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Sample records for hiv-1 vif interacts

  1. Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBFβ that is critical for Vif function.

    PubMed

    Desimmie, Belete A; Smith, Jessica L; Matsuo, Hiroshi; Hu, Wei-Shau; Pathak, Vinay K

    2017-03-17

    HIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif-CBFβ interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFβ, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif-CBFβ interactions that are important for Vif function has not been performed. Here, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFβ and their ability to induce A3G degradation as well as rescue HIV-1 replication in the presence of A3G. We found that multiple Vif residues are involved in the extensive N-terminal Vif-CBFβ interaction and that the (5)WQVMIVW(11) region of Vif is the major determinant. A minimum of three alanine substitutions are required to completely abrogate the Vif-CBFβ interaction and Vif's ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFβ revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction with W5 of Vif to maintain a stable and functional Vif-CBFβ complex. We also determined that CBFβ amino acids (73)WQGEQR(78), which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif. Our results provide detailed insight into the Vif-CBFβ interactions that are critical for Vif function and may contribute to the rational design of HIV-1 inhibitors that block Vif-mediated degradation of A3 proteins.

  2. Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production.

    PubMed

    Zheng, Wenwen; Ling, Limian; Li, Zhaolong; Wang, Hong; Rui, Yajuan; Gao, Wenying; Wang, Shaohua; Su, Xing; Wei, Wei; Yu, Xiao-Fang

    2017-04-01

    The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55(Gag)) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55(Gag) compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55(Gag) interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55(Gag), which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4(+) T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55(Gag) interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55(Gag)) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55(Gag) although HIV-1 Vif proteins

  3. Mapping Region of Human Restriction Factor APOBEC3H Critical for Interaction with HIV-1 Vif.

    PubMed

    Nakashima, Masaaki; Tsuzuki, Shinya; Awazu, Hiroaki; Hamano, Akiko; Okada, Ayaka; Ode, Hirotaka; Maejima, Masami; Hachiya, Atsuko; Yokomaku, Yoshiyuki; Watanabe, Nobuhisa; Akari, Hirofumi; Iwatani, Yasumasa

    2017-04-21

    The APOBEC3 (A3) family of cellular cytidine deaminases comprises seven members (A, B, C, D, F, G, and H) that potently inhibit retroviral replication. Human immunodeficiency virus type 1 (HIV-1) Vif is a small pleiotropic protein that specifically inactivates these enzymes, targeting them for ubiquitin-mediated proteasomal degradation. A3 Vif-interaction sites are presumed to fall into three distinct types: A3C/D/F, A3G, and A3H. To date, two types of A3G and A3C/D/F sites have been well characterized, whereas the A3H Vif-binding site remains poorly defined. Here, we explore the residues critical for the A3H-type Vif interaction. To avoid technical difficulties in performing experiments with human A3H haplotype II (hapII), which is relatively resistant to HIV-1 Vif, we employed its ortholog chimpanzee A3H (cA3H), which displays high Vif sensitivity, for a comparison of sensitivity with that of A3H hapII. The Vif susceptibility of A3H hapII-cA3H chimeras and their substitution mutants revealed a single residue at position 97 as a major determinant for the difference in their Vif sensitivities. We further surveyed critical residues by structure-guided mutagenesis using an A3H structural model and thus identified eight additional residues important for Vif sensitivity, which mapped to the α3 and α4 helices of A3H. Interestingly, this area is located on a surface adjacent to the A3G and A3C/D/F interfaces and is composed of negatively charged and hydrophobic patches. These findings suggest that HIV-1 Vif has evolved to utilize three dispersed surfaces for recognizing three types of interfaces on A3 proteins under certain structural constraints.

  4. Defining HIV-1 Vif residues that interact with CBFβ by site-directed mutagenesis

    PubMed Central

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Io, Katsuhiro; Tada, Kohei; Iwai, Fumie; Kobayashi, Masayuki; Kadowaki, Norimitsu; Harris, Reuben S; Takaori-Kondo, Akifumi

    2014-01-01

    Vif is essential for HIV-1 replication in T cells and macrophages. Vif recruits a host ubiquitin ligase complex to promote proteasomal degradation of the APOBEC3 restriction factors by poly-ubiquitination. The cellular transcription cofactor CBFβ is required for Vif function by stabilizing the Vif protein and promoting recruitment of a cellular Cullin5-RING ubiquitin ligase complex. Interaction between Vif and CBFβ is a promising therapeutic target, but little is known about the interfacial residues. We now demonstrate that Vif conserved residues E88/W89 are crucial for CBFβ binding. Substitution of E88/W89 to alanines impaired binding to CBFβ, degradation of APOBEC3, and virus infectivity in the presence of APOBEC3 in single-cycle infection. In spreading infection, NL4-3 with Vif E88A/W89A mutation replicated comparably to wild-type virus in permissive CEM-SS cells, but not in multiple APOBEC3 expressing non-permissive CEM cells. These results support a model in which HIV-1 Vif residues E88/W89 may participate in binding CBFβ. PMID:24418540

  5. HIV-1 Accessory Proteins: Vpu and Vif

    PubMed Central

    Andrew, Amy; Strebel, Klaus

    2014-01-01

    HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins. PMID:24158820

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

    PubMed

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

    2010-04-01

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

  7. Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCF(cyclin F) E3 Ligase Machinery.

    PubMed

    Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis

    2017-03-31

    Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCF(cyclin F) E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4(+) T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCF(cyclin F) E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.

  8. 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.

  9. 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

  10. 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.

  11. The Structural Interface between HIV-1 Vif and Human APOBEC3H.

    PubMed

    Ooms, Marcel; Letko, Michael; Simon, Viviana

    2017-03-01

    Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif β-sheet (β2-β5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif β-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV

  12. 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

    2007-09-07

    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.

  13. The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein.

    PubMed

    Hassaïne, G; Courcoul, M; Bessou, G; Barthalay, Y; Picard, C; Olive, D; Collette, Y; Vigne, R; Decroly, E

    2001-05-18

    The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.

  14. 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.

  15. Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

    PubMed Central

    Feng, Yuqing; Baig, Tayyba T.; Love, Robin P.; Chelico, Linda

    2014-01-01

    The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective. PMID:25206352

  16. 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.

  17. Multiple APOBEC3 Restriction Factors for HIV-1 and One Vif to Rule Them All

    PubMed Central

    Desimmie, Belete A.; Delviks-Frankenberry, Krista A.; Burdick, Ryan; Qi, Dongfei; Izumi, Taisuke; Pathak, Vinay K.

    2013-01-01

    Several members of the APOBEC3 family of cellular restriction factors provide intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection against HIV-1Δvif through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif, which contains distinct domains that specifically interact with these APOBEC3 proteins to ensure their proteasomal degradation, allowing virus replication to proceed. Here, we review our current understanding of APOBEC3 structure, editing and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3 interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3 proteins to restriction and control of HIV-1 replication in infected patients. PMID:24189052

  18. Crystal structure of the DNA cytosine deaminase APOBEC3F: the catalytically active and HIV-1 Vif-binding domain.

    PubMed

    Bohn, Markus-Frederik; Shandilya, Shivender M D; Albin, John S; Kouno, Takahide; Anderson, Brett D; McDougle, Rebecca M; Carpenter, Michael A; Rathore, Anurag; Evans, Leah; Davis, Ahkillah N; Zhang, Jingying; Lu, Yongjian; Somasundaran, Mohan; Matsuo, Hiroshi; Harris, Reuben S; Schiffer, Celia A

    2013-06-04

    Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.

  19. Multiple lysines combined in HIV-1 Vif determines the responsiveness to CBF-β.

    PubMed

    Ai, Youwei; Ma, Jianzhang

    2015-02-13

    The Vif (viral infectivity factor) protein of human immunodeficiency virus type-1 (HIV-1) is critical for HIV-1 infectivity. CBF-β is required for HIV-1 Vif function, as it increases the steady-state level of the HIV-1 Vif protein to promote host restriction factor APOBEC3 degradation. However, the precise mechanism by which CBF-β promotes HIV-1 Vif levels remains unclear. In the present study, we provided evidences that CBF-β promoted steady-state levels of HIV-1 Vif by inhibiting the degradation of HIV-1 Vif through the proteasome pathway. Our results reveal a new mechanism by which a cellular protein supports viral infectivity by inhibiting viral protein degradation.

  20. CBFβ enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G.

    PubMed

    Miyagi, Eri; Kao, Sandra; Yedavalli, Venkat; Strebel, Klaus

    2014-05-01

    Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. In this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature

  1. Running Loose or Getting Lost: How HIV-1 Counters and Capitalizes on APOBEC3-Induced Mutagenesis through Its Vif Protein

    PubMed Central

    Münk, Carsten; Jensen, Björn-Erik O.; Zielonka, Jörg; Häussinger, Dieter; Kamp, Christel

    2012-01-01

    Human immunodeficiency virus-1 (HIV-1) dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif) protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race. PMID:23202519

  2. Evolution of HIV-1 isolates that use a novel Vif-independent mechanism to resist restriction by human APOBEC3G.

    PubMed

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

    2008-06-03

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

  3. Productive replication of Vif-chimeric HIV-1 in feline cells.

    PubMed

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

    2010-07-01

    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-1(VF) and HIV-1(VS)) 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.

  4. 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.

  5. 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

  6. Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    PubMed Central

    Zhang, Wenyan; Chen, Gongying; Niewiadomska, Anna Maria; Xu, Rongzhen; Yu, Xiao-Fang

    2008-01-01

    Background APOBEC3G (A3G) and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized. Methods and Findings In the present study, we have demonstrated that the regions of APOBEC3F (A3F) that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD) of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE. Conclusions Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors. PMID:19088851

  7. HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

    PubMed Central

    Goila-Gaur, Ritu; Khan, Mohammad A.; Miyagi, Eri; Kao, Sandra; Opi, Sandrine; Takeuchi, Hiroaki; Strebel, Klaus

    2008-01-01

    HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat-denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expressed in HeLa cells from a low molecular mass conformation to puromycin-sensitive high molecular mass (HMM) complexes. In the absence of Vif or the presence of biologically inactive Vif APO3G failed to form HMM complexes. Our results expose a novel function of Vif that promotes the assembly of APO3G into presumably packaging-incompetent HMM complexes and may explain how Vif can overcome the APO3G-imposed block to HIV replication under conditions of no or inefficient APO3G degradation. PMID:18023836

  8. 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.

  9. Anti-APOBEC3G Activity of HIV-1 Vif Protein Is Attenuated in Elite Controllers

    PubMed Central

    Kikuchi, Tadashi; Iwabu, Yukie; Tada, Takuya; Kawana-Tachikawa, Ai; Koga, Michiko; Hosoya, Noriaki; Nomura, Shigeru; Brumme, Zabrina L.; Jessen, Heiko; Pereyra, Florencia; Trocha, Alicja; Walker, Bruce D.; Iwamoto, Aikichi

    2015-01-01

    ABSTRACT HIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. The HIV-1 accessory protein Vif (virion infectivity factor) enhances viral infectivity through anti-retroviral factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) degradation; however, little is known regarding Vif function in EC. Here, the anti-APOBEC3G activities of clonal, plasma HIV RNA-derived Vif sequences from 46 EC, 46 noncontrollers (NC), and 44 individuals with acute infection (AI) were compared. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viruses were generated by cotransfecting 293T cells with expression plasmids encoding patient-derived Vif, human APOBEC3G, VSV-G, and a vif/env-deficient luciferase-reporter HIV-1 proviral DNA clone. Viral stocks were used to infect 293T cells, and Vif anti-APOBEC3G activity was quantified in terms of luciferase signal. On average, the anti-APOBEC3G activities of EC-derived Vif sequences (median log10 relative light units [RLU], 4.54 [interquartile range {IQR}, 4.30 to 4.66]) were significantly lower than those of sequences derived from NC (4.75 [4.60 to 4.92], P < 0.0001) and AI (4.74 [4.62 to 4.94], P < 0.0001). Reduced Vif activities were not associated with particular HLA class I alleles expressed by the host. Vif functional motifs were highly conserved in all patient groups. No single viral polymorphism could explain the reduced anti-APOBEC3G activity of EC-derived Vif, suggesting that various combinations of minor polymorphisms may underlie these effects. These results further support the idea of relative attenuation of viral protein function in EC-derived HIV sequences. IMPORTANCE HIV-1 elite controllers (EC) are rare individuals who are able to control plasma viremia to undetectable levels without antiretroviral therapy. Understanding the

  10. Core-binding factor β increases the affinity between human Cullin 5 and HIV-1 Vif within an E3 ligase complex.

    PubMed

    Salter, Jason D; Lippa, Geoffrey M; Belashov, Ivan A; Wedekind, Joseph E

    2012-11-06

    HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.

  11. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Deletion, insertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patients.

    PubMed

    Rangel, Héctor Rafael; Garzaro, Domingo; Rodríguez, Anny Karely; Ramírez, Alvaro Hernán; Ameli, Gladys; Del Rosario Gutiérrez, Cristina; Pujol, Flor Helene

    2009-08-30

    Variable progression towards AIDS has been described and has been related to viral and host factors. Around 10% of the HIV-1 infected patients are slow progressors (SP), not presenting with AIDS disease signs even after more than 10 years of infection. Viral gene defects have been associated with the disease progression but more studies are still needed. The sequence of vif and nef were analyzed for HIV-1 infecting 14 SP and 46 normal progressors (NP) patients. Co-circulation of a strain carrying vif deleted gene with the wild type strain was detected in an SP patient with more than 10 years of infection. Other mutations (insertion in aa 63 in one strain, two premature stop codons in another one) were found in viruses infecting two other patients. Except for the SP8 strain, which exhibited a premature stop codon in nef, no gross deletions or insertions were observed in nef genes of both NPs and SPs strains analyzed. Different kind of mutation: deletion, insertion and stop codon, were detected in 3/14 samples from SP, with co-circulation of a 195 bp vif deletion virus with a wild type in one of these patients. Although vif defects do not seem to be a frequent feature in SPs, this study illustrates the importance of analysing this gene, in addition to the multiple factors associated with the long-term non progression to AIDS.

  13. The tyrosine kinases Fyn and Hck favor the recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles.

    PubMed

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

    2005-04-15

    The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.

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

    PubMed

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

    1999-06-11

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

  15. 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

  16. Unusual substitutions in HIV-1 vif from children infected perinatally without progression to AIDS for more than 8 years without therapy.

    PubMed

    De Maio, Federico A; Rocco, Carlos A; Aulicino, Paula C; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

    2012-12-01

    The HIV-1 vif gene encodes for an accessory protein that is central for virus replication due mainly to its capacity to counteract the antiviral action of host APOBEC3 restriction factors. In order to evaluate whether HIV-1 vif alterations account for a delayed progression to AIDS in children infected perinatally, the vif genes from a group of 11 patients who exhibited an extremely slow disease progression (slow progressors) were studied by direct sequencing. In addition, the vif genes from a group of 93 children with typical disease progression (typical progressors) were analyzed for comparison. Phylogenetic analysis indicated that sequences from slow progressors did not have a common origin, discarding a shared ancestor of reduced virulence. There were no differences in the diversity between the vif genes from slow and typical progressors. No gross defects showing a clear distinction among sequences from both groups of children were found. However, in the deduced Vif proteins, changes V13I, V55T, and L81M were observed only in sequences from slow progressors. By analyzing sequences stored in databases, these mutations were determined as unusual substitutions occurring at highly conserved Vif sites across different HIV-1 clades, but were observed with an increased frequency in sequences from elite controllers. These mutations were in the Vif regions reported as relevant for protein activity. These findings suggest that the Vif sequences from slow progressors carry unusual substitutions, which may alter the protein function and may contribute to viral attenuation.

  17. Interactions of host APOBEC3 restriction factors with HIV-1 in vivo: implications for therapeutics.

    PubMed

    Albin, John S; Harris, Reuben S

    2010-01-22

    Restriction factors are natural cellular proteins that defend individual cells from viral infection. These factors include the APOBEC3 family of DNA cytidine deaminases, which restrict the infectivity of HIV-1 by hypermutating viral cDNA and inhibiting reverse transcription and integration. HIV-1 thwarts this restriction activity through its accessory protein virion infectivity factor (Vif), which uses multiple mechanisms to prevent APOBEC3 proteins such as APOBEC3G and APOBEC3F from entering viral particles. Here, we review the basic biology of the interactions between human APOBEC3 proteins and HIV-1 Vif. We also summarise, for the first time, current clinical data on the in vivo effects of APOBEC3 proteins, and survey strategies and progress towards developing therapeutics aimed at the APOBEC3-Vif axis.

