUC(0-8 h)] (P < 0.0001) and dihydroartemisinin AUC(60-68 h) (P = 0.01) were lower in the nevirapine group. Combined artemether and dihydroartemisinin exposure decreased over time only in the nevirapine group (geometric mean ratio [GMR], 0.76 [95% confidence interval {CI}, 0.65 to 0.90]; P < 0.0001) and increased with the weight-adjusted artemether dose (GMR, 2.12 [95% CI, 1.31 to 3.45]; P = 0.002). Adverse events were similar between groups, with no difference in electrocardiographic Fridericia corrected QT and P-R intervals at the expected time of maximum lumefantrine concentration (Tmax). Nevirapine-based ART decreased artemether and dihydroartemisinin

  3. Interaction between artemether-lumefantrine and nevirapine-based antiretroviral therapy in HIV-1-infected patients.

    PubMed

    Kredo, T; Mauff, K; Van der Walt, J S; Wiesner, L; Maartens, G; Cohen, K; Smith, P; Barnes, K I

    2011-12-01

    Artemether-lumefantrine and nevirapine-based antiretroviral therapy (ART) are the most commonly recommended first-line treatments for malaria and HIV, respectively, in Africa. Artemether, lumefantrine, and nevirapine are metabolized by the cytochrome P450 3A4 enzyme system, which nevirapine induces, creating potential for important drug interactions. In a parallel-design pharmacokinetic study, concentration-time profiles were obtained in two groups of HIV-infected patients: ART-naïve patients and those stable on nevirapine-based therapy. Both groups received the recommended artemether-lumefantrine dose. Patients were admitted for intense pharmacokinetic sampling (0 to 72 h) with outpatient sampling until 21 days. Concentrations of lumefantrine, artemether, dihydroartemisinin, and nevirapine were determined by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The primary outcome was observed day 7 lumefantrine concentrations, as these are associated with therapeutic response in malaria. We enrolled 36 patients (32 females). Median (range) day 7 lumefantrine concentrations were 622 ng/ml (185 to 2,040 ng/ml) and 336 ng/ml (29 to 934 ng/ml) in the nevirapine and ART-naïve groups, respectively (P = 0.0002). The median artemether area under the plasma concentration-time curve from 0 to 8 h [AUC((0-8 h))] (P < 0.0001) and dihydroartemisinin AUC((60-68 h)) (P = 0.01) were lower in the nevirapine group. Combined artemether and dihydroartemisinin exposure decreased over time only in the nevirapine group (geometric mean ratio [GMR], 0.76 [95% confidence interval {CI}, 0.65 to 0.90]; P < 0.0001) and increased with the weight-adjusted artemether dose (GMR, 2.12 [95% CI, 1.31 to 3.45]; P = 0.002). Adverse events were similar between groups, with no difference in electrocardiographic Fridericia corrected QT and P-R intervals at the expected time of maximum lumefantrine concentration (T(max)). Nevirapine-based ART decreased artemether and

  4. Tumultuous Relationship between the Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) and the Human APOBEC-3G and APOBEC-3F Restriction Factors

    PubMed Central

    Henriet, Simon; Mercenne, Gaëlle; Bernacchi, Serena; Paillart, Jean-Christophe; Marquet, Roland

    2009-01-01

    Summary: The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55Gag, by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F. PMID:19487726

  5. Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors.

    PubMed

    Henriet, Simon; Mercenne, Gaëlle; Bernacchi, Serena; Paillart, Jean-Christophe; Marquet, Roland

    2009-06-01

    The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55(Gag), by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F. PMID:19487726

  6. Analysis of human immunodeficiency virus type 1 Vif gene sequences among men who have sex with men in Heilongjiang province of China.

    PubMed

    Shao, Bing; Li, Hang; Liu, Sheng-Yuan; Li, Wen-Jing; Huang, Chao-Qun; Lin, Yuan-Long; Wang, Fu-Xiang; Wang, Bin-You

    2013-05-01

    To identify the current prevalent subtypes and to study the genetic variation of HIV-1 strains in men who have sex with men (MSM) residing in Heilongjiang province, China. We analyzed the characteristics of the nucleotide sequences and the corresponding deduced protein of Vif of HIV-1 strains isolated from 17 drug-naive HIV-1-seropositive MSM. Subtypes B (7.65%) and B' (Thailand B) (11.76%), CRF07_BC (47.06%), and CRF01_AE (23.53%) were identified. Phylogenetic analysis showed that there was a close relationship between our strains and those from the same MSM population in Hebei province, which is geographically close to Heilongjiang. Most of the documented Vif functional motifs are well conserved in the majority of our analyzed sequences. Taken together, our results suggest that there might be multiple introductions of HIV in Heilongjiang MSM and frequent sexual communications with other geographically nearby MSM populations. PMID:23231069

  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. 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.