  18. Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag-Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing.

    PubMed

    Bardy, M; Gay, B; Pébernard, S; Chazal, N; Courcoul, M; Vigne, R; Decroly, E; Boulanger, P

    2001-11-01

    Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag-Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Deltap6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag-Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag-Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA-CA and CA-NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag-Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag-Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag-Pol precursor.

  19. The resistance of human APOBEC3H to HIV-1 NL4-3 molecular clone is determined by a single amino acid in Vif.

    PubMed

    Ooms, Marcel; Letko, Michael; Binka, Mawuena; Simon, Viviana

    2013-01-01

    Some human APOBEC3 cytidine deaminases have antiviral activity against HIV-1 and other retroviruses. The single deaminase domain APOBEC3H (A3H) enzyme is highly polymorphic and multiple A3H haplotypes have been identified. A3H haplotype II (A3H-hapII) possesses the strongest activity against HIV-1. There remains, however, uncertainty regarding the extent to which A3H-hapII is sensitive to HIV-1 Vif mediated degradation. We tested, therefore, the two different reference Vif proteins widely used in previous studies. We show that A3H-hapII is resistant to NL4-3 Vif while it is efficiently degraded by LAI Vif. Co-immunoprecipitation assays demonstrate that LAI Vif, but not NL4-3 Vif associates with A3H-hapII. Chimeras between NL4-3 and LAI Vif identify the amino acid responsible for the differential degradation activity: A histidine at position 48 in Vif confers activity against A3H-hapII, while an asparagine abolishes its anti-A3H activity. Furthermore, the amino acid identity at position 48 only affects the degradation of A3H-hapII, whereas recognition of and activity against human A3D, A3F and A3G are only minimally affected. NL4-3 encoding 48H replicates better than NL4-3 WT (48N) in T cell-lines stably expressing A3H hapII, whereas there is no fitness difference in the absence of APOBEC3. These studies provide an explanation for the conflicting reports regarding A3H resistance to Vif mediated degradation.

  20. Translational regulation of APOBEC3G mRNA by Vif requires its 5′UTR and contributes to restoring HIV-1 infectivity

    PubMed Central

    Guerrero, Santiago; Libre, Camille; Batisse, Julien; Mercenne, Gaëlle; Richer, Delphine; Laumond, Géraldine; Decoville, Thomas; Moog, Christiane; Marquet, Roland; Paillart, Jean-Christophe

    2016-01-01

    The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5′-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G. PMID:27996044

  1. Translational regulation of APOBEC3G mRNA by Vif requires its 5'UTR and contributes to restoring HIV-1 infectivity.

    PubMed

    Guerrero, Santiago; Libre, Camille; Batisse, Julien; Mercenne, Gaëlle; Richer, Delphine; Laumond, Géraldine; Decoville, Thomas; Moog, Christiane; Marquet, Roland; Paillart, Jean-Christophe

    2016-12-20

    The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5'-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.

  2. Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

    PubMed

    Refsland, Eric W; Hultquist, Judd F; Luengas, Elizabeth M; Ikeda, Terumasa; Shaban, Nadine M; Law, Emily K; Brown, William L; Reilly, Cavan; Emerman, Michael; Harris, Reuben S

    2014-11-01

    The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

  3. gag, vif, and nef Genes Contribute to the Homologous Viral Interference Induced by a Nonproducer Human Immunodeficiency Virus Type 1 (HIV-1) Variant: Identification of Novel HIV-1-Inhibiting Viral Protein Mutants

    PubMed Central

    D’Aloja, Paola; Olivetta, Eleonora; Bona, Roberta; Nappi, Filomena; Pedacchia, Daniela; Pugliese, Katherina; Ferrari, Giuliana; Verani, Paola; Federico, Maurizio

    1998-01-01

    We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed

  4. An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

    PubMed

    Bennett, Ryan P; Stewart, Ryan A; Hogan, Priscilla A; Ptak, Roger G; Mankowski, Marie K; Hartman, Tracy L; Buckheit, Robert W; Snyder, Beth A; Salter, Jason D; Morales, Guillermo A; Smith, Harold C

    2016-12-01

    Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Molecular docking analysis of RN18 and VEC5 in A3G-Vif inhibition

    PubMed Central

    Sinha, Chanda; Nischal, Anuradha; Pant, Kamlesh K; Bandaru, Srinivas; Nayarisseri, Anuraj; Khattri, Sanjay

    2014-01-01

    The HIV-1 protein Vif is essential for in vivo viral replication that targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses. The Vif-A3G interactions are believed to be important targets for antiviral drug development. Since the interactions of A3G and Vif evade the ubiquitination pathways in human host, the viral replication precedes which otherwise spreads infection. In this study, two potent Vif inhibitors RN 18 and VEC5 have been evaluated for their inhibitory potential employing ligand receptor and protein-protein interactions studies. VEC 5 showed better interaction with Vif than RN18. Predicted data show that VEC5 bound Vif and RN18 bound Vif showed diminished interaction to A3G compared to inhibitor unbound Vif. However, this should be further validated using in vitro studies. PMID:25489169

  6. Molecular docking analysis of RN18 and VEC5 in A3G-Vif inhibition.

    PubMed

    Sinha, Chanda; Nischal, Anuradha; Pant, Kamlesh K; Bandaru, Srinivas; Nayarisseri, Anuraj; Khattri, Sanjay

    2014-01-01

    The HIV-1 protein Vif is essential for in vivo viral replication that targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses. The Vif-A3G interactions are believed to be important targets for antiviral drug development. Since the interactions of A3G and Vif evade the ubiquitination pathways in human host, the viral replication precedes which otherwise spreads infection. In this study, two potent Vif inhibitors RN 18 and VEC5 have been evaluated for their inhibitory potential employing ligand receptor and protein-protein interactions studies. VEC 5 showed better interaction with Vif than RN18. Predicted data show that VEC5 bound Vif and RN18 bound Vif showed diminished interaction to A3G compared to inhibitor unbound Vif. However, this should be further validated using in vitro studies.

  7. GANP interacts with APOBEC3G and facilitates its encapsidation into the virions to reduce HIV-1 infectivity

    PubMed Central

    Singh, Shailendra Kumar; Eid, Mohammed Mansour Abbas; Shimoda, Mayuko; Ikeda, Terumasa; Koito, Atsushi; Pham, Phuong; Goodman, Myron F.; Sakaguchi, Nobuo

    2013-01-01

    The single-stranded DNA-dependent deoxycytidine deaminase APOBEC3G (A3G) is a potent restrictive factor against HIV-1 virus lacking viral-encoded infectivity factor (Vif) in CD4+ T cells. A3G antiretroviral activity requires its encapsulation into HIV-1 virions. Here we show that germinal center-associated nuclear protein (GANP) is induced in activated CD4+ T cells and physically interacts with A3G. Overexpression of GANP augments the A3G encapsidation into the virion-like particles and ΔVif HIV-1 virions. GANP is encapsidated in HIV-1 virion and modulates A3G packaging into the cores together with cellular RNAs including 7SL RNA, and with unspliced HIV-1 genomic RNA. GANP upregulation leads to a significant increase in A3G-catalyzed G→A hypermutation in the viral genome and suppression of HIV-1 infectivity in a single-round viral infection assay. Conversely, GANP knockdown caused a marked increase in HIV-1 infectivity in a multiple rounds infection assay. The data suggest that GANP is a cellular factor that facilitates A3G encapsidation into HIV-1 virions to inhibit the viral infectivity. PMID:24198285

  8. 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

  9. Viral diversity and diversification of major non-structural genes vif, vpr, vpu, tat exon 1 and rev exon 1 during primary HIV-1 subtype C infection.

    PubMed

    Rossenkhan, Raabya; Novitsky, Vladimir; Sebunya, Theresa K; Musonda, Rosemary; Gashe, Berhanu A; Essex, M

    2012-01-01

    To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; -0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

  10. Human and Rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H Demonstrate a Conserved Capacity To Restrict Vif-Deficient HIV-1 ▿ †

    PubMed Central

    Hultquist, Judd F.; Lengyel, Joy A.; Refsland, Eric W.; LaRue, Rebecca S.; Lackey, Lela; Brown, William L.; Harris, Reuben S.

    2011-01-01

    Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4+ T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4+ T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins—APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H—in HIV-1 restriction. PMID:21835787

  11. 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

  12. Small molecules that inhibit Vif-induced degradation of APOBEC3G

    PubMed Central

    2014-01-01

    Background HIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the Vif-APOBEC3G interaction has emerged as a promising HIV-1 therapeutic target. Methods 20,000 small molecules were used in live-cell screens for those that preserve EGFP-APOBEC3G fluorescence and luciferase-APOBEC3G luminescence in the presence of HIV-1 Vif. Results 2 compounds with similar core structures preserved APOBEC3G levels in the presence of Vif. 10 μM of compound restored APOBEC3G to levels sufficient for incorporation into vif-proficient virus particles and restriction of virus infectivity. Vif-dependent APOBEC3G polyubiquitination and general proteasomal activity were unaffected at the same concentration. Conclusions The small molecules described here preserve APOBEC3G levels and activity in the presence of Vif. These molecules are starting points for further development as antiretrovirals. PMID:24986077

  13. The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin.

    PubMed

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

    2004-02-27

    The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.

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

    PubMed Central

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

    2006-01-01

    Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4+ T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)5-C-(Xaa)17–18-C-(Xaa)3–5-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif–Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif, as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein–protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation. PMID:17132731

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Core Binding Factor Beta Plays a Critical Role by Facilitating the Assembly of the Vif-Cullin 5 E3 Ubiquitin Ligase

    PubMed Central

    Fribourgh, Jennifer L.; Nguyen, Henry C.; Wolfe, Leslie S.; DeWitt, David C.; Zhang, Wenyan; Yu, Xiao-Fang; Rhoades, Elizabeth

    2014-01-01

    ABSTRACT The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) for degradation via the host ubiquitin-proteasome pathway. Vif recruits a cellular E3 ubiquitin ligase to polyubiquitinate A3G/F. The activity of Vif critically depends on the cellular core binding factor beta (CBFβ). In this study, we investigated the Vif-CBFβ interaction and the role of CBFβ in the E3 ligase assembly. Vif-CBFβ interaction requires an extensive region of Vif spanning most of its amino terminus and zinc finger region, and cullin 5 (Cul5) binding enhances the stability of the Vif-CBFβ interaction. Our results further demonstrate that CBFβ plays a critical role in facilitating Cul5 binding to the Vif/elongin B/elongin C complex. Vif, with or without bound substrate, is unable to bind Cul5 in the absence of CBFβ. These studies support the notion that CBFβ serves as a molecular chaperone to facilitate Vif-E3 ligase assembly. IMPORTANCE The host antiviral restriction factors A3G/F inhibit viral replication. The HIV-1 protein Vif targets A3G/F for degradation. This immune evasion activity of Vif is dependent on the cellular factor CBFβ. Multiple regions of Vif are known to be important for Vif function, but the mechanisms are unclear. The studies described here provide important information about the Vif-CBFβ interaction interface and the function of CBFβ in E3 ligase assembly. In particular, our comprehensive Vif-CBFβ interface mapping results help to delineate the role of various Vif regions, determining if they are important for binding CBFβ or A3G/F. Furthermore, our studies reveal an important potential mechanism of CBFβ that has not been shown before. Our results suggest that CBFβ may serve as a molecular chaperone to enable Vif to adopt an appropriate conformation for interaction with the Cul5-based E3 ligase. This study advances our understanding of how CBFβ facilitates the Vif-mediated degradation of

  18. The role of Siglec-1 in HIV-1/macrophage interaction

    PubMed Central

    Jobe, Ousman; Kim, Jiae; Rao, Mangala

    2016-01-01

    Although CD4 T-cells are a major target for HIV, recent work has demonstrated the ability of macrophages despite expressing relatively low levels of CD4, to be a target of the virus. Our recent study has found that the presence of growth factors not only play a role in the phenotype of these monocyte-derived-macrophages, but also are an important aspect of the permissiveness of these cells to infection. The work utilized cellular and biophysical methods to examine Siglec-1 on macrophages as a primary receptor in HIV-1 infection. These findings support the notion that Siglec-1 and macrophages and their interactions with the HIV-1 envelope should be considered in HIV-1 vaccine development.

  19. A patch of positively charged amino acids surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G.

    PubMed

    Chen, Gongying; He, Zhiwen; Wang, Tao; Xu, Rongzhen; Yu, Xiao-Fang

    2009-09-01

    The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K(22)x(3)K(26)x(3)Y(30)x(9)YRHHY(44)) and a cluster of hydrophobic residues (V(55)xIPLx(4-5)LxPhix2YWxL(72)) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.

  20. HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin.

    PubMed

    Astiazaran, Paulina; Bueno, Murilo Td; Morales, Elisa; Kugelman, Jeffrey R; Garcia-Rivera, Jose A; Llano, Manuel

    2011-04-21

    Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD). Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

  1. War and peace between microbes: HIV-1 interactions with coinfecting viruses.

    PubMed

    Lisco, Andrea; Vanpouille, Christophe; Margolis, Leonid

    2009-11-19

    HIV-1 disrupts the homeostatic equilibrium between the host and coinfecting microbes, facilitating reactivation of persistent viruses and invasion by new viruses. These viruses usually accelerate HIV disease but occasionally create conditions detrimental for HIV-1. Understanding these phenomena may lead to anti-HIV-1 strategies that specifically target interactions between HIV-1 and coinfecting viruses.

  2. Vpr-host interactions during HIV-1 viral life cycle.

    PubMed

    Zhao, Richard Y; Li, Ge; Bukrinsky, Michael I

    2011-06-01

    Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a multifunctional viral protein that plays important role at multiple stages of the HIV-1 viral life cycle. Although the molecular mechanisms underlying these activities are subject of ongoing investigations, overall, these activities have been linked to promotion of viral replication and impairment of anti-HIV immunity. Importantly, functional defects of Vpr have been correlated with slow disease progression of HIV-infected patients. Vpr is required for efficient viral replication in non-dividing cells such as macrophages, and it promotes, to some extent, viral replication in proliferating CD4+ T cells. The specific activities of Vpr include modulation of fidelity of viral reverse transcription, nuclear import of the HIV-1 pre-integration complex, transactivation of the HIV-1 LTR promoter, induction of cell cycle G2 arrest and cell death via apoptosis. In this review, we focus on description of the cellular proteins that specifically interact with Vpr and discuss their significance with regard to the known Vpr activities at each step of the viral life cycle in proliferating and non-proliferating cells.

  3. Quantum study of HIV-1 protease-bridge water interaction

    NASA Astrophysics Data System (ADS)

    Duan, Li L.; Tong, Yan; Mei, Ye; Zhang, Qing G.; Zhang, John Z. H.

    2007-10-01

    We present a fully quantum mechanical calculation for binding interaction between HIV-1 protease (PR) and the water molecule W301 which bridges the flaps of the protease with the inhibitors of PR. The quantum calculation is made possible by applying a recently developed molecular fractionation with conjugate caps (MFCC) method which divides a protein molecule into capped amino acid-based fragments and their conjugate caps. These individual fragments are properly treated to preserve the chemical property of bonds that are cut. Ab initio methods at HF, B3LYP, and MP2 levels with a fixed basis set 6-31+G* have been employed in the present calculation. The MFCC calculation produces a quantum mechanical interaction "map" representing interactions between individual residues of PR and W301. This enables a detailed quantitative analysis on binding of W301 to specific residues of PR at quantum mechanical level.

  4. Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction

    PubMed Central

    Lehmann, Martin; Rocha, Susana; Mangeat, Bastien; Blanchet, Fabien; Uji-i, Hiroshi; Hofkens, Johan; Piguet, Vincent

    2011-01-01

    Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions. PMID:22194693

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

    PubMed

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

    2014-05-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. 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.

  8. Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif▿

    PubMed Central

    Russell, Rebecca A.; Smith, Jessica; Barr, Rebekah; Bhattacharyya, Darshana; Pathak, Vinay K.

    2009-01-01

    Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their degradation. Thus, the Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development, as inhibiting these interactions could allow the host defense mechanism to control HIV-1 replication. Recently, it has been reported that amino acids 105 to 156 of A3G are involved in the interaction with Vif; however, to date, the region of A3F involved in Vif binding has not been identified. Using our previously reported Vif mutants that are capable of binding to only A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that the A3G domain that interacts with the Vif YRHHY region is located between amino acids 126 and 132 of A3G, which is consistent with the conclusions reported in previous studies. The A3F domain that interacts with the Vif DRMR region did not occur in the homologous domain but instead was located between amino acids 283 and 300 of A3F. These studies are the first to identify the A3F domain that interacts with the Vif DRMR region and show that distinct domains of A3G and A3F interact with different Vif regions. Pharmacological inhibition of either or both of these Vif-A3 interactions should prevent the degradation of the APOBEC3 proteins and could be used as a therapy against HIV-1. PMID:19036809

  9. 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.

  10. 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

  11. 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.

  12. Convergence and Divergence in the Evolution of the APOBEC3G-Vif Interaction Reveal Ancient Origins of Simian Immunodeficiency Viruses

    PubMed Central

    Compton, Alex A.; Emerman, Michael

    2013-01-01

    Naturally circulating lentiviruses are abundant in African primate species today, yet their origins and history of transmitting between hosts remain obscure. As a means to better understand the age of primate lentiviruses, we analyzed primate genomes for signatures of lentivirus-driven evolution. Specifically, we studied the adaptive evolution of host restriction factor APOBEC3G (A3G) in Old World Monkey (OWM) species. We find recurrent mutation of A3G in multiple primate lineages at sites that determine susceptibility to antagonism by the lentiviral accessory protein Vif. Using a broad panel of SIV Vif isolates, we demonstrate that natural variation in OWM A3G confers resistance to Vif-mediated degradation, suggesting that adaptive variants of the host factor were selected upon exposure to pathogenic lentiviruses at least 5–6 million years ago (MYA). Furthermore, in members of the divergent Colobinae subfamily of OWM, a multi-residue insertion event in A3G that arose at least 12 MYA blocks the activity of Vif, suggesting an even more ancient origin of SIV. Moreover, analysis of the lentiviruses associated with Colobinae monkeys reveal that the interface of the A3G-Vif interaction has shifted and given rise to a second genetic conflict. Our analysis of virus-driven evolution describes an ancient yet ongoing genetic conflict between simian primates and lentiviruses on a million-year time scale. PMID:23359341

  13. Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

    PubMed Central

    Ma, X Y; Sova, P; Chao, W; Volsky, D J

    1994-01-01

    The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity. Images PMID:8107232

  14. 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.

  15. 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

  16. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer.

    PubMed

    Liu, Qingbo; Acharya, Priyamvada; Dolan, Michael A; Zhang, Peng; Guzzo, Christina; Lu, Jacky; Kwon, Alice; Gururani, Deepali; Miao, Huiyi; Bylund, Tatsiana; Chuang, Gwo-Yu; Druz, Aliaksandr; Zhou, Tongqing; Rice, William J; Wigge, Christoph; Carragher, Bridget; Potter, Clinton S; Kwong, Peter D; Lusso, Paolo

    2017-04-01

    Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-Å resolution. A single CD4 molecule is embraced by a quaternary HIV-1-Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.

  17. HIV-1 and Morphine Regulation of Autophagy in Microglia: Limited Interactions in the Context of HIV-1 Infection and Opioid Abuse

    PubMed Central

    Rodriguez, Myosotys; Dever, Seth M.; Masvekar, Ruturaj R.; Gewirtz, David A.; Shacka, John J.

    2014-01-01

    ABSTRACT Microglia are the predominant resident central nervous system (CNS) cell type productively infected by HIV-1, and play a key role in the progression of HIV-associated dementia (HAD). Moreover, neural dysfunction and progression to HAD are accelerated in opiate drug abusers. In the present study, we examined the role of the autophagy pathway in the neuropathogenesis of HIV-1 using primary human microglial cells and determined whether opiates converge at this point. Infection of microglia with the HIV-1SF162 macrophage-tropic strain resulted in increased Beclin1 expression, accompanied by an increase of LC3 protein levels and accumulation of LC3 reporter RFP+ GFP+ (yellow) puncta, suggesting that HIV-1 infection triggers autophagosome formation without promoting protein degradation by the lysosome. Conversely, coexposure with HIV-1 and morphine significantly decreased virus-induced Beclin1 expression and autophagosome formation. Exploration of the possible mechanism(s) used by morphine to disrupt the autophagic process unveiled a significant increase in intracellular pH, which coincided with a reduction in the formation of acidic vesicular organelles and in autophagolysosome formation. Small interfering RNA targeting BECN1, a gene critical for autophagosome formation, significantly reduced viral replication and the virus-induced inflammatory responses. Conversely, morphine-enhanced viral replication and inflammatory responses were not affected by gene silencing with siBeclin1, suggesting that the interactive effect of morphine in HIV-1 pathogenesis is mediated through a Beclin1-independent mechanism. These novel findings may have important implications on the connections between autophagy and HIV-1 pathogenesis mediated by microglial cells in opioid-abusing individuals. IMPORTANCE About 50% of individuals infected with HIV-1 will develop some sort of neurocognitive impairment that cannot be prevented nor eradicated by antiretroviral therapy. The

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

    PubMed

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

    2016-10-19

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

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

    PubMed Central

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

    2016-01-01

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

  20. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein

    PubMed Central

    Miyakawa, Kei; Nishi, Mayuko; Matsunaga, Satoko; Okayama, Akiko; Anraku, Masaki; Kudoh, Ayumi; Hirano, Hisashi; Kimura, Hirokazu; Morikawa, Yuko; Yamamoto, Naoki; Ono, Akira; Ryo, Akihide

    2017-01-01

    Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1. PMID:28134256

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

    PubMed Central

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

    2016-01-01

    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 CRL4DCAF1 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 CRL4DCAF1 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 CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 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

  2. Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

    PubMed Central

    König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

    2008-01-01

    Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

  3. Comparative analysis of protein interaction networks reveals that conserved pathways are susceptible to HIV-1 interception.

    PubMed

    Qian, Xiaoning; Yoon, Byung-Jun

    2011-02-15

    Human immunodeficiency virus type one (HIV-1) is the major pathogen that causes the acquired immune deficiency syndrome (AIDS). With the availability of large-scale protein-protein interaction (PPI) measurements, comparative network analysis can provide a promising way to study the host-virus interactions and their functional significance in the pathogenesis of AIDS. Until now, there have been a large number of HIV studies based on various animal models. In this paper, we present a novel framework for studying the host-HIV interactions through comparative network analysis across different species. Based on the proposed framework, we test our hypothesis that HIV-1 attacks essential biological pathways that are conserved across species. We selected the Homo sapiens and Mus musculus PPI networks with the largest coverage among the PPI networks that are available from public databases. By using a local network alignment algorithm based on hidden Markov models (HMMs), we first identified the pathways that are conserved in both networks. Next, we analyzed the HIV-1 susceptibility of these pathways, in comparison with random pathways in the human PPI network. Our analysis shows that the conserved pathways have a significantly higher probability of being intercepted by HIV-1. Furthermore, Gene Ontology (GO) enrichment analysis shows that most of the enriched GO terms are related to signal transduction, which has been conjectured to be one of the major mechanisms targeted by HIV-1 for the takeover of the host cell. This proof-of-concept study clearly shows that the comparative analysis of PPI networks across different species can provide important insights into the host-HIV interactions and the detailed mechanisms of HIV-1. We expect that comparative multiple network analysis of various species that have different levels of susceptibility to similar lentiviruses may provide a very effective framework for generating novel, and experimentally verifiable hypotheses on the

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

    PubMed

    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/.

  5. Molecular Mechanism of HIV-1 Tat Interacting with Human Dopamine Transporter

    PubMed Central

    Midde, Narasimha M.; Quizon, Pamela M.; Sun, Wei-Lun; Zhu, Jun; Zhan, Chang-Guo

    2015-01-01

    Nearly 70% of HIV-infected individuals suffer from HIV-associated neurocognitive disorders (HAND). HIV-1 transactivator of transcription (Tat) protein is known to synergize with abused drugs and exacerbate the progression of central nervous system (CNS) pathology. Cumulative evidences suggest that the HIV-1 Tat protein exerts the neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. Through computational modeling and molecular dynamics (MD) simulations, we develop a three-dimensional (3D) structural model for HIV-1 Tat binding with hDAT. The model provides novel mechanistic insights concerning how HIV-1 Tat interacts with hDAT and inhibits dopamine uptake in hDAT. In particular, according to the computational modeling, Tat binds most favorably with the outward-open state of hDAT. Residues Y88, K92, and Y470 of hDAT are predicted to be key residues involved in the interaction between hDAT and Tat. The roles of these hDAT residues in the interaction with Tat are validated by experimental tests through site-directed mutagensis and DA uptake assays. The agreement between the computational and experimental data suggests that the computationally predicted hDAT-Tat binding mode and mechanistic insights are reasonable and provide a new starting point to design further pharmacological studies on the molecular mechanism of HIV-associated neurocognitive disorders. PMID:25695767

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

    PubMed Central

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

    2014-01-01

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

  7. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    PubMed Central

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-01-01

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

  8. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries.

    PubMed

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-11-23

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N⁶-methyladenosine (m⁶A), allowing the recruitment of YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2), an m⁶A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  9. Interaction between herpesvirus entry mediator and HSV-2 glycoproteins mediates HIV-1 entry of HSV-2-infected epithelial cells.

    PubMed

    Hu, Kai; He, Siyi; Xiao, Juhua; Li, Mei; Luo, Sukun; Zhang, Mudan; Hu, Qinxue

    2017-09-01

    Herpes simplex virus type 2 (HSV-2) increases human immunodeficiency virus type 1 (HIV-1) acquisition and transmission via unclear mechanisms. Herpesvirus entry mediator (HVEM), an HSV-2 entry receptor, is highly expressed on HIV-1 target cells (CD4+ T cells) and may be incorporated into HIV-1 virions, while HSV-2 glycoproteins can be present on the infected cell surface. Since HVEM-gD interaction together with gB/gH/gL is essential for HSV-2 entry, HVEM-bearing HIV-1 (HIV-1/HVEM) may enter HSV-2-infected cells through such interactions. To test this hypothesis, we first confirmed the presence of HVEM on HIV-1 virions and glycoproteins on the HSV-2-infected cell surface. Additional studies showed that HIV-1/HVEM bound to the HSV-2-infected cell surface in an HSV-2 infection-time-dependent manner via HVEM-gD interaction. HIV-1/HVEM entry of HSV-2-infected cells was dependent on HVEM-gD interaction and the presence of gB/gH/gL, and was inhibited by azidothymidine. Furthermore, peripheral blood mononuclear cell-derived HIV-1 infected HSV-2-infected primary foreskin epithelial cells and the infection was inhibited by anti-HVEM/gD antibodies. Together, our results indicate that HIV-1 produced from CD4+ T cells bears HSV-2 receptor HVEM and can bind to and enter HSV-2-infected epithelial cells depending on HVEM-gD interaction and the presence of gB/gH/gL. Our findings provide a potential new mechanism underlying HSV-2 infection-enhanced HIV-1 mucosal transmission and may shed light on HIV-1 prevention.

  10. Overlapping Regions in HIV-1 Genome Act as Potential Sites for Host–Virus Interaction

    PubMed Central

    Saha, Deeya; Podder, Soumita; Ghosh, Tapash C.

    2016-01-01

    More than a decade, overlapping genes in RNA viruses became a subject of research which has explored various effect of gene overlapping on the evolution and function of viral genomes like genome size compaction. Additionally, overlapping regions (OVRs) are also reported to encode elevated degree of protein intrinsic disorder (PID) in unspliced RNA viruses. With the aim to explore the roles of OVRs in HIV-1 pathogenesis, we have carried out an in-depth analysis on the association of gene overlapping with PID in 35 HIV1- M subtypes. Our study reveals an over representation of PID in OVR of HIV-1 genomes. These disordered residues endure several vital, structural features like short linear motifs (SLiMs) and protein phosphorylation (PP) sites which are previously shown to be involved in massive host–virus interaction. Moreover, SLiMs in OVRs are noticed to be more functionally potential as compared to that of non-overlapping region. Although, density of experimentally verified SLiMs, resided in 9 HIV-1 genes, involved in host–virus interaction do not show any bias toward clustering into OVR, tat and rev two important proteins mediates host–pathogen interaction by their experimentally verified SLiMs, which are mostly localized in OVR. Finally, our analysis suggests that the acquisition of SLiMs in OVR is mutually exclusive of the occurrence of disordered residues, while the enrichment of PPs in OVR is solely dependent on PID and not on overlapping coding frames. Thus, OVRs of HIV-1 genomes could be demarcated as potential molecular recognition sites during host–virus interaction. PMID:27867372

  11. 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.

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

    PubMed

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

    2017-08-15

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

  13. HIV-1 p6-Another viral interaction partner to the host cellular protein cyclophilin A.

    PubMed

    Solbak, Sara M Ø; Reksten, Tove R; Röder, Rene; Wray, Victor; Horvli, Ole; Raae, Arnt J; Henklein, Petra; Henklein, Peter; Fossen, Torgils

    2012-04-01

    The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D (1)H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl-prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme-substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

  14. HIV-1 interacts with human endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes.

    PubMed

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

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  16. Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

    PubMed

    Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

    2011-01-01

    HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors.

  17. Crystal Structures of HIV-1 Reverse Transcriptase with Picomolar Inhibitors Reveal Key Interactions for Drug Design

    PubMed Central

    Frey, Kathleen M.; Bollini, Mariela; Mislak, Andrea C.; Cisneros, José A.; Gallardo-Macias, Ricardo; Jorgensen, William L.; Anderson, Karen S.

    2012-01-01

    X-ray crystal structures at 2.9 Å resolution are reported for complexes of catechol diethers 1 and 2 with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles. PMID:23163887

  18. Intermolecular interactions in the crystal structures of potential HIV-1 integrase inhibitors.

    PubMed

    Majerz-Maniecka, Katarzyna; Musiol, Robert; Nitek, Wojciech; Oleksyn, Barbara J; Mouscadet, Jean-Francois; Le Bret, Marc; Polanski, Jaroslaw

    2006-02-15

    2-[(2,5-dichloro-4-nitro-phenylamino)-methoxy-methyl]-8-hydroxy-quinoline 1 and 2-methyl-quinoline-5,8-dione-5-oxime 2 were obtained as potential HIV-1 integrase inhibitors and analyzed by X-ray crystallography. Semiempirical theoretical calculations of energy preferred conformations were also carried out. The crystal structures of both compounds are stabilized via hydrogen bonds and pi-pi stacking interactions. The planarity of compound 1 is caused by intramolecular hydrogen bonds.

  19. 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.

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

    SciTech Connect

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

    2006-05-25

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

  1. HIV-1 replication.

    PubMed

    Freed, E O

    2001-11-01

    surface (SU) Env glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. gp120 contains the determinants that interact with receptor and coreceptor, while gp41 not only anchors the gp120/gp41 complex in the membrane (Fig. 2), but also contains domains that are critical for catalyzing the membrane fusion reaction between viral and host lipid bilayers during virus entry. Comparison of env sequences from a large number of virus isolates revealed that gp120 is organized into five conserved regions (C1-C5) and five highly variable domains (V1-V5). The variable regions tend to be located in disulfide-linked loops. gp41 is composed of three major domains: the ectodomain (which contains determinants essential for membrane fusion), the transmembrane anchor sequence, and the cytoplasmic tail. In addition to the gag, pol, and env genes, HIV-1 also encodes a number of regulatory and accessory proteins. Tat is critical for transcription from the HIV-1 LTR and Rev plays a major [figure: see text] role in the transport of viral RNAs from the nucleus to the cytoplasm. Vpu, Vif, Vpr and Nef have been termed "accessory" or "auxiliary" proteins to reflect the fact that they are not uniformly required for virus replication. The functions of these very interesting proteins will be discussed in more detail at the end of this chapter. HIV replication proceeds in a series of events that can be divided into two overall phases: "early" and "late" (Fig. 3). Although some events occur in a concerted or simultaneous fashion, the replication cycle can be viewed most simply as proceeding in an ordered, step-wise manner. In this chapter, each step in virus replication will be considered; additional information can be obtained from the more detailed reviews and primary references that are cited.