  9. 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

  10. 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

  11. 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

  12. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

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

  13. 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

  14. 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

  15. 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

  16. 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…

  17. Revisiting HIV-1 uncoating.

    PubMed

    Arhel, Nathalie

    2010-01-01

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

  18. 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

  19. 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

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

    PubMed

    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

  1. Genome sequence of a novel HIV-1 circulating recombinant form 54_01B from Malaysia.

    PubMed

    Ng, Kim Tien; Ong, Lai Yee; Takebe, Yutaka; Kamarulzaman, Adeeba; Tee, Kok Keng

    2012-10-01

    We report here the first novel HIV-1 circulating recombinant form (CRF) 54_01B (CRF54_01B) isolated from three epidemiologically unlinked subjects of different risk groups in Malaysia. These recently sampled recombinants showed a complex genome organization composed of parental subtype B' and CRF01_AE, with identical recombination breakpoints observed in the gag, pol, and vif genes. Such a discovery highlights the ongoing active generation and spread of intersubtype recombinants involving the subtype B' and CRF01_AE lineages and indicates the potential of the new CRF in bridging HIV-1 transmission among different risk groups in Southeast Asia. PMID:22997423

  2. APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

    PubMed

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-07-29

    Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  3. APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

    PubMed Central

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-01-01

    Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  4. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    PubMed

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein. PMID:27109379

  5. 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.

  6. 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.

  7. 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

  8. Probing the influence of hypermodified residues within the tRNA3(Lys) anticodon stem loop interacting with the A-loop primer sequence from HIV-1.

    PubMed

    Galindo-Murillo, Rodrigo; Davis, Darrell R; Cheatham, Thomas E

    2016-03-01

    Replication of the HIV-1 virus requires reverse transcription of the viral RNA genome, a process that is specifically initiated by human tRNA3(Lys) packaged within the infectious virion. The primary binding site for the tRNA involves the 3' 18 nucleotides with an additional interaction between an adenine rich loop (A-loop) in the template and the anticodon stem-loop region of the tRNA3(Lys). The loop of the tRNA primer contains two hypermodified base residues and a pseudouridine that are required for a proper binding and activity. Here, we investigate the influence on the structure, dynamics and binding stability of the three modified residues (mnm(5)s(2)U34, t(6)A37 and Ψ39) using extensive molecular dynamics and Quantum Theory of Atoms in Molecules (QTAIM) analysis. Consistent with experiment, the results suggest that the three modified residues are required for faithful binding. Residues mnm(5)s(2)U34 and Ψ39 have a major influence in stabilizing the anticodon loop whereas mnm(5)s(2)U34 and t(6)A37 appear to stabilize the formation of the complex of tRNA3(Lys) with the HIV-1 A-loop. PMID:26655694

  9. Uneven Genetic Robustness of HIV-1 Integrase

    PubMed Central

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

    2014-01-01

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

  10. 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. Structural Convergence between CryoEM and NMR Reveals Novel Intersubunit Interactions Critical for HIV-1 Capsid Function

    PubMed Central

    Byeon, In-Ja L.; Meng, Xin; Jung, Jinwon; Zhao, Gongpu; Yang, Ruifeng; Ahn, Jinwoo; Shi, Jiong; Concel, Jason; Aiken, Christopher; Zhang, Peijun; Gronenborn, Angela M.

    2009-01-01

    Summary Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two and three-dimensional arrays provided a molecular model of the capsid protein (CA) hexamer and revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits extremely well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a novel CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly. PMID:19914170

  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. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

    PubMed

    Wilson, S A; Sieiro-Vazquez, C; Edwards, N J; Iourin, O; Byles, E D; Kotsopoulou, E; Adamson, C S; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    1999-08-15

    The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans. PMID:10432305

  14. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

    PubMed Central

    Wilson, S A; Sieiro-Vazquez, C; Edwards, N J; Iourin, O; Byles, E D; Kotsopoulou, E; Adamson, C S; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    1999-01-01

    The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans. PMID:10432305

  15. TNPO3 Is Required for HIV-1 Replication after Nuclear Import but prior to Integration and Binds the HIV-1 Core

    PubMed Central

    Valle-Casuso, Jose Carlos; Di Nunzio, Francesca; Yang, Yang; Reszka, Natalia; Lienlaf, Maritza; Arhel, Nathalie; Perez, Patricio; Brass, Abraham L.

    2012-01-01

    TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import. PMID:22398280

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  18. HIV-1 diversity in infected individuals in Suzhou and Suqian, China.

    PubMed

    Qin, Chenhao; Zhang, Ping; Zhu, Weiguang; Hao, Fangyuan; Gu, Aiping; Fen, Ping; Zhu, Xueming; Du, Hong