  2. 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

  3. HIV-1 and GBV-C co-infection in Venezuela.

    PubMed

    Rodríguez, Anny Karely; Garzaro, Domingo José; Loureiro, Carmen Luisa; Gutiérrez, Cristina R; Ameli, Gladys; Jaspe, Rossana Celeste; Porto, Leticia; Monsalve, Francisca; Pozada, Ángela; Vázquez, Luzmary; Quiñones-Mateu, Miguel E; Pujol, Flor Helene; Rangel, Héctor Rafael

    2014-07-14

    Co-infection with GB virus C (GBV-C) in patients infected with human immunodeficiency virus 1 (HIV-1) has been associated with prolonged survival. The aim of this study was to evaluate the prevalence of GBV-C infection among HIV-1-infected patients in Venezuela, and to determine the effects of the co-infection on the levels of relevant cytokines. Plasma samples were collected from 270 HIV-1-seronegative and 255 HIV-1-seropositive individuals. GBV-C infection was determined by RT-PCR of the NS5 region and genotyped by sequence analysis of the 5´UTR region. HIV-1 strains were characterized by sequence analysis of pol, vif, env, and nef genes. Selected cytokines were evaluated by ELISA. Ninety-seven of 525 (18.5%) plasma samples tested positive for GBV-C RNA. A significantly higher prevalence of GBV-C was found among HIV-1 patients compared to HIV-1-seronegative individuals (67/255, 26% versus 30/270, 11%; p < 0.001). Statistical difference was observed in the viral load between HIV-1+GBV-C+ and HIV-1+GBV-C- (p = 0.014), although no differences in CD4+ cell counts were found between both groups. TNFα concentration was higher in HIV-1+GBV-C- than in HIV-1+GBV-C+ patients (25.9 pg/mL versus 17.3 pg/mL; p = 0.02); RANTES expression levels were more variable in GBV-C co-infected patients and more frequently elevated in HIV-1 mono-infected patients compared to patients co-infected with GBV-C. The previously observed beneficial effect of co-infection with HIV-1 and GBV-C on disease progression is complex and might be due in part to a change in the cytokine environment. More studies are required to understand the interaction between both viruses.

  4. NMR detection of intermolecular interaction sites in the dimeric 5'-leader of the HIV-1 genome.

    PubMed

    Keane, Sarah C; Van, Verna; Frank, Heather M; Sciandra, Carly A; McCowin, Sayo; Santos, Justin; Heng, Xiao; Summers, Michael F

    2016-11-15

    HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5'-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a (2)H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5'-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5' (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a (2)H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-11-24

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

  7. 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

  8. 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).

  9. Theoretical investigation on nevirapine and HIV-1 reverse transcriptase binding site interaction, based on ONIOM method

    NASA Astrophysics Data System (ADS)

    Kuno, Mayuso; Hannongbua, Supa; Morokuma, Keiji

    2003-10-01

    The ONIOM method was applied to the interaction of nevirapine with the HIV-1 reverse transcriptase binding site. The isolated complex of pyridine (part of nevirapine) and methyl phenol (part of Tyr181) was found at the MP2/6-31+G(d) level to have stacking interaction with 8.8 kcal/mol binding energy. Optimization of nevirapine and Tyr181 geometry in the pocket of 16 amino acid residues at the ONIOM3(MP2/6-31G(d):HF/3-21G:PM3) level gave the complex structure with weak hydrogen bonding but without stacking interaction. The binding energy of 8.9 kcal/mol comes almost entirely from the interaction of nevirapine with amino acid residues other than Tyr181.

  10. CD4-gp120 interaction interface - a gateway for HIV-1 infection in human: molecular network, modeling and docking studies.

    PubMed

    Pandey, Deeksha; Podder, Avijit; Pandit, Mansi; Latha, Narayanan

    2017-09-01

    The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4-gp120 interaction interface has been studied through a comprehensive protein-protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.

  11. Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

    Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

  12. Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

    PubMed Central

    Ignatovich, Anna; Reiter, Jochen; Dittmar, Matthias T.; Wain-Hobson, Simon; Meyerhans, Andreas

    2011-01-01

    Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1) resistance to azidothymidine (AZT) requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41–215 cluster) of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions. PMID:21483787

  13. HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein.

    PubMed

    Solbak, Sara Marie Øie; Reksten, Tove Ragna; Hahn, Friedrich; Wray, Victor; Henklein, Petra; Henklein, Peter; Halskau, Øyvind; Schubert, Ulrich; Fossen, Torgils

    2013-02-01

    The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p6(23-52) S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.

  14. Determining confidence of predicted interactions between HIV-1 and human proteins using conformal method.

    PubMed

    Nouretdinov, Ilia; Gammerman, Alex; Qi, Yanjun; Klein-Seetharaman, Judith

    2012-01-01

    Identifying protein-protein interactions (PPI's) is critical for understanding virtually all cellular molecular mechanisms. Previously, predicting PPI's was treated as a binary classification task and has commonly been solved in a supervised setting which requires a positive labeled set of known PPI's and a negative labeled set of non-interacting protein pairs. In those methods, the learner provides the likelihood of the predicted interaction, but without a confidence level associated with each prediction. Here, we apply a conformal prediction framework to make predictions and estimate confidence of the predictions. The conformal predictor uses a function measuring relative 'strangeness' interacting pairs to check whether prediction of a new example added to the sequence of already known PPI's would conform to the 'exchangeability' assumption: distribution of interacting pairs is invariant with any permutations of the pairs. In fact, this is the only assumption we make about the data. Another advantage is that the user can control a number of errors by providing a desirable confidence level. This feature of CP is very useful for a ranking list of possible interactive pairs. In this paper, the conformal method has been developed to deal with just one class - class interactive proteins - while there is not clearly defined of 'non-interactive'pairs. The confidence level helps the biologist in the interpretation of the results, and better assists the choices of pairs for experimental validation. We apply the proposed conformal framework to improve the identification of interacting pairs between HIV-1 and human proteins.

  15. Pharmacokinetic interaction between zidovudine and trimethoprim/sulphamethoxazole in HIV-1 infected children.

    PubMed

    Dallas, S; E Read, S; King, S; Koren, G; Bendayan, R

    2000-09-01

    To evaluate the effect of the antimicrobial agent trimethoprim/sulphamethoxazole (TMP/SMX) on the pharmacokinetic properties of the antiretroviral drug zidovudine (ZDV). This single dose, open label, crossover study involved the oral administration of ZDV (150 mg/m²) alone and in combination with oral TMP/SMX (2.5 mg/kg) on two separate occasions. Serial blood samples (0 to 8 h) were collected, and concentrations of ZDV and its glucuronide metabolite were quantified using a radioimmunoassay. ZDV pharmacokinetics were determined by noncompartmental analysis. Six HIV-1 infected children aged four months to five years were recruited from the HIV clinic at The Hospital for Sick Children, Toronto, Ontario. Only three patients completed both study phases and were included in the pharmacokinetic analysis. With TMP/SMX therapy, no statistically significant changes were observed in ZDV pharmacokinetic parameters. However, there was a trend towards increased ZDV half-life and area under the concentration versus time curve, as well as decreased apparent oral clearance. Similarly, a trend towards an increased half-life of the ZDV-glucuronide metabolite was also observed. The changes in ZDV pharmacokinetics in the presence of TMP/SMX did not reach statistical significance, most likely due to the limited number of patients involved. Despite the limited data, a possible interaction between ZDV and TMP/SMX in young HIV-1 infected children should be considered, and patients may require close clinical monitoring.

  16. Biologic interactions between HSV-2 and HIV-1 and possible implications for HSV vaccine development.

    PubMed

    Schiffer, Joshua T; Gottlieb, Sami L

    2017-09-25

    Development of a safe and effective vaccine against herpes simplex virus type 2 (HSV-2) has the potential to limit the global burden of HSV-2 infection and disease, including genital ulcer disease and neonatal herpes, and is a global sexual and reproductive health priority. Another important potential benefit of an HSV-2 vaccine would be to decrease HIV infections, as HSV-2 increases the risk of HIV-1 acquisition several-fold. Acute and chronic HSV-2 infection creates ulcerations and draws dendritic cells and activated CD4+ T cells into genital mucosa. These cells are targets for HIV entry and replication. Prophylactic HSV-2 vaccines (to prevent infection) and therapeutic vaccines (to modify or treat existing infections) are currently under development. By preventing or modifying infection, an effective HSV-2 vaccine could limit HSV-associated genital mucosal inflammation and thus HIV risk. However, a vaccine might have competing effects on HIV risk depending on its mechanism of action and cell populations generated in the genital mucosa. In this article, we review biologic interactions between HSV-2 and HIV-1, consider HSV-2 vaccine development in the context of HIV risk, and discuss implications and research needs for future HSV vaccine development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Interaction of HIV-1 gag and membranes in a cell-free system.

    PubMed

    Yang, Liuzhan; Ratner, Lee

    2002-10-10

    A coupled transcription-translation (TNT) reticulocyte lysate system was used to examine posttranslational alterations in HIV-1 Gag upon addition of Jurkat T cell membranes. Incubation of the Gag precursor protein, Pr55gag, with membranes resulted in a time-dependent alteration in Gag resulting in partial resistance to trypsin treatment. Treatment of membranes and TNT extract with apyrase or pretreatment of membranes with trypsin prevented this posttranslational alteration of Gag. In contrast, this activity was not disrupted by pretreatment of membranes with Triton X-100 at 4 degrees C, under conditions which do not solubilize raft-associated proteins. Flotation studies revealed that acquisition of trypsin-resistance was accompanied by Gag binding to membranes. The myristylation signal and nucleocapsid domain were found to mediate Gag binding to membranes. The posttranslational alteration of Gag accompanying membrane interaction may represent a conformational change, oligomerization, and/or association with or envelopment by membranes. These findings provide new clues to the stepwise process of HIV-1 assembly.

  18. 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

  19. Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide

    PubMed Central

    Dennison, S. Moses; Anasti, Kara M.; Jaeger, Frederick H.; Stewart, Shelley M.; Pollara, Justin; Liu, Pinghuang; Kunz, Erika L.; Zhang, Ruijun; Vandergrift, Nathan; Permar, Sallie; Ferrari, Guido; Tomaras, Georgia D.; Bonsignori, Mattia; Michael, Nelson L.; Kim, Jerome H.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Liao, Hua-Xin

    2014-01-01

    ABSTRACT Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site. IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1

  20. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins.

    PubMed

    Suryawanshi, Gajendra W; Hoffmann, Alexander

    2015-12-07

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target

  1. 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.

  2. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Veillette, Maxime; Désormeaux, Anik; Medjahed, Halima; Gharsallah, Nour-Elhouda; Coutu, Mathieu; Baalwa, Joshua; Guan, Yongjun; Lewis, George; Ferrari, Guido; Hahn, Beatrice H; Haynes, Barton F; Robinson, James E; Kaufmann, Daniel E; Bonsignori, Mattia; Sodroski, Joseph; Finzi, Andrés

    2014-03-01

    Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

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

    PubMed Central

    Hetti Arachchilage, Madara; Piontkivska, Helen

    2016-01-01

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

  4. The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

    PubMed

    Harrison, Angela T; Kriel, Frederik H; Papathanasopoulos, Maria A; Mosebi, Salerwe; Abrahams, Shaakira; Hewer, Raymond

    2015-03-01

    Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 μm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 μm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

  5. Possible Footprints of APOBEC3F and/or Other APOBEC3 Deaminases, but Not APOBEC3G, on HIV-1 from Patients with Acute/Early and Chronic Infections

    PubMed Central

    Armitage, Andrew E.; Deforche, Koen; Welch, John J.; Van Laethem, Kristel; Camacho, Ricardo; Rambaut, Andrew

    2014-01-01

    ABSTRACT Members of the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3 (APOBEC3) innate cellular cytidine deaminase family, particularly APOBEC3F and APOBEC3G, can cause extensive and lethal G-to-A mutations in HIV-1 plus-strand DNA (termed hypermutation). It is unclear if APOBEC3-induced mutations in vivo are always lethal or can occur at sublethal levels that increase HIV-1 diversification and viral adaptation to the host. The viral accessory protein Vif counteracts APOBEC3 activity by binding to APOBEC3 and promoting proteasome degradation; however, the efficiency of this interaction varies, since a range of hypermutation frequencies are observed in HIV-1 patient DNA. Therefore, we examined “footprints” of APOBEC3G and APOBEC3F activity in longitudinal HIV-1 RNA pol sequences from approximately 3,000 chronically infected patients by determining whether G-to-A mutations occurred in motifs that were favored or disfavored by these deaminases. G-to-A mutations were more frequent in APOBEC3G-disfavored than in APOBEC3G-favored contexts. In contrast, mutations in APOBEC3F-disfavored contexts were relatively rare, whereas mutations in contexts favoring APOBEC3F (and possibly other deaminases) occurred 16% more often than average G-to-A mutations. These results were supported by analyses of >500 HIV-1 env sequences from acute/early infection. IMPORTANCE Collectively, our results suggest that APOBEC3G-induced mutagenesis is lethal to HIV-1, whereas mutagenesis caused by APOBEC3F and/or other deaminases may result in sublethal mutations that might facilitate viral diversification. Therefore, Vif-specific cytotoxic T lymphocyte (CTL) responses and drugs that manipulate the interplay between Vif and APOBEC3 may have beneficial or detrimental clinical effects depending on how they affect the binding of Vif to various members of the APOBEC3 family. PMID:25165112

  6. Structural Basis of the Allosteric Inhibitor Interaction on the HIV-1 Reverse Transcriptase RNase H domain

    PubMed Central

    Christen, Martin T.; Menon, Lakshmi; Myshakina, Nataliya A.; Ahn, Jinwoo; Parniak, Michael A.; Ishima, Rieko

    2012-01-01

    HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH. PMID:22846652

  7. DETERMINING CONFIDENCE OF PREDICTED INTERACTIONS BETWEEN HIV-1 AND HUMAN PROTEINS USING CONFORMAL METHOD

    PubMed Central

    Nouretdinov, Ilia; Gammerman, Alex; Qi, Yanjun; Klein-Seetharaman, Judith

    2011-01-01

    Identifying protein-protein interactions (PPI’s) is critical for understanding virtually all cellular molecular mechanisms. Previously, predicting PPI’s was treated as a binary classification task and has commonly been solved in a supervised setting which requires a positive labeled set of known PPI’s and a negative labeled set of non-interacting protein pairs. In those methods, the learner provides the likelihood of the predicted interaction, but without a confidence level associated with each prediction. Here, we apply a conformal prediction framework to make predictions and estimate confidence of the predictions. The conformal predictor uses a function measuring relative ’strangeness’ interacting pairs to check whether prediction of a new example added to the sequence of already known PPI’s would conform to the ’exchangeability’ assumption: distribution of interacting pairs is invariant with any permutations of the pairs. In fact, this is the only assumption we make about the data. Another advantage is that the user can control a number of errors by providing a desirable confidence level. This feature of CP is very useful for a ranking list of possible interactive pairs. In this paper, the conformal method has been developed to deal with just one class - class interactive proteins - while there is not clearly defined of ’non-interactive’ pairs. The confidence level helps the biologist in the interpretation of the results, and better assists the choices of pairs for experimental validation. We apply the proposed conformal framework to improve the identification of interacting pairs between HIV-1 and human proteins. PMID:22174286

  8. 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

  9. HIV-1 protease inhibits its homologous reverse transcriptase by protein-protein interaction.

    PubMed Central

    Böttcher, M; Grosse, F

    1997-01-01

    The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells. The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase. Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR. Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell. Physical interactions were confirmed with purified components by means of an ELISA assay. Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected. The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM. A possible physiological significance of this type of interaction is discussed. PMID:9108151

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

    PubMed Central

    2013-01-01

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

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

    PubMed

    Bastian, Arangassery Rosemary; Contarino, Mark; Bailey, Lauren D; Aneja, Rachna; Moreira, Diogo Rodrigo Magalhaes; Freedman, Kevin; McFadden, Karyn; Duffy, Caitlin; Emileh, Ali; Leslie, George; Jacobson, Jeffrey M; Hoxie, James A; Chaiken, Irwin

    2013-12-13

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

  12. LINE-1 Retrotransposable Element DNA Accumulates in HIV-1-Infected Cells

    PubMed Central

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

    2013-01-01

    Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4+ cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity. PMID:24089548

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

    PubMed

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

    2013-12-01

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

  14. Vif of Feline Immunodeficiency Virus from Domestic Cats Protects against APOBEC3 Restriction Factors from Many Felids▿

    PubMed Central

    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-01-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Δvif FIV, felid A3Z2s did not show any antiviral activity against Δvif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether VifFIV can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.VifFIV was constructed. This HIV-1.VifFIV was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor. PMID:20444897

  15. 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

  16. Structural plasticity in the topology of the membrane-interacting domain of HIV-1 gp41.

    PubMed

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

    2014-02-04

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-26

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

  19. 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.