    2016-01-01

    Jiangsu is one province with severe HIV-1 epidemic in China. However, the molecular epidemiological characterizations of HIV-1 in many cities of Jiangsu remain unclear. A molecular epidemiological investigation was performed based on 38 HIV-positive samples collected from Suzhou and Suqian during 2011-2013. Five HIV-1 genomic fragments, p17, pol, vif-vpr, vpr-env, and C2V3 were amplified and sequenced from these samples. HIV-1 group M subtype of each sample was determined by phylogenetic analyses with the standard reference sequences. Among these infected individuals, 81.6 % (31/38) self-reported to be infected via sexual contacts, including 50.0 % (19/38) via heterosexual contact and 31.6 % (12/38) via homosexual contact. Among 34 samples with available pol or vif-env sequence, 19 (55.9 %) CRF01_AE, 7 (20.6 %) CRF07_BC, 3 (8.8 %) CRF08_BC, and 5 (14.7 %) inter-subtype recombinants were identified. No pure B, B' and C subtypes were found in this cohort. The five recombinants contain one B/C, three CRF01/B and one CRF01/B/C recombinants. These results suggest that CRF01_AE was the most predominant HIV-1 group M subtype and CRF01_AE-involved recombinants were the major recombinant forms. Comparison showed that there was no obvious difference in HIV-1 group M subtype distribution between Jiangsu (including Suzhou and Suqian) and the surrounding provinces (e.g., Shanghai, Anhui, and Shandong). CRF01_AE and CRF07_BC were the top two predominant HIV-1 genotypes in Jiangsu, and less and/or no pure subtype B and C was currently circulating here. We predicted that more CRF01/CRF07 recombinants, but fewer B/C recombinants will be generated in Jiangsu in future. PMID:27386334

  19. Impacts of Humanized Mouse Models on the Investigation of HIV-1 Infection: Illuminating the Roles of Viral Accessory Proteins in Vivo

    PubMed Central

    Yamada, Eri; Yoshikawa, Rokusuke; Nakano, Yusuke; Misawa, Naoko; Koyanagi, Yoshio; Sato, Kei

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) encodes four accessory genes: vif, vpu, vpr, and nef. Recent investigations using in vitro cell culture systems have shed light on the roles of these HIV-1 accessory proteins, Vif, Vpr, Vpu, and Nef, in counteracting, modulating, and evading various cellular factors that are responsible for anti-HIV-1 intrinsic immunity. However, since humans are the exclusive target for HIV-1 infection, conventional animal models are incapable of mimicking the dynamics of HIV-1 infection in vivo. Moreover, the effects of HIV-1 accessory proteins on viral infection in vivo remain unclear. To elucidate the roles of HIV-1 accessory proteins in the dynamics of viral infection in vivo, humanized mouse models, in which the mice are xenotransplanted with human hematopoietic stem cells, has been utilized. This review describes the current knowledge of the roles of HIV-1 accessory proteins in viral infection, replication, and pathogenicity in vivo, which are revealed by the studies using humanized mouse models. PMID:25807049

  20. 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

  1. 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

  2. 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.

  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. 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

  5. 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.

  6. HIV-1 Fusion Assay

    PubMed Central

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

    2016-01-01

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

  7. Molecular dynamics simulation and experimental verification of the interaction between cyclin T1 and HIV-1 Tat proteins.

    PubMed

    Asamitsu, Kaori; Hirokawa, Takatsugu; Hibi, Yurina; Okamoto, Takashi

    2015-01-01

    The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2' helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1' and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound. PMID:25781978

  8. Molecular Dynamics Simulation and Experimental Verification of the Interaction between Cyclin T1 and HIV-1 Tat Proteins

    PubMed Central

    Asamitsu, Kaori; Hibi, Yurina

    2015-01-01

    The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2’ helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1’ and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound. PMID:25781978

  9. Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

    NASA Astrophysics Data System (ADS)

    Mely, Yves; Azoulay, Joel; Beltz, Herve; Clamme, Jean-Pierre; Bernacchi, Serena; Ficheux, Damien; Roques, Bernard P.; Darlix, Jean-Luc

    2004-09-01

    The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3" terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

  10. 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.

  11. 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

  12. TIM-family proteins inhibit HIV-1 release

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  14. TopoisomeraseIIβ in HIV-1 transactivation.

    PubMed

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

    2016-03-01

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

  15. 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

  16. 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

  17. Opiate Addiction Therapies and HIV-1 Tat: Interactive Effects on Glial [Ca2+]i, Oxyradical and Neuroinflammatory Chemokine Production and Correlative Neurotoxicity

    PubMed Central

    Fitting, Sylvia; Zou, Shiping; El-Hage1, 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. PMID:25760046

  18. Protein–protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein

    PubMed Central

    Betzi, Stéphane; Restouin, Audrey; Opi, Sandrine; Arold, Stefan T.; Parrot, Isabelle; Guerlesquin, Françoise; Morelli, Xavier; Collette, Yves

    2007-01-01

    Protein–protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein–protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of “hot spots” and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef–SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules. PMID:18042718