  20. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

  1. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes

    PubMed Central

    Singh, Parmit Kumar; Plumb, Matthew R.; Ferris, Andrea L.; Iben, James R.; Wu, Xiaolin; Fadel, Hind J.; Luke, Brian T.; Esnault, Caroline; Poeschla, Eric M.; Hughes, Stephen H.; Kvaratskhelia, Mamuka; Levin, Henry L.

    2015-01-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced. PMID:26545813

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

    NASA Astrophysics Data System (ADS)

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

    2005-03-01

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

  3. Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch.

    PubMed

    Datta, Siddhartha A K; Zhao, Zhuojun; Clark, Patrick K; Tarasov, Sergey; Alexandratos, Jerry N; Campbell, Stephen J; Kvaratskhelia, Mamuka; Lebowitz, Jacob; Rein, Alan

    2007-01-19

    Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

  4. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4(+) T cell apoptosis.

    PubMed

    Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Dirk, Brennan S; Van Nynatten, Logan R; Haeryfar, S M Mansour; Dikeakos, Jimmy D

    2017-09-01

    Acquired Immune Deficiency Syndrome is characterized by a decline in CD4(+) T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4(+) T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (NefM20A or NefEEEE62-65AAAA) or Nef:AP-2 (NefLL164/165AA), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (NefH89A or NefF191A) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Crystal structure of the neutralizing Llama V(HH) D7 and its mode of HIV-1 gp120 interaction.

    PubMed

    Hinz, Andreas; Lutje Hulsik, David; Forsman, Anna; Koh, Willie Wee-Lee; Belrhali, Hassan; Gorlani, Andrea; de Haard, Hans; Weiss, Robin A; Verrips, Theo; Weissenhorn, Winfried

    2010-05-05

    HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment V(HH) D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 V(HH) at 1.5 A resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site.

  6. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

    PubMed

    Exline, C M; Feng, Z; Stoltzfus, C M

    2008-04-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

  7. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

    PubMed Central

    2010-01-01

    Background Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data

  8. 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.

  9. 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

  10. 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.

  11. Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction.

    PubMed

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

    2010-11-10

    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 1.4-1.6 Å 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. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2011-01-01

    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 1.4 to 1.6 Å 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. PMID:21070952

  13. Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7.

    PubMed

    Plotnik, David; Guo, Wenjin; Cleveland, Brad; von Haller, Priska; Eng, Jimmy K; Guttman, Miklos; Lee, Kelly K; Arthos, James; Hu, Shiu-Lok

    2017-08-16

    Gut-homing α4β7(high) CD4(+) T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α4β7, and that this likely contributes to the infection of α4β7(high) T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions in a similar manner to CHO fibronectin. These findings provide an explanation for the apparent contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4(+) T cells, into gut lymphoid tissues. Previous reports

  14. A new approach to an AIDS vaccine: creating antibodies to HIV vif will enable apobec3G to turn HIV-infection into a benign problem.

    PubMed

    Jeffrey Fessel, W

    2005-01-01

    For a decade, attempts to produce a vaccine that prevents HIV infection have been fruitless, and fresh approaches are required. Apobec3G is a natural defensin and a cytidine deaminase. Apobec3G induces a high rate of dC to dU mutation in the first minus strand of cDNA, causing degradation throughout the HIV genome that renders the virus effete. The viral infectivity factor (vif) of HIV is essential for efficient replication of that virus. Vif binds to apobec3G and induces its polyubiquitination, which enables HIV to evade apobec3G. This suggests that a vif-based vaccine which induced anti-vif antibodies, would prevent the neutralizing action of vif upon apobec3G. Then, with HIV-vif ineffective, apobec3G could act without hindrance to create a less aggressive, non-lethal HIV infection. Mutated vif impedes HIV infection. Slow progressors with vif 132S had 4-fold lower viral loads than those with vif 132R; and introducing vif 132S into HIV-1 caused a 5-fold decrease in viral replication. And in the absence of vif, HIV virions accumulate multiple defects in structural, enzymatic, and regulatory viral proteins. The success of a vif-based vaccine depends upon (1) a vif-antibody response, and (2) vif antibodies entering the cells that harbor HIV. First, antibodies to vif have been seen in frequencies ranging between 25% and 100% in patients infected with HIV-1. Second, transport of anti-vif antibodies into cells might occur via several mechanisms. Likeliest is that in viremic persons, antibodies would attach to cell-free virions which would piggyback the antibodies into CD4+ cells. Alternatively, a fusion protein between vif and a cell-surface receptor, e.g., CD4 or CCR5, might be used as vaccine antigen. Also, anti-vif antibodies might internalize after ligation of HIV virions budding on the cell surface, in the same way as monoclonal antibodies against porcine pseudorabies virus induced viral glycoproteins on the cell surface to internalize. Finally, monoclonal

  15. The Human Immunodeficiency Virus Type 1 Vif Protein Reduces Intracellular Expression and Inhibits Packaging of APOBEC3G (CEM15), a Cellular Inhibitor of Virus Infectivity

    PubMed Central

    Kao, Sandra; Khan, Mohammad A.; Miyagi, Eri; Plishka, Ron; Buckler-White, Alicia; Strebel, Klaus

    2003-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles. PMID:14557625

  16. Towards semi-automated curation: using text mining to recreate the HIV-1, human protein interaction database.

    PubMed

    Jamieson, Daniel G; Gerner, Martin; Sarafraz, Farzaneh; Nenadic, Goran; Robertson, David L

    2012-01-01

    Manual curation has long been used for extracting key information found within the primary literature for input into biological databases. The human immunodeficiency virus type 1 (HIV-1), human protein interaction database (HHPID), for example, contains 2589 manually extracted interactions, linked to 14,312 mentions in 3090 articles. The advancement of text-mining (TM) techniques has offered a possibility to rapidly retrieve such data from large volumes of text to a high degree of accuracy. Here, we present a recreation of the HHPID using the current state of the art in TM. To retrieve interactions, we performed gene/protein named entity recognition (NER) and applied two molecular event extraction tools on all abstracts and titles cited in the HHPID. Our best NER scores for precision, recall and F-score were 87.5%, 90.0% and 88.6%, respectively, while event extraction achieved 76.4%, 84.2% and 80.1%, respectively. We demonstrate that over 50% of the HHPID interactions can be recreated from abstracts and titles. Furthermore, from 49 available open-access full-text articles, we extracted a total of 237 unique HIV-1-human interactions, as opposed to 187 interactions recorded in the HHPID from the same articles. On average, we extracted 23 times more mentions of interactions and events from a full-text article than from an abstract and title, with a 6-fold increase in the number of unique interactions. We further demonstrated that more frequently occurring interactions extracted by TM are more likely to be true positives. Overall, the results demonstrate that TM was able to recover a large proportion of interactions, many of which were found within the HHPID, making TM a useful assistant in the manual curation process. Finally, we also retrieved other types of interactions in the context of HIV-1 that are not currently present in the HHPID, thus, expanding the scope of this data set. All data is available at http://gnode1.mib.man.ac.uk/HIV1-text-mining.

  17. Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

    PubMed Central

    Saito, Akatsuki; Henning, Matthew S.; Serrao, Erik; Dubose, Brittany N.; Teng, Samantha; Huang, Jing; Li, Xiangming; Saito, Namiko; Roy, Saumendra Prasad; Siddiqui, Mohammad Adnan; Ahn, Jinwoo; Tsuji, Moriya; Hatziioannou, Theodora; Engelman, Alan N.

    2016-01-01

    ABSTRACT Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCE CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for

  18. Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

    PubMed Central

    DeAnda, Felix; Hightower, Kendra E.; Nolte, Robert T.; Hattori, Kazunari; Yoshinaga, Tomokazu; Kawasuji, Takashi; Underwood, Mark R.

    2013-01-01

    Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic. PMID:24146996

  19. 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

  20. NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

    PubMed Central

    Keane, Sarah C.; Van, Verna; Frank, Heather M.; Sciandra, Carly A.; McCowin, Sayo; Santos, Justin; Heng, Xiao; Summers, Michael F.

    2016-01-01

    HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones. PMID:27791166

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

    PubMed

    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.

  2. 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.

  3. 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.

  4. Vif of feline immunodeficiency virus from domestic cats protects against APOBEC3 restriction factors from many felids.

    PubMed

    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-07-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

  5. Optimal translation initiation enables Vif-deficient human immunodeficiency virus type 1 to escape restriction by APOBEC3G.

    PubMed

    Haché, Guylaine; Abbink, Truus E M; Berkhout, Ben; Harris, Reuben S

    2009-06-01

    APOBEC3G restricts Vif-deficient human immunodeficiency virus type 1 (HIV-1) by deaminating viral cDNA cytosines to uracils. This promutagenic activity is counteracted by HIV-1 Vif, which is a natural APOBEC3G antagonist. However, we previously reported that Vif-deficient HIV-1 could evolve resistance to APOBEC3G by a novel mechanism requiring an A200-to-C/T transition mutation and Vpr inactivation. A pyrimidine at nucleotide 200 in the untranslated leader region contributed to resistance by increasing virus particle production, which resulted in fewer APOBEC3G molecules per particle. Here we show that the A200-to-C/T mutation functions posttranscriptionally by inactivating an upstream start codon, which in turn enables optimal viral mRNA translation from canonical start codons.

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

    PubMed Central

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

    2006-01-01

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

  7. Host genetics and viral load in primary HIV-1 infection: clear evidence for gene by sex interactions.

    PubMed

    Li, Xuelin; Price, Matthew A; He, Dongning; Kamali, Anatoli; Karita, Etienne; Lakhi, Shabir; Sanders, Eduard J; Anzala, Omu; Amornkul, Pauli N; Allen, Susan; Hunter, Eric; Kaslow, Richard A; Gilmour, Jill; Tang, Jianming

    2014-09-01

    Research in the past two decades has generated unequivocal evidence that host genetic variations substantially account for the heterogeneous outcomes following human immunodeficiency virus type 1 (HIV-1) infection. In particular, genes encoding human leukocyte antigens (HLA) have various alleles, haplotypes, or specific motifs that can dictate the set-point (a relatively steady state) of plasma viral load (VL), although rapid viral evolution driven by innate and acquired immune responses can obscure the long-term relationships between HLA genotypes and HIV-1-related outcomes. In our analyses of VL data from 521 recent HIV-1 seroconverters enrolled from eastern and southern Africa, HLA-A*03:01 was strongly and persistently associated with low VL in women (frequency = 11.3 %, P < 0.0001) but not in men (frequency = 7.7 %, P = 0.66). This novel sex by HLA interaction (P = 0.003, q = 0.090) did not extend to other frequent HLA class I alleles (n = 34), although HLA-C*18:01 also showed a weak association with low VL in women only (frequency = 9.3 %, P = 0.042, q > 0.50). In a reduced multivariable model, age, sex, geography (clinical sites), previously identified HLA factors (HLA-B*18, B*45, B*53, and B*57), and the interaction term for female sex and HLA-A*03:01 collectively explained 17.0 % of the overall variance in geometric mean VL over a 3-year follow-up period (P < 0.0001). Multiple sensitivity analyses of longitudinal and cross-sectional VL data yielded consistent results. These findings can serve as a proof of principle that the gap of "missing heritability" in quantitative genetics can be partially bridged by a systematic evaluation of sex-specific associations.

  8. Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes.

    PubMed

    Barrero-Villar, Marta; Cabrero, José Román; Gordón-Alonso, Mónica; Barroso-González, Jonathan; Alvarez-Losada, Susana; Muñoz-Fernández, M Angeles; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín

    2009-01-01

    The human immunodeficiency virus 1 (HIV-1) envelope regulates the initial attachment of viral particles to target cells through its association with CD4 and either CXCR4 or CCR5. Although F-actin is required for CD4 and CXCR4 redistribution, little is known about the molecular mechanisms underlying this fundamental process in HIV infection. Using CD4(+) CXCR4(+) permissive human leukemic CEM T cells and primary lymphocytes, we have investigated whether HIV-1 Env might promote viral entry and infection by activating ERM (ezrin-radixin-moesin) proteins to regulate F-actin reorganization and CD4/CXCR4 co-clustering. The interaction of the X4-tropic protein HIV-1 gp120 with CD4 augments ezrin and moesin phosphorylation in human permissive T cells, thereby regulating ezrin-moesin activation. Moreover, the association and clustering of CD4-CXCR4 induced by HIV-1 gp120 requires moesin-mediated anchoring of actin in the plasma membrane. Suppression of moesin expression with dominant-negative N-moesin or specific moesin silencing impedes reorganization of F-actin and HIV-1 entry and infection mediated by the HIV-1 envelope protein complex. Therefore, we propose that activated moesin promotes F-actin redistribution and CD4-CXCR4 clustering and is also required for efficient X4-tropic HIV-1 infection in permissive lymphocytes.

  9. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    PubMed

    Kammula, Ellen C; Mötter, Jessica; Gorgels, Alexandra; Jonas, Esther; Hoffmann, Silke; Willbold, Dieter

    2012-01-01

    HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  10. Interactions of Pluronic Block Copolymers on P-gp Efflux Activity: Experience With HIV-1 Protease Inhibitors

    PubMed Central

    SHAIK, NAVEED; PAN, GUOYU; ELMQUIST, WILLIAM F.

    2016-01-01

    The objective was to examine the influence of Pluronic block-copolymers on the interaction between the drug efflux transporter, P-glycoprotein and HIV-1 protease inhibitors (PIs). The ATPase assay determined the effect of various Pluronics on PI-stimulated P-gp ATPase activity. Cellular accumulation studies were conducted using MDCKII and LLC-PK1 cells transfected with human MDR1 to assess Pluronic modulation of PI efflux. Pluronic P85 inhibited both basal and nelfinavir-stimulated P-gp ATPase activity, while Pluronic F127 had no effect. In cell accumulation studies, Pluronic P85 restored the accumulation of nelfinavir in MDCKII-MDR1 cells while Pluronic F127 and F88 had no effect. Pluronic P85 increased saquinavir accumulation in wild-type and MDR1-transfected cells in both the MDCKII and LLC-PK1 cell models, suggesting inhibition of multiple transporters, including MRPs. In conclusion, this study provides evidence that a block-copolymer, Pluronic P85, effectively inhibits the interaction of P-gp with nelfinavir and saquinavir. These data indicate that effective inhibition of HIV-1 PI efflux by Pluronic P85 may influence the distribution of antiretroviral agents to sites protected by efflux mechanisms, such as the blood–brain barrier, and possibly increase the brain exposure of these drugs resulting in suppression of viral replication and reduction in the incidence of drug resistant mutants. PMID:18393290

  11. A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors

    PubMed Central

    Hoffman, Trevor L.; Canziani, Gabriela; Jia, Li; Rucker, Joseph; Doms, Robert W.

    2000-01-01

    The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor, which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env–coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced, stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env–receptor binding constants to viral tropism and pathogenesis. PMID:11005830

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

    PubMed

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

    2015-10-01

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

  13. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

    PubMed

    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

    2016-12-01

    Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z

  14. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s

    PubMed Central

    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter

    2016-01-01

    ABSTRACT Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. IMPORTANCE Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with

  15. Secretion modification region-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release.

    PubMed

    Shelton, Martin N; Huang, Ming-Bo; Ali, Syed A; Powell, Michael D; Bond, Vincent C

    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.

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

    DOE PAGES

    Do Kwon, Young; Pancera, Marie; Acharya, Priyamvada; ...

    2015-06-22

    As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, 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 boundmore » by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, 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.« less

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

    SciTech Connect

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

    2015-06-22

    As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, 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. Finally, 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.