  19. Evidence for Vpr-dependent HIV-1 Replication in Human CD4+ CEM.NKR T-Cells

    PubMed Central

    2012-01-01

    Background Vpr is exclusively expressed in primate lentiviruses and contributes to viral replication and disease progression in vivo. HIV-1 Vpr has two major activities in vitro: arrest of cell cycle in the G2 phase (G2 arrest), and enhancement of viral replication in macrophages. Previously, we reported a potent HIV-1 restriction in the human CD4+ CEM.NKR (NKR) T cells, where wild-type (WT) HIV-1 replication was inhibited by almost 1,000-fold. From the parental NKR cells, we isolated eight clones by limiting dilution. These clones showed three levels of resistance to the WT HIV-1 infection: non-permissive (NP), semi-permissive (SP), and permissive (P). Here, we compared the replication of WT, Vif-defective, Vpr-defective, and Vpu-defective viruses in these cells. Results Although both WT and Vpu-defective viruses could replicate in the permissive and semi-permissive clones, the replication of Vif-defective and Vpr-defective viruses was completely restricted. The expression of APOBEC3G (A3G) cytidine deaminase in NKR cells explains why Vif, but not Vpr, was required for HIV-1 replication. When the Vpr-defective virus life cycle was compared with the WT virus life cycle in the semi-permissive cells, it was found that the Vpr-defective virus could enter the cell and produce virions containing properly processed Gag and Env proteins, but these virions showed much less efficiency for reverse transcription during the next-round of infection. In addition, although viral replication was restricted in the non-permissive cells, treatment with arsenic trioxide (As2O3) could completely restore WT, but not Vpr-defective virus replication. Moreover, disruption of Vpr binding to its cofactor DCAF1 and/or induction of G2 arrest activity did not disrupt the Vpr activity in enhancing HIV-1 replication in NKR cells. Conclusions These results demonstrate that HIV-1 replication in NKR cells is Vpr-dependent. Vpr promotes HIV-1 replication from the 2nd cycle likely by overcoming a

  20. Polymorphisms of CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals.

    PubMed

    An, Ping; Duggal, Priya; Wang, Li Hua; O'Brien, Stephen J; Donfield, Sharyne; Goedert, James J; Phair, John; Buchbinder, Susan; Kirk, Gregory D; Winkler, Cheryl A

    2007-01-26

    Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) antiretroviral factors cause hypermutation of proviral DNA leading to degradation or replication-incompetent HIV-1. However, HIV-1 viral infectivity factor (Vif) suppresses Apobec3 activity through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase complex. We examined the effect of genetic polymorphisms in the CUL5 gene (encoding Cullin 5 protein) on AIDS disease progression in five HIV-1 longitudinal cohorts. A total of 12 single nucleotide polymorphisms (SNPs) spanning 93 kb in the CUL5 locus were genotyped and their haplotypes inferred. A phylogenetic network analysis revealed that CUL5 haplotypes were grouped into two clusters of evolutionarily related haplotypes. Cox survival analysis and mixed effects models were used to assess time to AIDS outcomes and CD4(+) T cell trajectories, respectively. Relative to cluster I haplotypes, the collective cluster II haplotypes were associated with more rapid CD4(+) T cell loss (relative hazards [RH] = 1.47 and p = 0.009), in a dose-dependent fashion. This effect was mainly attributable to a single cluster II haplotype (Hap10) (RH = 2.49 and p = 0.00001), possibly due to differential nuclear protein-binding efficiencies of a Hap10-specifying SNP as indicated by a gel shift assay. Consistent effects were observed for CD4(+) T cell counts and HIV-1 viral load trajectories over time. The findings of both functional and genetic epidemiologic consequences of CUL5 polymorphism on CD4(+) T cell and HIV-1 levels point to a role for Cullin 5 in HIV-1 pathogenesis and suggest interference with the Vif-Cullin 5 pathway as a possible anti-HIV-1 therapeutic strategy. PMID:17257057

  1. A solution NMR study of the interactions of oligomannosides and the anti-HIV-1 2G12 antibody reveals distinct binding modes for branched ligands.

    PubMed

    Enríquez-Navas, Pedro M; Marradi, Marco; Padro, Daniel; Angulo, Jesús; Penadés, Soledad

    2011-02-01

    The structural and affinity details of the interactions of synthetic oligomannosides, linear (di-, tri-, and tetra-) and branched (penta- and hepta-), with the broadly neutralizing anti-HIV-1 antibody 2G12 (HIV=human immunodeficiency virus) have been investigated in solution by using ligand-based NMR techniques, specifically saturation transfer difference (STD) NMR spectroscopy and transferred NOE experiments. Linear oligomannosides show similar binding modes to the antibody, with the nonreducing terminal disaccharide Manα(1→2)Man (Man=mannose) making the closest protein/ligand contacts in the bound state. In contrast, the branched pentamannoside shows two alternate binding modes, involving both ligand arms (D2- and D3-like), a dual binding description of the molecular recognition of this ligand by 2G12 in solution that differs from the single binding mode deduced from X-ray studies. On the contrary, the antibody shows an unexpected selectivity for one arm (D1-like) of the other branched ligand (heptamannoside). This result explains the previously reported lack of affinity enhancement relative to that of the D1-like tetramannoside. Single-ligand STD NMR titration experiments revealed noticeable differences in binding affinities among the linear and branched ligands in solution, with the latter showing decreased affinity. Among the analyzed series of ligands, the strongest 2G12 binders were the linear tri- and tetramannosides because both show similar affinity for the antibody. These results demonstrate that NMR spectroscopic techniques can deliver abundant structural, dynamics, and affinity information for the characterization of oligomannose-2G12 binding in solution, thus complementing, and, as in the case of the pentamannoside, extending, the structural view from X-ray crystallography. This information is of key importance for the development of multivalent synthetic gp120 high-mannose glycoconjugate mimics in the context of vaccine development. PMID:21268157

  2. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

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

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

    PubMed

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

    2015-05-01

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

  4. Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

    PubMed Central

    Fackler, Oliver T.

    2015-01-01

    The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef. PMID:26703715

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. 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

  8. 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.

  9. 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

  10. Role of individual’s T-cell immunome in controlling HIV-1 progression

    PubMed Central

    Grifoni, Alba; Montesano, Carla; Palma, Paolo; Giovannetti, Marta; Castelli-Gattinara, Guido; Ciccozzi, Massimo; Mattei, Maurizio; Mancino, Giorgio; Salerno, Alfredo; Colizzi, Vittorio; Amicosante, Massimo

    2014-01-01

    Viral and host factors can influence HIV-1 progression, among them human leucocyte antigen (HLA) has shown the strongest effect. However, studies on the functional contribution of HLA in controlling HIV progression toward AIDS are limited by multiple issues, including the viral strain variability within the study subjects. In this study, in a cohort of children infected with a monophyletic strain (CRF02_AG) during an outbreak, we evaluated the HIV-1 Gag, Vif, Vpr, Tat and hepatitis C virus E1/E2 (as control) proteins circulating in a cohort for the capability to be presented by the HLA molecules in the same population. A total of 70 Non-progressors and 37 Progressors to AIDS were evaluated. In the presence of a constant capability of HIV-1 to mutate in the region containing epitopes of Gag protein, the number of epitopes recognized in silico by the combination of the HLA alleles along the Gag consensus sequence is significantly higher in the Non-progressors compared with Progressors (HLA-A: Non-progressors = 1·532 ± 1·211, Progressors = 0·7714± 1·031, P = 0·0016; HLA-B: Non-progressors = 1·556 ± 1·298, Progressors = 1·000 ± 0·817, P = 0·0319; HLA-DR: Non-progressors = 13·30± 9·488, Progressors = 7·294 ± 6·952, P = 0·0006). Similar results were obtained for the other HIV-1 proteins Vif and Vpr, whereas no differences were obtained in the number of epitopes for the hepatitis C virus E1/E2 protein sequence or for the scrambled HIV-1 sequence. Finally, the results were confirmed also in a subgroup of subjects where both HLA typing and Gag sequence were available. In conclusion, in the absence of bias due to viral strain diversity, it is the overall fitting of the HLA molecules that are capable of better binding HIV-1 proteins in determining the major role in the control of HIV-1 replication and progression to AIDS. PMID:24954875

  11. 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

  12. 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.

  13. 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

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

    PubMed Central

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

    2008-01-01

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

  15. Chemically Programmed Antibodies As HIV-1 Attachment Inhibitors

    PubMed Central

    2013-01-01

    Herein, we describe the design and application of two small-molecule anti-HIV compounds for the creation of chemically programmed antibodies. N-Acyl-β-lactam derivatives of two previously described molecules BMS-378806 and BMS-488043 that inhibit the interaction between HIV-1 gp120 and T-cells were synthesized and used to program the binding activity of aldolase antibody 38C2. Discovery of a successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1 gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel bispecific antibodies and topical microbicides. PMID:23750312

  16. 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

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

    PubMed Central

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

    2011-01-01

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

  18. Inhibiting early-stage events in HIV-1 replication by small-molecule targeting of the HIV-1 capsid.

    PubMed

    Kortagere, Sandhya; Madani, Navid; Mankowski, Marie K; Schön, Arne; Zentner, Isaac; Swaminathan, Gokul; Princiotto, Amy; Anthony, Kevin; Oza, Apara; Sierra, Luz-Jeannette; Passic, Shendra R; Wang, Xiaozhao; Jones, David M; Stavale, Eric; Krebs, Fred C; Martín-García, Julio; Freire, Ernesto; Ptak, Roger G; Sodroski, Joseph; Cocklin, Simon; Smith, Amos B

    2012-08-01

    The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4'-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity. PMID:22647699

  19. 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. PMID:24482324

  20. 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. PMID:27067385

  1. 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

  2. A Case of Seronegative HIV-1 Infection

    PubMed Central

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

    2009-01-01

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

  3. HIV-1 assembly in macrophages

    PubMed Central

    2010-01-01

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

  4. 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

  5. HIV-1 Vpu accessory protein induces caspase-mediated cleavage of IRF3 transcription factor.

    PubMed

    Park, Sang Yoon; Waheed, Abdul A; Zhang, Zai-Rong; Freed, Eric O; Bonifacino, Juan S

    2014-12-19

    Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis. PMID:25352594

  6. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

    PubMed Central

    Benko, Zsigmond; Elder, Robert T.; Li, Ge; Liang, Dong; Zhao, Richard Y.