  18. 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

  19. Role of protein interactions in defining HIV-1 viral capsid shape and stability: a coarse-grained analysis.

    PubMed

    Krishna, Vinod; Ayton, Gary S; Voth, Gregory A

    2010-01-06

    Coarse-grained models of the HIV-1 CA dimer are constructed based on all-atom molecular dynamics simulations. Coarse-grained representations of the capsid shell, which is composed of approximately 1500 copies of CA proteins, are constructed and their stability is examined. A key interaction between carboxyl and hexameric amino terminal domains is shown to generate the curvature of the capsid shell. It is demonstrated that variation of the strength of this interaction for different subunits in the lattice can cause formation of asymmetric, conical-shaped closed capsid shells, and it is proposed that variations, in the structure of the additional carboxyl-amino terminal binding interface during self-assembly, are important aspects of capsid cone formation. These results are in agreement with recent structural studies of the capsid hexamer subunit, which suggest that variability in the binding interface is a cause of the differences in subunit environments that exist in a conical structure.

  20. Role of Protein Interactions in Defining HIV-1 Viral Capsid Shape and Stability: A Coarse-Grained Analysis

    PubMed Central

    Krishna, Vinod; Ayton, Gary S.; Voth, Gregory A.

    2010-01-01

    Abstract Coarse-grained models of the HIV-1 CA dimer are constructed based on all-atom molecular dynamics simulations. Coarse-grained representations of the capsid shell, which is composed of ∼1500 copies of CA proteins, are constructed and their stability is examined. A key interaction between carboxyl and hexameric amino terminal domains is shown to generate the curvature of the capsid shell. It is demonstrated that variation of the strength of this interaction for different subunits in the lattice can cause formation of asymmetric, conical-shaped closed capsid shells, and it is proposed that variations, in the structure of the additional carboxyl-amino terminal binding interface during self-assembly, are important aspects of capsid cone formation. These results are in agreement with recent structural studies of the capsid hexamer subunit, which suggest that variability in the binding interface is a cause of the differences in subunit environments that exist in a conical structure. PMID:20085716

  1. 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.

  2. Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach.

    PubMed

    Franquelim, Henri G; Gaspar, Diana; Veiga, A Salomé; Santos, Nuno C; Castanho, Miguel A R B

    2013-08-01

    Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides.

  3. 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.

  4. Productive replication and evolution of HIV-1 in ferret cells.

    PubMed

    Fadel, Hind J; Saenz, Dyana T; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary; Poeschla, Eric M

    2012-02-01

    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

  5. 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

  6. Molecular structural formulas as one-electron density and hamiltonian operators: the VIF method extended.

    PubMed

    Alia, Joseph D

    2007-03-29

    The valency interaction formula (VIF) method is given a broader and more general interpretation in which these simple molecular structural formulas implicitly include all overlaps between valence atomic orbitals even for interactions not drawn in the VIF picture. This applies for VIF pictures as one-electron Hamiltonian operators as well as VIF pictures as one-electron density operators that constitute a new implementation of the VIF method simpler in its application and more accurate in its results than previous approaches. A procedure for estimating elements of the effective charge density-bond order matrix, Pmunu, from electron configurations in atoms is presented, and it is shown how these lead to loop and line constants in the VIF picture. From these structural formulas, one finds the number of singly, doubly, and unoccupied molecular orbitals, as well as the number of molecular orbitals with energy lower, equal, and higher than -1/2Eh, the negative of the hydrogen atom's ionization energy. The VIF results for water are in qualitative agreement with MP2/6311++G3df3pd, MO energy levels where the simple VIF for water presented in the earlier literature does not agree with computed energy levels. The method presented here gives the simplest accurate VIF pictures for hydrocarbons. It is shown how VIF can be used to predict thermal barriers to chemical reactions. Insertion of singlet carbene into H2 is given as an example. VIF pictures as one-electron density operators describe the ground-state multiplicities of B2, N2, and O2 molecules and as one-electron Hamiltonian operators give the correct electronegativity trend across period two. Previous implementations of VIF do not indicate singly occupied molecular orbitals directly from the pictorial VIF rules for these examples. The direct comparison between structural formulas that represent electron density and those that represent energy is supported by comparison of a simple electronegativity scale, chiD=N/n2, with

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

    PubMed Central

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

    2016-01-01

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

  8. Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

    PubMed

    Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

    2010-12-12

    Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

  9. Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck

    PubMed Central

    Olszewski, Allison; Sato, Ken; Aron, Zachary D.; Cohen, Frederick; Harris, Aleishia; McDougall, Brenda R.; Robinson, W. Edward; Overman, Larry E.; Weiss, Gregory A.

    2004-01-01

    With current anti-HIV treatments targeting only 4 of the 15 HIV proteins, many potential viral vulnerabilities remain unexploited. We report small-molecule inhibitors of the HIV-1 protein Nef. In addition to expanding the anti-HIV arsenal, small-molecule inhibitors against untargeted HIV proteins could be used to dissect key events in the HIV lifecycle. Numerous incompletely characterized interactions between Nef and cellular ligands, for example, present a challenge to understanding molecular events during HIV progression to AIDS. Assays with phage-displayed Nef from HIVNL4-3 were used to identify a series of guanidine alkaloid-based inhibitors of Nef interactions with p53, actin, and p56lck. The guanidines, synthetic analogs of batzellidine and crambescidin natural products, inhibit the Nef–ligand interactions with IC50 values in the low micromolar range. In addition, sensitive in vivo assays for Nef inhibition are reported. Although compounds that are effective in vitro proved to be too cytotoxic for cellular assays, the reported Nef inhibitors provide proof-of-concept for disrupting a new HIV target and offer useful leads for drug development. PMID:15371598

  10. Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56lck.

    PubMed

    Olszewski, Allison; Sato, Ken; Aron, Zachary D; Cohen, Frederick; Harris, Aleishia; McDougall, Brenda R; Robinson, W Edward; Overman, Larry E; Weiss, Gregory A

    2004-09-28

    With current anti-HIV treatments targeting only 4 of the 15 HIV proteins, many potential viral vulnerabilities remain unexploited. We report small-molecule inhibitors of the HIV-1 protein Nef. In addition to expanding the anti-HIV arsenal, small-molecule inhibitors against untargeted HIV proteins could be used to dissect key events in the HIV lifecycle. Numerous incompletely characterized interactions between Nef and cellular ligands, for example, present a challenge to understanding molecular events during HIV progression to AIDS. Assays with phage-displayed Nef from HIV(NL4-3) were used to identify a series of guanidine alkaloid-based inhibitors of Nef interactions with p53, actin, and p56(lck). The guanidines, synthetic analogs of batzellidine and crambescidin natural products, inhibit the Nef-ligand interactions with IC(50) values in the low micromolar range. In addition, sensitive in vivo assays for Nef inhibition are reported. Although compounds that are effective in vitro proved to be too cytotoxic for cellular assays, the reported Nef inhibitors provide proof-of-concept for disrupting a new HIV target and offer useful leads for drug development.

  11. Interaction between HIV-1 Nef and calnexin: from modeling to small molecule inhibitors reversing HIV-induced lipid accumulation

    PubMed Central

    Hunegnaw, Ruth; Vassylyeva, Marina; Dubrovsky, Larisa; Pushkarsky, Tatiana; Sviridov, Dmitri; Anashkina, Anastasia A.; Üren, Aykut; Brichacek, Beda; Vassylyev, Dmitry; Adzhubei, Alexei A.; Bukrinsky, Michael

    2016-01-01

    Objective HIV-infected patients are at an increased risk of developing atherosclerosis, in part due to downmodulation and functional impairment of ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an endoplasmic reticulum (ER) chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting translational impact of this finding. Approach and results Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nM measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with affinity of 9.4 nM. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the NCI library of compounds, we identified a compound, 1[(7-Oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. Conclusion This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects. PMID:27470515

  12. 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

  13. 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

  14. P20A inhibits HIV-1 fusion through its electrostatic interaction with the distal region of the gp41 fusion core.

    PubMed

    Fu, Shushu; Tong, Pei; Tan, Yue; Zhu, Yun; Chen, Ying-Hua

    2015-09-01

    We previously identified an HIV-1 fusion inhibitor P20A targeting HIV-1 gp41 6-HB fusion core. Using alanine scanning mutagenesis, we investigated the effect of 6-HB surface residue mutations on the binding affinity between P20A and 6-HB. Substitution of positively or negatively charged residues in the distal region of 6-HB with alanines resulted in significant decrease or increase of its binding affinity to P20A, respectively. The 6-HB with E630K, D632K, or E634K mutation exhibited enhanced binding affinity with P20A, suggesting that P20A blocks HIV-1 fusion through electrostatic interaction with the positively charged residues in the distal region of the gp41 fusion core.

  15. 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.

  16. Vif Substitution Enables Persistent Infection of Pig-Tailed Macaques by Human Immunodeficiency Virus Type 1 ▿

    PubMed Central

    Thippeshappa, Rajesh; Polacino, Patricia; Yu Kimata, Monica T.; Siwak, Edward B.; Anderson, David; Wang, Weiming; Sherwood, Laura; Arora, Reetakshi; Wen, Michael; Zhou, Paul; Hu, Shiu-Lok; Kimata, Jason T.

    2011-01-01

    Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4+ T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease. PMID:21289128

  17. Interactive role of human immunodeficiency virus type 1 (HIV-1) clade-specific Tat protein and cocaine in blood-brain barrier dysfunction: implications for HIV-1-associated neurocognitive disorder.

    PubMed

    Gandhi, Nimisha; Saiyed, Zainulabedin M; Napuri, Jessica; Samikkannu, Thangavel; Reddy, Pichili V B; Agudelo, Marisela; Khatavkar, Pradnya; Saxena, Shailendra K; Nair, Madhavan P N

    2010-07-01

    In recent years, increasing interest has emerged to assess the human immunodeficiency virus type 1 (HIV-1) clade C viral pathogenesis due to its anticipated spread in the United States and other western countries. Previous studies suggest that clade C is less neuropathogenic than clade B; however, the underlying mechanism is poorly understood. Additionally, the interactive role of drugs of abuse such as cocaine on clade C-associated neuropathogenesis has not been reported. In the current study, we hypothesize that HIV-1 clade-specific Tat proteins exert differential effects on blood-brain barrier (BBB) integrity and cocaine further differentially aggravates the BBB dysfunction. We evaluated the effect of Tat B and Tat C and/or cocaine on the BBB integrity using an in vitro model constructed with primary human brain microvascular endothelial cells (HBMECs) and astrocytes. The BBB membrane integrity was measured by transendothelial electrical resistance (TEER) and paracellular permeability was measured by fluorescein isothiocyanate (FITC)-dextran transport assay and monocytes transmigration across the BBB. Results indicate that Tat B disrupts BBB integrity to a greater extent compared to Tat C and cocaine further differentially exacerbates the BBB dysfunction. This BBB dysfunction was associated with altered expression of tight junction proteins zona occuldens (ZO-1) and junctional adhesion molecule (JAM)-2. Thus, these results for the first time delineate the differential role of Tat B and Tat C and/or cocaine in BBB dysfunction, which may be correlated with the clade-specific differences observed in HIV-1-associated neurological disorders.

  18. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins

    PubMed Central

    Suryawanshi, Gajendra W.; Hoffmann, Alexander

    2015-01-01

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif’s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that

  19. Probability Weighted Ensemble Transfer Learning for Predicting Interactions between HIV-1 and Human Proteins

    PubMed Central

    Mei, Suyu

    2013-01-01

    Reconstruction of host-pathogen protein interaction networks is of great significance to reveal the underlying microbic pathogenesis. However, the current experimentally-derived networks are generally small and should be augmented by computational methods for less-biased biological inference. From the point of view of computational modelling, data scarcity, data unavailability and negative data sampling are the three major problems for host-pathogen protein interaction networks reconstruction. In this work, we are motivated to address the three concerns and propose a probability weighted ensemble transfer learning model for HIV-human protein interaction prediction (PWEN-TLM), where support vector machine (SVM) is adopted as the individual classifier of the ensemble model. In the model, data scarcity and data unavailability are tackled by homolog knowledge transfer. The importance of homolog knowledge is measured by the ROC-AUC metric of the individual classifiers, whose outputs are probability weighted to yield the final decision. In addition, we further validate the assumption that only the homolog knowledge is sufficient to train a satisfactory model for host-pathogen protein interaction prediction. Thus the model is more robust against data unavailability with less demanding data constraint. As regards with negative data construction, experiments show that exclusiveness of subcellular co-localized proteins is unbiased and more reliable than random sampling. Last, we conduct analysis of overlapped predictions between our model and the existing models, and apply the model to novel host-pathogen PPIs recognition for further biological research. PMID:24260261

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

    PubMed

    Mei, Suyu

    2013-01-01

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

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

    PubMed

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

    2015-03-01

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

  2. Protein–Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer

    PubMed Central

    Dodagatta-Marri, Eswari; Mitchell, Daniel A.; Pandit, Hrishikesh; Sonawani, Archana; Murugaiah, Valarmathy; Idicula-Thomas, Susan; Nal, Béatrice; Al-Mozaini, Maha M.; Kaur, Anuvinder; Madan, Taruna; Kishore, Uday

    2017-01-01

    Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein–protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1. PMID:28824609

  3. Protein-Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer.

    PubMed

    Dodagatta-Marri, Eswari; Mitchell, Daniel A; Pandit, Hrishikesh; Sonawani, Archana; Murugaiah, Valarmathy; Idicula-Thomas, Susan; Nal, Béatrice; Al-Mozaini, Maha M; Kaur, Anuvinder; Madan, Taruna; Kishore, Uday

    2017-01-01

    Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein-protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4(+) T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

  4. Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿

    PubMed Central

    Exline, C. M.; Feng, Z.; Stoltzfus, C. M.

    2008-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication. PMID:18272582

  5. 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.

  6. 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

  7. 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

  8. Double-labelled HIV-1 particles for study of virus-cell interaction

    SciTech Connect

    Lampe, Marko; Briggs, John A.G.; Endress, Thomas; Glass, Baerbel; Riegelsberger, Stefan; Kraeusslich, Hans-Georg; Lamb, Don C.; Braeuchle, Christoph; Mueller, Barbara . E-mail: Barbara_Mueller@med.uni-heidelberg.de

    2007-03-30

    Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the viral envelope with a cellular membrane. While the viral and cellular proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the viral matrix protein combined with a red label fused to the viral Vpr protein designed to distinguish between complete virions and subviral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Unusual Fusion Proteins of HIV-1

    PubMed Central

    Langer, Simon; Sauter, Daniel

    2017-01-01

    Despite its small genome size, the Human Immunodeficiency Virus 1 (HIV-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. In total, HIV-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. Expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, and equips the virus to evade or counteract host immune responses. In addition to the canonically expressed proteins, a growing number of publications describe the existence of non-canonical fusion proteins in HIV-1 infected cells. Most of them are encoded by the tat-env-rev locus. While the majority of these fusion proteins (e.g., TNV/p28tev, p186Drev, Tat1-Rev2, Tat^8c, p17tev, or Ref) are the result of alternative splicing events, Tat-T/Vpt is produced upon programmed ribosomal frameshifting, and a Rev1-Vpu fusion protein is expressed due to a nucleotide polymorphism that is unique to certain HIV-1 clade A and C strains. A better understanding of the expression and activity of these non-canonical viral proteins will help to dissect their potential role in viral replication and reveal how HIV-1 optimized the coding potential of its genes. The goal of this review is to provide an overview of previously described HIV-1 fusion proteins and to summarize our current knowledge of their expression patterns and putative functions. PMID:28119676

  14. Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors.

    PubMed

    Zhang, Zeli; Gu, Qinyong; Jaguva Vasudevan, Ananda Ayyappan; Hain, Anika; Kloke, Björn-Philipp; Hasheminasab, Sascha; Mulnaes, Daniel; Sato, Kei; Cichutek, Klaus; Häussinger, Dieter; Bravo, Ignacio G; Smits, Sander H J; Gohlke, Holger; Münk, Carsten

    2016-07-01

    Feline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3. To identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains. Here we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was

  15. Exploring the molecular mechanism of cross-resistance to HIV-1 integrase strand transfer inhibitors by molecular dynamics simulation and residue interaction network analysis.

    PubMed

    Xue, Weiwei; Jin, Xiaojie; Ning, Lulu; Wang, Meixia; Liu, Huanxiang; Yao, Xiaojun

    2013-01-28

    The rapid emergence of cross-resistance to the integrase strand transfer inhibitors (INSTIs) has become a serious problem in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Understanding the detailed molecular mechanism of INSTIs cross-resistance is therefore critical for the development of new effective therapy against cross-resistance. On the basis of the homology modeling constructed structure of tetrameric HIV-1 intasome, the detailed molecular mechanism of the cross-resistance mutation E138K/Q148K to three important INSTIs (Raltegravir (RAL, FDA approved in 2007), Elvitegravir (EVG, FDA approved in 2012), and Dolutegravir (DTG, phase III clinical trials)) was investigated by using molecular dynamics (MD) simulation and residue interaction network (RIN) analysis. The results from conformation analysis and binding free energy calculation can provide some useful information about the detailed binding mode and cross-resistance mechanism for the three INSTIs to HIV-1 intasome. Binding free energy decomposition analysis revealed that Pro145 residue in the 140s 1oop (Gly140 to Gly149) of the HIV-1 intasome had strong hydrophobic interactions with INSTIs and played an important role in the binding of INSTIs to HIV-1 intasome active site. A systematic comparison and analysis of the RIN proves that the communications between the residues in the resistance mutant is increased when compared with that of the wild-type HIV-1 intasome. Further analysis indicates that residue Pro145 may play an important role and is relevant to the structure rearrangement in HIV-1 intasome active site. In addition, the chelating ability of the oxygen atoms in INSTIs (e.g., RAL and EVG) to Mg(2+) in the active site of the mutated intasome was reduced due to this conformational change and is also responsible for the cross-resistance mechanism. Notably, the cross-resistance mechanism we proposed could give some important information for the future rational design of novel

  16. Pulsed Stable Isotope Labeling of Amino Acids in Cell Culture Uncovers the Dynamic Interactions between HIV-1 and the Monocyte-Derived Macrophage

    PubMed Central

    2011-01-01

    Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host–cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus–cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in de novo protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection. PMID:21500866

  17. HIV-1 Control by NK Cells via Reduced Interaction between KIR2DL2 and HLA-C(∗)12:02/C(∗)14:03.

    PubMed

    Lin, Zhansong; Kuroki, Kimiko; Kuse, Nozomi; Sun, Xiaoming; Akahoshi, Tomohiro; Qi, Ying; Chikata, Takayuki; Naruto, Takuya; Koyanagi, Madoka; Murakoshi, Hayato; Gatanaga, Hiroyuki; Oka, Shinichi; Carrington, Mary; Maenaka, Katsumi; Takiguchi, Masafumi

    2016-11-22

    Natural killer (NK) cells control viral infection in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands. We investigated 504 anti-retroviral (ART)-free Japanese patients chronically infected with HIV-1 and identified two KIR/HLA combinations, KIR2DL2/HLA-C(∗)12:02 and KIR2DL2/HLA-C(∗)14:03, that impact suppression of HIV-1 replication. KIR2DL2(+) NK cells suppressed viral replication in HLA-C(∗)14:03(+) or HLA-C(∗)12:02(+) cells to a significantly greater extent than did KIR2DL2(-) NK cells in vitro. Functional analysis showed that the binding between HIV-1-derived peptide and HLA-C(∗)14:03 or HLA-C(∗)12:02 influenced KIR2DL2(+) NK cell activity through reduced expression of the peptide-HLA (pHLA) complex on the cell surface (i.e., reduced KIR2DL2 ligand expression), rather than through reduced binding affinity of KIR2DL2 to the respective pHLA complexes. Thus, KIR2DL2/HLA-C(∗)12:02 and KIR2DL2/HLA-C(∗)14:03 compound genotypes have protective effects on control of HIV-1 through a mechanism involving KIR2DL2-mediated NK cell recognition of virus-infected cells, providing additional understanding of NK cells in HIV-1 infection.

  18. Design and SAR of new substituted purines bearing aryl groups at N9 position as HIV-1 Tat-TAR interaction inhibitors.

    PubMed

    Pang, Ruifang; Zhang, Chunlei; Yuan, Dekai; Yang, Ming

    2008-09-01

    Twenty-four purine derivatives bearing aryl groups at N9 position were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Ten of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells at a concentration of 30 microM, indicating effective inhibitory activities of blocking the Tat-TAR interaction. The aryl groups at N9 position affected the binding affinities between compounds and TAR RNA, showing some specificities of aryl groups to TAR RNA.

  19. 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. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  20. Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis.

    PubMed

    Ziegler, André; Blatter, Xiaochun Li; Seelig, Anna; Seelig, Joachim

    2003-08-05

    Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K

  1. Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR

    PubMed Central

    Konagaya, Yuta; Miyakawa, Rina; Sato, Masumi; Matsugami, Akimasa; Watanabe, Satoru; Hayashi, Fumiaki; Kigawa, Takanori; Nishimura, Chiaki

    2016-01-01

    To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551–9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly. PMID:27907055

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

    PubMed

    Abayev, Meital; Moseri, Adi; Tchaicheeyan, Oren; Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-05-01

    C-C chemokine receptor 5 (CCR5) serves as a co-receptor for 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 extracellular loops 1 and 3 (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 found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-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 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 that are 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. The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505. © 2015 FEBS.

  3. Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission.

    PubMed

    Pednekar, Lina; Pandit, Hrishikesh; Paudyal, Basudev; Kaur, Anuvinder; Al-Mozaini, Maha Ahmed; Kouser, Lubna; Ghebrehiwet, Berhane; Mitchell, Daniel A; Madan, Taruna; Kishore, Uday

    2016-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells capable of priming naïve T-cells. Its C-type lectin receptor, DC-SIGN, regulates a wide range of immune functions. Along with its role in HIV-1 pathogenesis through complement opsonization of the virus, DC-SIGN has recently emerged as an adaptor for complement protein C1q on the surface of immature DCs via a trimeric complex involving gC1qR, a receptor for the globular domain of C1q. Here, we have examined the nature of interaction between C1q and DC-SIGN in terms of domain localization, and implications of C1q-DC-SIGN-gC1qR complex formation on HIV-1 transmission. We first expressed and purified recombinant extracellular domains of DC-SIGN and its homologue DC-SIGNR as tetramers comprising of the entire extra cellular domain including the α-helical neck region and monomers comprising of the carbohydrate recognition domain only. Direct binding studies revealed that both DC-SIGN and DC-SIGNR were able to bind independently to the recombinant globular head modules ghA, ghB, and ghC, with ghB being the preferential binder. C1q appeared to interact with DC-SIGN or DC-SIGNR in a manner similar to IgG. Mutational analysis using single amino acid substitutions within the globular head modules showed that Tyr(B175) and Lys(B136) were critical for the C1q-DC-SIGN/DC-SIGNR interaction. Competitive studies revealed that gC1qR and ghB shared overlapping binding sites on DC-SIGN, implying that HIV-1 transmission by DCs could be modulated due to the interplay of gC1qR-C1q with DC-SIGN. Since C1q, gC1qR, and DC-SIGN can individually bind HIV-1, we examined how C1q and gC1qR modulated HIV-1-DC-SIGN interaction in an infection assay. Here, we report, for the first time, that C1q suppressed DC-SIGN-mediated transfer of HIV-1 to activated pooled peripheral blood mononuclear cells, although the globular head modules did not. The protective effect of C1q was negated by the addition of gC1qR. In fact, gC1qR enhanced DC

  4. 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

  5. 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.

  6. Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

    PubMed Central

    Pednekar, Lina; Pandit, Hrishikesh; Paudyal, Basudev; Kaur, Anuvinder; Al-Mozaini, Maha Ahmed; Kouser, Lubna; Ghebrehiwet, Berhane; Mitchell, Daniel A.; Madan, Taruna; Kishore, Uday

    2016-01-01

    Dendritic cells (DCs) are the most potent antigen-presenting cells capable of priming naïve T-cells. Its C-type lectin receptor, DC-SIGN, regulates a wide range of immune functions. Along with its role in HIV-1 pathogenesis through complement opsonization of the virus, DC-SIGN has recently emerged as an adaptor for complement protein C1q on the surface of immature DCs via a trimeric complex involving gC1qR, a receptor for the globular domain of C1q. Here, we have examined the nature of interaction between C1q and DC-SIGN in terms of domain localization, and implications of C1q–DC-SIGN-gC1qR complex formation on HIV-1 transmission. We first expressed and purified recombinant extracellular domains of DC-SIGN and its homologue DC-SIGNR as tetramers comprising of the entire extra cellular domain including the α-helical neck region and monomers comprising of the carbohydrate recognition domain only. Direct binding studies revealed that both DC-SIGN and DC-SIGNR were able to bind independently to the recombinant globular head modules ghA, ghB, and ghC, with ghB being the preferential binder. C1q appeared to interact with DC-SIGN or DC-SIGNR in a manner similar to IgG. Mutational analysis using single amino acid substitutions within the globular head modules showed that TyrB175 and LysB136 were critical for the C1q–DC-SIGN/DC-SIGNR interaction. Competitive studies revealed that gC1qR and ghB shared overlapping binding sites on DC-SIGN, implying that HIV-1 transmission by DCs could be modulated due to the interplay of gC1qR-C1q with DC-SIGN. Since C1q, gC1qR, and DC-SIGN can individually bind HIV-1, we examined how C1q and gC1qR modulated HIV-1–DC-SIGN interaction in an infection assay. Here, we report, for the first time, that C1q suppressed DC-SIGN-mediated transfer of HIV-1 to activated pooled peripheral blood mononuclear cells, although the globular head modules did not. The protective effect of C1q was negated by the addition of gC1qR. In fact, gC1qR enhanced

  7. The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

    PubMed Central

    2011-01-01

    Background Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear. Results Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA. Conclusions For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the

  8. Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2-V3 interaction and modulates neutralization sensitivity.

    PubMed

    Cimbro, Raffaello; Gallant, Thomas R; Dolan, Michael A; Guzzo, Christina; Zhang, Peng; Lin, Yin; Miao, Huiyi; Van Ryk, Donald; Arthos, James; Gorshkova, Inna; Brown, Patrick H; Hurt, Darrell E; Lusso, Paolo

    2014-02-25

    Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4(+) T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.

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

    PubMed

    Ziegler, André; Seelig, Joachim

    2004-01-01

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

  10. Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F

    PubMed Central

    Albin, John S.; Anderson, John S.; Johnson, Jeffrey R.; Harjes, Elena; Matsuo, Hiroshi; Krogan, Nevan J.; Harris, Reuben S.

    2013-01-01

    APOBEC3F and APOBEC3G are DNA cytosine deaminases that potently restrict Human Immunodeficiency Virus-type 1 replication when the virus is deprived of its accessory protein Vif. Vif counteracts these restriction factors by recruiting APOBEC3F and APOBEC3G to an E3 ubiquitin ligase complex that mediates their polyubiquitination and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream ubiquitin acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in APOBEC3G and proposed an antiparallel model of APOBEC3G interaction with the Vif-E3 ubiquitin ligase complex wherein Vif binding at one terminus of APOBEC3G orients the opposite terminus for polyubiquitination [Iwatani Y, et al. (2009) PNAS 106(46):19539–19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in APOBEC3F and used a complementary, unbiased proteomic approach to identify ubiquitin acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant ubiquitin acceptor sites in both APOBEC3F and APOBEC3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyubiquitination of APOBEC3F and APOBEC3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 ubiquitin ligase complex and diminish enthusiasm for the amenability of APOBEC3 ubiquitin acceptor sites to therapeutic intervention. PMID:23318957

  11. 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.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

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

    2011-10-01

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

  14. HIV-1 Prevention for HIV-1 Serodiscordant Couples

    PubMed Central

    Curran, Kathryn; Baeten, Jared M.; Coates, Thomas J.; Kurth, Ann; Mugo, Nelly R.

    2013-01-01

    A substantial proportion of HIV-1-infected individuals in sub-Saharan Africa are in stable relationships with HIV-1-uninfected partners, and HIV-1 serodiscordant couples thus represent an important target population for HIV-1 prevention. Couple-based HIV-1 testing and counseling facilitates identification of HIV-1 serodiscordant couples, counseling about risk reduction, and referrals to HIV-1 treatment, reproductive health services, and support services. Maximizing HIV-1 prevention for HIV-1 serodiscordant couples requires a combination of strategies, including counseling about condoms, sexual risk, fertility, contraception, and the clinical and prevention benefits of antiretroviral therapy (ART) for the HIV-1-infected partner; provision of clinical care and ART for the HIV-1-infected partner; antenatal care and services to prevent mother to child transmission for HIV-1- infected pregnant women; male circumcision for HIV-1-uninfected men; and, pending guidelines and demonstration projects, oral pre-exposure prophylaxis (PrEP) for HIV-1-uninfected partners. PMID:22415473

  15. 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

  16. 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

  17. 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

  18. Lack of Pharmacokinetic Interaction between Amdoxovir and Reduced- and Standard-Dose Zidovudine in HIV-1-Infected Individuals▿

    PubMed Central

    Hurwitz, Selwyn J.; Asif, Ghazia; Fromentin, Emilie; Tharnish, Phillip M.; Schinazi, Raymond F.

    2010-01-01

    Amdoxovir (AMDX) inhibits HIV-1 containing the M184V/I mutation and is rapidly absorbed and deaminated to its active metabolite, β-d-dioxolane guanosine (DXG). DXG is synergistic with zidovudine (ZDV) in HIV-1-infected primary human lymphocytes. A recent in silico pharmacokinetic (PK)/enzyme kinetic study suggested that ZDV at 200 mg twice a day (b.i.d.) may reduce toxicity without compromising efficacy relative to the standard 300-mg b.i.d. dose. Therefore, an intense PK clinical study was conducted using AMDX/placebo, with or without ZDV, in 24 subjects randomized to receive oral AMDX at 500 mg b.i.d., AMDX at 500 mg plus ZDV at 200 or 300 mg b.i.d., or ZDV at 200 or 300 mg b.i.d. for 10 days. Full plasma PK profiles were collected on days 1 and 10, and complete urine sampling was performed on day 9. Plasma and urine concentrations of AMDX, DXG, ZDV, and ZDV-5′-O-glucuronide (GZDV) were measured using a validated liquid chromatography-tandem mass spectrometry method. Data were analyzed using noncompartmental methods, and multiple comparisons were performed on the log-transformed parameters, at steady state. Coadministration of AMDX with ZDV did not significantly change either of the plasma PK parameters or percent recovery in the urine of AMDX, DXG, or ZDV/GZDV. Larger studies with AMDX/ZDV, with a longer duration, are warranted. PMID:20038617

  19. 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

    2016-06-02

    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.

  20. HIV-1 envelope glycoprotein

    DOEpatents

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  1. 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…

  2. Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.

    PubMed

    Pan, Ting; He, Xin; Chen, Bing; Chen, Hui; Geng, Guannan; Luo, Haihua; Zhang, Hui; Bai, Chuan

    2015-05-05

    Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

  3. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    PubMed

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  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

    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.

  5. Identifying interaction sites in "recalcitrant" proteins: predicted protein and RNA binding sites in rev proteins of HIV-1 and EIAV agree with experimental data.

    PubMed

    Terribilini, Michael; Lee, Jae-Hyung; Yan, Changhui; Jernigan, Robert L; Carpenter, Susan; Honavar, Vasant; Dobbs, Drena

    2006-01-01

    Protein-protein and protein nucleic acid interactions are vitally important for a wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses. We have developed machine learning approaches for predicting which amino acids of a protein participate in its interactions with other proteins and/or nucleic acids, using only the protein sequence as input. In this paper, we describe an application of classifiers trained on datasets of well-characterized protein-protein and protein-RNA complexes for which experimental structures are available. We apply these classifiers to the problem of predicting protein and RNA binding sites in the sequence of a clinically important protein for which the structure is not known: the regulatory protein Rev, essential for the replication of HIV-1 and other lentiviruses. We compare our predictions with published biochemical, genetic and partial structural information for HIV-1 and EIAV Rev and with our own published experimental mapping of RNA binding sites in EIAV Rev. The predicted and experimentally determined binding sites are in very good agreement. The ability to predict reliably the residues of a protein that directly contribute to specific binding events--without the requirement for structural information regarding either the protein or complexes in which it participates--can potentially generate new disease intervention strategies.

  6. 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.

  7. Design of improved protein inhibitors of HIV-1 cell entry: Optimization of electrostatic interactions at the binding interface.