    2016-01-01

    Background HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. Results A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. Conclusions This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings. PMID:26982200

  7. HIV-1 evades innate immune recognition through specific cofactor recruitment.

    PubMed

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J; Price, Amanda J; Blondeau, Caroline; Hilditch, Laura; Jacques, David A; Selwood, David L; James, Leo C; Noursadeghi, Mahdad; Towers, Greg J

    2013-11-21

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages. PMID:24196705

  8. 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.

  9. Interactive HIV-1 Tat and Morphine-Induced Synaptodendritic Injury Is Triggered through Focal Disruptions in Na+ Influx, Mitochondrial Instability, and Ca2+ Overload

    PubMed Central

    Knapp, Pamela E.; Zou, Shiping; Marks, William D.; Bowers, M. Scott; Akbarali, Hamid I.; Hauser, Kurt F.

    2014-01-01

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca2+]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca2+]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na+]i, mitochondrial instability, excessive Ca2+ influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca2+]i and by further disrupting [Ca2+]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS. PMID:25232120

  10. Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant Form Originated from CRF01_AE and CRF08_BC in Dali Prefecture of Yunnan Province, China.

    PubMed

    Li, Jianjian; Li, Lin; Li, Huiqin; Li, Jingyun; Yang, Shaomin; Zhang, Mi; Ouyang, Hongmei

    2016-06-01

    Yunnan seems to be a "hot spot" region of HIV-1 recombination. CRF01_AE and subtype CRF08_BC are two main HIV-1 clades circulating in Yunnan. We report here a novel HIV-1 second-generation recombinant form originated from CRF01_AE and CRF08_BC. The strain (12YN10551) was isolated from a HIV-positive male infected through heterosexual contact in Dali prefecture of Yunnan province, China. This is the first report of HIV-1 near full-length genomic sequence in Dali. Recombinant analysis shows that 12YN10551 was composed of two well-established circulating recombinant forms (CRF01_AE and CRF08_BC). Two CRF01_AE recombinant fragments were inserted into the CRF08_BC backbone genome in the pol/vif/vpr/tat/rev and nef gene regions, respectively. The discovery and characterization of this new recombinant indicate that intersubtype recombination is continuously generating new forms of HIV-1. More work is needed to better monitor the genetic diversity of HIV-1 in this region. PMID:26885777

  11. A new functional role of HIV-1 integrase during uncoating of the viral core.

    PubMed

    Briones, Marisa S; Chow, Samson A

    2010-12-01

    An early and critical event of the human immunodeficiency virus type 1 (HIV-1) life cycle is uncoating of the viral core. Uncoating involves the disassembly of HIV-1 capsid (CA). The underlying mechanisms governing uncoating are poorly defined, and the role of viral and host factors in uncoating is not well understood. Cyclophilin A and TRIM5α are two cellular factors that interact with CA in exerting their effects on HIV-1 replication. Here, we review the current understanding of uncoating and the new functional role of HIV-1 IN during uncoating. PMID:20721640

  12. Novel Approaches to Inhibiting HIV-1 Replication

    PubMed Central

    Adamson, Catherine S.; Freed, Eric O.

    2009-01-01

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

  13. Novel approaches to inhibiting HIV-1 replication.

    PubMed

    Adamson, Catherine S; Freed, Eric O

    2010-01-01

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

  14. 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

  15. 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

  16. In Vivo Functions of CPSF6 for HIV-1 as Revealed by HIV-1 Capsid Evolution in HLA-B27-Positive Subjects

    PubMed Central

    Henning, Matthew S.; Dubose, Brittany N.; Burse, Mallori J.; Aiken, Christopher; Yamashita, Masahiro

    2014-01-01

    The host protein CPSF6 possesses a domain that can interact with the HIV-1 capsid (CA) protein. CPSF6 has been implicated in regulating HIV-1 nuclear entry. However, its functional significance for HIV-1 replication has yet to be firmly established. Here we provide evidence for two divergent functions of CPSF6 for HIV-1 replication in vivo. We demonstrate that endogenous CPSF6 exerts an inhibitory effect on naturally occurring HIV-1 variants in individuals carrying the HLA-B27 allele. Conversely, we find a strong selective pressure in these individuals to preserve CPSF6 binding, while escaping from the restrictive activity by CPSF6. This active maintenance of CPSF6 binding during HIV-1 CA evolution in vivo contrasts with the in vitro viral evolution, which can reduce CPSF6 binding to evade from CPSF6-mediated restriction. Thus, these observations argue for a beneficial role of CPSF6 for HIV-1 in vivo. CPSF6-mediated restriction renders HIV-1 less dependent or independent from TNPO3, RanBP2 and Nup153, host factors implicated in HIV-1 nuclear entry. However, viral evolution that maintains CPSF6 binding in HLA-B27+ subjects invariably restores the ability to utilize these host factors, which may be the major selective pressure for CPSF6 binding in vivo. Our study uncovers two opposing CA-dependent functions of CPSF6 in HIV-1 replication in vivo; however, the benefit for binding CPSF6 appears to outweigh the cost, providing support for a vital function of CPSF6 during HIV-1 replication in vivo. PMID:24415937

  17. 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

  18. Role and modulation of drug transporters in HIV-1 therapy.

    PubMed

    Alam, Camille; Whyte-Allman, Sana-Kay; Omeragic, Amila; Bendayan, Reina

    2016-08-01

    Current treatment of human immunodeficiency virus type-1 (HIV-1) infection involves a combination of antiretroviral drugs (ARVs) that target different stages of the HIV-1 life cycle. This strategy is commonly referred to as highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART). Membrane-associated drug transporters expressed ubiquitously in mammalian systems play a crucial role in modulating ARV disposition during HIV-1 infection. Members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter superfamilies have been shown to interact with ARVs, including those that are used as part of first-line treatment regimens. As a result, the functional expression of drug transporters can influence the distribution of ARVs at specific sites of infection. In addition, pathological factors related to HIV-1 infection and/or ARV therapy itself can alter transporter expression and activity, thus further contributing to changes in ARV disposition and the effectiveness of HAART. This review summarizes current knowledge on the role of drug transporters in regulating ARV transport in the context of HIV-1 infection. PMID:27181050

  19. 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

  20. Slower uncoating is associated with impaired replicative capability of simian-tropic HIV-1.

    PubMed

    Kono, Ken; Takeda, Eri; Tsutsui, Hiromi; Kuroishi, Ayumu; Hulme, Amy E; Hope, Thomas J; Nakayama, Emi E; Shioda, Tatsuo

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116(th) position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus. PMID:23967315

  1. Strategies for Eliciting HIV-1 Inhibitory Antibodies

    PubMed Central

    Tomaras, Georgia D.; Haynes, Barton F.

    2012-01-01

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

  2. 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. PMID:24888904

  3. Differentially-Expressed Pseudogenes in HIV-1 Infection

    PubMed Central

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

    2015-01-01

    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. PMID:26426037

  4. HIV-1 proteins accelerate HPA axis habituation in female rats.

    PubMed

    Panagiotakopoulos, Leonidas; Kelly, Sean; Neigh, Gretchen N

    2015-10-15

    Congenital infection by the Human Immunodeficiency Virus (HIV) has been shown to lead to multiple co-morbidities, and people living with HIV have a higher incidence of affective and anxiety disorders. A marked increase in mood disorders is evident during the sensitive phase of adolescence and this is further pronounced in females. Depression has been linked to dysfunction of the intracellular response system to corticosteroids at the level of the hippocampus (HC) and prefrontal cortex (PFC) with a notable role of the glucocorticoid receptor (GR) and its co-chaperones (FKBP5 and FKBP4). The current study examined the extent to which HIV protein expression in adolescent female rats altered the stress response at both the level of corticosterone output and molecular regulation of the glucocorticoid receptor in the brain. WT and HIV-1 genotype female rats were randomly allocated in control, acute stress and repeat stress groups. Corticosterone plasma levels and expression of GR, FKBP4, and FKBP5 in the HC and PFC were measured. The presence of HIV-1 proteins facilitates habituation of the corticosterone response to repeated stressors, such that HIV-1 TG rats habituated to repeated restraint and WT rats did not. This was reflected by interactions between stress exposure and HIV-1 protein expression at the level of GR co-chaperones. Although expression of the GR was similarly reduced after acute and repeat stress in both genotypes, expression of FKBP5 and FKBP4 was altered in a brain-region specific manner depending on the duration of the stress exposure and the presence or absence of HIV-1 proteins. Collectively, the data presented demonstrate that HIV-1 proteins accelerate habituation to repeated stressors and modify the influence of acute and repeat stressors on GR co-chaperones in a brain region-specific manner. PMID:25666308

  5. 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.

  6. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure

    PubMed Central

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG. PMID:26779399

  7. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure.

    PubMed

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG. PMID:26779399

  8. 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

  9. 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. PMID:24521207

  10. Interplay between HIV-1 and Toll-like receptors in human myeloid cells: friend or foe in HIV-1 pathogenesis?

    PubMed

    Donninelli, Gloria; Gessani, Sandra; Del Cornò, Manuela

    2016-01-01

    The Toll-like receptors are the first line of the host response to pathogens, representing an essential component of the innate and adaptive immune response. They recognize different pathogens and trigger responses directed at eliminating the invader and at developing immunologic long-term memory, ultimately affecting viral pathogenesis. In viral infections, sensing of nucleic acids and/or viral structural proteins generally induces a protective immune response. Thus, it is not surprising that many viruses have developed strategies to evade or counteract signaling through the Toll-like receptor pathways, to survive the host defense machinery and ensure propagation. Thus, Toll-like receptor engagement can also be part of viral pathogenic mechanisms. Evidence for a direct interaction of Toll-like receptors with human immunodeficiency virus type 1 (HIV-1) structures has started to be achieved, and alterations of their expression and function have been described in HIV-1-positive subjects. Furthermore, Toll-like receptor triggering by bacterial and viral ligands have been described to modulate HIV-1 replication and host response, leading to protective or detrimental effects. This review covers major advances in the field of HIV-1 interplay with Toll-like receptors, focusing on human myeloid cells (e.g., monocytes/macrophages and dendritic cells). The role of this interaction in the dysregulation of myeloid cell function and in dictating aspects of the multifaceted pathogenesis of acquired immunodeficiency syndrome will be discussed. PMID:26307548

  11. 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

  12. The HIV-1 associated protein, Tat1–86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: a no-net-flux microdialysis study

    PubMed Central

    Ferris, Mark J.; Frederick-Duus, Danielle; Fadel, Jim; Mactutus, Charles F.; Booze, Rosemarie M.

    2009-01-01

    Injection drug use accounts for approximately one-third of HIV-infections in the United States. HIV associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 hours of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 hrs post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharamacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to DA systems occurring in NeuroAIDS. PMID:19344635

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

    PubMed

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao; He, Yuxian; Liu, Keliang

    2015-08-01

    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

  14. Buried surface analysis of HIV-1 reverse transcriptase p66/p51 heterodimer and its interaction with dsDNA template/primer.

    PubMed

    Ding, J; Jacobo-Molina, A; Tantillo, C; Lu, X; Nanni, R G; Arnold, E

    1994-06-01

    The p66/p51 human immunodeficiency virus type 1 reverse transcriptase is a heterodimer with identical N-terminal amino acid sequences. The enzyme contains two polymerization domains and one RNase H domain, which is located at the C-terminus of the p66 subunit. Both polymerization domains fold into four individual subdomains that are not arranged in a similar fashion, forming an unusually asymmetric dimer. The complexity of the RT p66/p51 heterodimer structure is simplified using solvent-accessibility surface areas to describe the buried surface area of contact among the different subdomains. In addition, the RT/DNA contacts in the recently published RT/DNA/Fab structure [Jacobo-Molina et al., Proc. Natl Acad. Sci. USA, 90, 6320-6324 (1993)] are described using the same approach. Finally, the RT/DNA complex is compared with other dimeric DNA-binding proteins. It was found that the size of the protein and the extent of the dimer interface were not directly related to the extent of contact between the protein and the DNA. Furthermore, RT, the only protein that is not a sequence-specific DNA binding protein in this analysis, had the largest surface of interaction with the nucleic acid. PMID:7530020

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

    PubMed

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

    2016-06-01

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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization

    PubMed Central

    McCoy, Laura E.; Quigley, Anna Forsman; Strokappe, Nika M.; Bulmer-Thomas, Bianca; Seaman, Michael S.; Mortier, Daniella; Rutten, Lucy; Chander, Nikita; Edwards, Carolyn J.; Ketteler, Robin; Davis, David; Verrips, Theo

    2012-01-01

    Llamas (Lama glama) naturally produce heavy chain–only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain–only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti–HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell–based vaccines and supports the development of VHH as anti–HIV-1 microbicides. PMID:22641382

  1. Cytoplasmic dynein promotes HIV-1 uncoating.

    PubMed

    Pawlica, Paulina; Berthoux, Lionel

    2014-11-01

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

  2. In vitro Uncoating of HIV-1 Cores

    PubMed Central

    Shah, Vaibhav B.; Aiken, Christopher

    2011-01-01

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

  3. In vitro uncoating of HIV-1 cores.

    PubMed

    Shah, Vaibhav B; Aiken, Christopher

    2011-01-01

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

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

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-01

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

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

    PubMed Central

    Kline, Erik R.; Sutliff, Roy L.

    2008-01-01

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

  6. 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

  7. Structure based activity prediction of HIV-1 reverse transcriptase inhibitors.

    PubMed

    de Jonge, Marc R; Koymans, Lucien M H; Vinkers, H Maarten; Daeyaert, Frits F D; Heeres, Jan; Lewi, Paul J; Janssen, Paul A J

    2005-03-24

    We have developed a fast and robust computational method for prediction of antiviral activity in automated de novo design of HIV-1 reverse transcriptase inhibitors. This is a structure-based approach that uses a linear relation between activity and interaction energy with discrete orientation sampling and with localized interaction energy terms. The localization allows for the analysis of mutations of the protein target and for the separation of inhibition and a specific binding to the enzyme. We apply the method to the prediction of pIC(50) of HIV-1 reverse transcriptase inhibitors. The model predicts the activity of an arbitrary compound with a q(2) of 0.681 and an average absolute error of 0.66 log value, and it is fast enough to be used in high-throughput computational applications. PMID:15771460

  8. ZASC1 Stimulates HIV-1 Transcription Elongation by Recruiting P-TEFb and TAT to the LTR Promoter

    PubMed Central

    Bruce, James W.; Reddington, Rachel; Mathieu, Elizabeth; Bracken, Megan; Young, John A. T.; Ahlquist, Paul

    2013-01-01

    Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter. PMID:24204263

  9. Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus

    PubMed Central

    Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.

    2012-01-01

    The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

  10. 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