    PubMed

    Green, David F; Tidor, Bruce

    2005-09-01

    Continuum electrostatic methods are a powerful tool for the analysis and design of biomolecular complexes, with methodologies that allow for the detailed analysis of the electrostatic contributions to binding affinities and procedures for computing the properties of electrostatically optimal ligands. We have applied these methods to the design of improved inhibitors of HIV-1 cell entry. HIV infection of a cell requires viral-cell membrane fusion, an event partially driven by a large-scale conformational change in the viral membrane glycoprotein gp41. This transformation involves the docking of a helix from the C-terminal region of three gp41 chains against a pre-formed trimeric-coiled coil; several protein constructs that inhibit membrane fusion act by binding to an isolated C-terminal helix and blocking the formation of the fusogenic structure. A detailed analysis of the electrostatic contributions to the binding of one such inhibitor (5-Helix) to a C-terminal helix was performed using the X-ray crystal structure of the core of the HIV-1 gp41 ectodomain as a structural model, and several residues on 5-Helix that make substantial contributions to binding, both favorable and unfavorable, were identified. An electrostatic affinity optimization methodology was applied to the side chains of 5-Helix, with the results showing that significant improvements in binding affinity are possible if the electrostatic contributions to the binding free energy are optimized. Several mutations accessible by experimental methods are suggested, with calculated improvements in binding affinity of as much as 500-fold and greater. Copyright 2005 Wiley-Liss, Inc.

  8. 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.

  9. Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

    PubMed

    Yoshikawa, Rokusuke; Takeuchi, Junko S; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Kimura, Yuichi; Ren, Fengrong; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

    2017-06-01

    The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease.IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3

  10. The HIV-1 Nef protein interacts with two components of the 40S small ribosomal subunit, the RPS10 protein and the 18S rRNA.

    PubMed

    Abbas, Wasim; Dichamp, Isabelle; Herbein, Georges

    2012-07-10

    Human immunodeficiency virus type 1 (HIV-1) Nef-encoded protein plays key functions at almost all stages of the viral life cycle, but its role in translation is largely unknown. To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay. We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes. Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.

  11. Studies on the intermolecular forces involved in the antibody-antigen interactions, using V3 synthetic peptides and sera from HIV1 seropositive patients.

    PubMed

    Măgureanu, C G; Diaconu, C; Alexandrescu, R; Tirdei, G; Cernescu, C

    1994-01-01

    The nature of physical forces responsible for the antibody-antigen (Ab-Ag) reaction was analyzed in an original system, represented by synthetic peptides derived from the V3 consensus sequences of some HIV1 subtypes gp 120 and HIV1 positive human serum. For locating antigenic determines, flexibility, hydrophilicity and hydrophobicity profiles of the V3 peptides were analysed. The hydrophilicity indicates that V3 apex borders are involved in the first stage of the reaction. The flexibility and hydrophobicity suggest that the apex of the V3 loop (GPGR/Q) is involved in the stabilization of the complex by hydrophobic interactions. Further, we followed up the influence of the dielectric constant and of the pH upon the forces established between Ab and Ag. Modifications in the dielectric constant and pH reveal a significant contribution of electrostatic and van der Waals forces in securing the intermolecular complementarity. D2O produces the highest augmentation of the antibody affinity for the most hydrophilic peptides, while a very slight one was recorded for the most hydrophobic sequence. A high affinity of antibodies for the peptides MN, R and Z was registered at an acid pH, when their His residue was protonated. On the contrary, no influence was recorded in the case of the peptide A, which does not contain any His residue.

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

    PubMed Central

    Sayer, Jane M.; Louis, John M.

    2008-01-01

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

  13. Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin.

    PubMed

    Sayer, Jane M; Louis, John M

    2009-05-15

    The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.

  14. Bispecific Engineered Antibody Domains (Nanoantibodies) That Interact Noncompetitively with an HIV-1 Neutralizing Epitope and FcRn

    PubMed Central

    Gong, Rui; Wang, Yanping; Ying, Tianlei; Dimitrov, Dimiter S.

    2012-01-01

    Libraries based on an isolated human immunoglobulin G1 (IgG1) constant domain 2 (CH2) have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd) (m01s) with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn) (Gong et al, JBC, 2009 and 2011). We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3)) onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences) by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn) can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs). Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics. PMID:22879932

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

    PubMed

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

    2013-07-17

    HIV-1 resulted from cross-species transmission of SIVcpz, a simian immunodeficiency virus that naturally infects chimpanzees. SIVcpz, in turn, is a recombinant between two SIV lineages from Old World monkeys. Lentiviral interspecies transmissions are partly driven by the evolution and capacity of viral accessory genes, such as vpx, vpr, and vif, to antagonize host antiviral factors, such as SAMHD1 and the APOBEC3 proteins. We show that vpx, which in other lentiviruses antagonizes SAMHD1, was deleted during the creation of SIVcpz. This genomic deletion resulted in the reconstruction of the overlapping vif gene by "overprinting," creating a unique vif that overlaps in its 3' end with the vpr gene and can antagonize hominid APOBEC3s. Moreover, passage of SIVs through chimpanzees facilitated the subsequent adaptation of HIV-1 to humans. Thus, HIV-1 originated through a series of gene loss and adaptation events that generated its chimpanzee precursor and lowered the species barrier to human infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses.

    PubMed

    Kirchhoff, Frank

    2010-07-22

    Retroviruses have evolved effective strategies to evade the host immune response, such as high variability and latent infection. In addition, primate lentiviruses, such as HIV-1, have acquired several "accessory" genes that antagonize antiviral host restriction factors and facilitate viral immune evasion, thereby allowing continuous and efficient viral replication despite apparently strong innate and acquired immune responses. Here, I summarize some of our current knowledge on the acquisition and function of the viral vif, vpr, vpu, and nef genes, with a particular focus on the evolution and specific properties of pandemic HIV-1 strains that may contribute to their efficient spread and high virulence. Copyright 2010 Elsevier Inc. All rights reserved.

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

    PubMed Central

    Campbell, Edward M.; Hope, Thomas J.

    2016-01-01

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

  18. 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.

  19. 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.

  20. 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.

  1. Possible allosteric interactions of monoindazole-substituted P2 cyclic urea analogues with wild-type and mutant HIV-1 protease

    NASA Astrophysics Data System (ADS)

    Garg, Rajni; Bhhatarai, Barun

    2008-10-01

    Our ongoing efforts to understand the difference in the binding pattern of HIV-1 protease inhibitor (HIVPI) with the wild-type and mutant HIV-1 protease (HIVPR) and to provide mechanistic insight are continued further. We report here the results of a recent quantitative structure-activity relationship (QSAR) study on monoindazole-substituted P2 analogues of cyclic urea HIVPIs. The QSAR models revealed an inverted parabolic relationship between biological activity and calculated molar refractivity (CMR). That is, biological activity first decreases with increase in CMR and at a certain minimum point (inversion point) it suddenly changes and increases with further increase in CMR. CMR is a measure of volume-dependent-polarizability and is an indication of the polar interactions between ligand and receptor. The results seem to be best rationalized by larger molecules inducing a change in a receptor unit that allows for a new mode of interaction. Similar QSAR models were also observed for the biological activity of these molecules tested against a panel of mutant viruses including mutant strains with single amino acid substitution (I84V), double amino acid substitutions (I84V/V82F), and multiple amino acid changes corresponding to mutations observed in clinical isolates of patients treated with Ritonavir®. Interestingly the inversion points for these mutant strains were found larger than for wild-type. The subtle but significant difference in the inversion point indicates change in the shape and size of the binding pocket. Earlier QSAR studies have shown that the correlation of biological activity with an inverted parabola is an indicative of the `allosteric interaction' of the ligands with the receptor. This report presents a detail analysis of these observations.

  2. Effects of the Nature and Concentration of Salt on the Interaction of the HIV-1 Nucleocapsid Protein with SL3 RNA§

    PubMed Central

    Athavale, Shreyas S.; Ouyang, Wei; McPike, Mark P.; Hudson, Bruce S.

    2010-01-01

    The mature nucleocapsid protein of HIV-1, NCp7, and the NC-domains in gag-precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC-domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The specific interactions from NCp7-binding to loop bases of SL3 produce 1:1 complexes in solutions that have [NaCl] at or above 0.2 M, while the electrostatic interactions can dominate at and below 0.15 M NaCl, leading to complexes that have mainly 1:2 RNA:protein. Persistent, non-equilibrium mixtures of 1:1 and protein-excess complexes can exist at these lower salt concentrations, where the distribution of complexes depends on the order of addition of RNA and protein. Adding salt causes rapid rearrangement of metastable multi-protein complexes to 1:1. The stability of complexes is also affected by the nature of the added salt, with 0.018 M MgCl2 and 0.200 M added NaCl producing the same Kd (21 ± 2 nM); acetate ion stabilizes the 1:1 complex by more than a factor of two compared to the same concentration of chloride ion. Maintaining a salt concentration of 0.2 M NaCl or 18 mM MgCl2 is sufficient for experiments to distinguish drug candidates that disrupt the specific SL3-NCp7 interactions in the 1:1 complex. PMID:20359247

  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. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. 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

  6. 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

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

    PubMed

    Tsvetkov, Vladimir B; Serbin, Alexander V

    2014-06-01

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

  8. Asymmetric synthesis of water-soluble analogues of galactosylceramide, an HIV-1 receptor: new tools to study virus-glycolipid interactions.

    PubMed

    Villard, Renaud; Hammache, Djilali; Delapierre, Guillaume; Fotiadu, Frédéric; Buono, Gérard; Fantini, Jacques

    2002-06-03

    Galactosylceramide (GalCer) is a glycosphingolipid (GSL) receptor that allows HIV-1 infection of CD4-negative cells from neural and intestinal tissues. A water-soluble analogue of GalCer that features its polar head and the characteristic galactose-ceramide linkage but lacks the carbohydrate chains was prepared as a single enantiomer from (S)-serine. This analogue was not recognized in binding tests with the HIV-1 surface envelope glycoprotein gp120 in solution, which revealed the crucial importance of the ceramide alkyl chains. Two series of water-soluble GalCer analogues that contained either a hexanoic or a decanoic acyl unit and a saturated nine-carbon sphingosine moiety were designed by using molecular modeling results from natural GSLs and analogues with truncated alkyl chains. The longer chain compounds exhibit the characteristic fundamental conformation of GalCer. Seven analogues were prepared from Garner's aldehyde according to a straightforward and efficient asymmetric synthesis. All of these compounds proved to be water soluble but did not bind to gp120 in a solid-phase binding assay. These analogues were thus tested by using surface pressure measurements on a monomolecular film of GalCer, which served as a model of the plasma membrane. The incorporation of analogues very similar to GalCer into a GalCer monolayer prevented the insertion of gp120, whereas a structurally different derivative was not active. Based on these data, the molecular bases for recognition of GSLs by gp120 were elucidated. The essential importance of the GSL conformation in the primary interaction event and the crucial role of the alkyl chains of the ceramide moiety in the secondary interactions and the insertion process were clearly established.

  9. 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

  10. 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.

  11. High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

    PubMed

    Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

    2015-04-24

    In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

  12. HIV-1 vaccines

    PubMed Central

    Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

    2014-01-01

    The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

  13. Elucidation of New Binding Interactions with the Tumor Susceptibility Gene 101 (Tsg101) Protein Using Modified HIV-1 Gag-p6 Derived Peptide Ligands.

    PubMed

    Kim, Sung-Eun; Liu, Fa; Im, Young Jun; Stephen, Andrew G; Fivash, Matthew J; Waheed, Abdul A; Freed, Eric O; Fisher, Robert J; Hurley, James H; Burke, Terrence R

    2011-05-12

    Targeting protein-protein interactions is gaining greater recognition as an attractive approach to therapeutic development. An example of this may be found with the human cellular protein encoded by the tumor susceptibility gene 101 (Tsg101), where interaction with the p6 C-terminal domain of the nascent viral Gag protein is required for HIV-1 particle budding and release. This association of Gag with Tsg101 is highly dependent on a "Pro-Thr-Ala-Pro" ("PTAP") peptide sequence within the p6 protein. Although p6-derived peptides offer potential starting points for developing Tsg101-binding inhibitors, the affinities of canonical peptides are outside the useful range (K(d) values greater than 50 μM). Reported herein are crystal structures of Tsg101 in complex with two structurally-modified PTAP-derived peptides. This data define new regions of ligand interaction not previously identified with canonical peptide sequences. This information could be highly useful in the design of Tsg101-binding antagonists.

  14. 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.

  15. Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity▿

    PubMed Central

    Dang, Ying; Davis, Roderick W.; York, Ian A.; Zheng, Yong-Hui

    2010-01-01

    The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, 81LGxGxSIEW89 and 171EDRWN175. Within the 81LGxGxSIEW89 sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues 86SIEW89 affect Vif binding to A3F, A3G, and Cul5. Accordingly, this 81LGxGxSIEW89 sequence was designated the 81LGxGxxIxW89 domain. Within the 171EDRWN175 sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated 171EDRW174. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, 81LGxGxxIxW89 and 171EDRW174 are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication. PMID:20335268

  16. HERV-K–specific T cells eliminate diverse HIV-1/2 and SIV primary isolates

    PubMed Central

    Jones, R. Brad; Garrison, Keith E.; Mujib, Shariq; Mihajlovic, Vesna; Aidarus, Nasra; Hunter, Diana V.; Martin, Eric; John, Vivek M.; Zhan, Wei; Faruk, Nabil F.; Gyenes, Gabor; Sheppard, Neil C.; Priumboom-Brees, Ingrid M.; Goodwin, David A.; Chen, Lianchun; Rieger, Melanie; Muscat-King, Sophie; Loudon, Peter T.; Stanley, Cole; Holditch, Sara J.; Wong, Jessica C.; Clayton, Kiera; Duan, Erick; Song, Haihan; Xu, Yang; SenGupta, Devi; Tandon, Ravi; Sacha, Jonah B.; Brockman, Mark A.; Benko, Erika; Kovacs, Colin; Nixon, Douglas F.; Ostrowski, Mario A.

    2012-01-01

    The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1–infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)–specific CD8+ T cells obtained from HIV-1–infected human subjects responded to HIV-1–infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)–specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)–specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)–targeted HIV-1 vaccines and immunotherapeutics. PMID:23143309

  17. Lipid interactions and angle of approach to the HIV-1 viral membrane of broadly neutralizing antibody 10E8: Insights for vaccine and therapeutic design

    PubMed Central

    Irimia, Adriana; Sarkar, Anita; Schiffner, Torben; Tingle, Ryan; Adachi, Yumiko; Deller, Marc C.; Burton, Dennis R.

    2017-01-01

    Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design. PMID:28225819

  18. Design of HIV-1 Protease Inhibitors with Pyrrolidinones and Oxazolidinones as Novel P1’-Ligands to Enhance Backbone-binding interactions with Protease: Synthesis, Biological Evaluation and Protein-ligand X-ray Studies

    PubMed Central

    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

    2009-01-01

    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′-ligands. These ligands are designed to interact with Gly-27′ carbonyl and Arg-8 side chain in the S1′-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 an (S)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. Inhibitor 19b 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′-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1’ and S2-subsites of HIV-1 protease. PMID:19473017

  19. 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.

  20. Evidence of at least two introductions of HIV-1 in the Amerindian Warao population from Venezuela.

    PubMed

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

    2012-01-01

    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. 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. 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.

  1. HIV-1 Reverse Transcription

    PubMed Central

    Hu, Wei-Shau; Hughes, Stephen H.

    2012-01-01

    Reverse transcription and integration are the defining features of the Retroviridae; the common name “retrovirus” derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT. PMID:23028129

  2. Differential binding of the HIV-1 envelope to phosphatidylserine receptors.

    PubMed

    Gu, Linlin; Sims, Brian; Krendelchtchikov, Alexandre; Tabengwa, Edlue; Matthews, Qiana L

    2017-10-01

    Prior work has shown that the HIV-1 envelope of the human immunodeficiency virus (HIV) interacts directly with T-cell immunoglobulin mucin (TIM) family proteins. Herein, we demonstrate that HIV-1 envelope glycoproteins from varying HIV-1 clades bind differentially to TIM proteins and functionally similar proteins acting as phosphatidylserine (PtdSer) receptors. Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology, we show that lysate containing HIV-1 envelope and recombinant HIV-1 envelope glycoproteins bind TIM-4 and advanced glycosylation end product-specific receptor (AGER). The complex binding of HIV-1 UG21 gp140 to TIM-4 or AGER suggests a biphasic interaction with these proteins. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. 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

  4. Subunit-selective mutational analysis and tissue culture evaluations of the interactions of the E138K and M184I mutations in HIV-1 reverse transcriptase.

    PubMed

    Xu, Hong-Tao; Oliveira, Maureen; Quashie, Peter K; McCallum, Matthew; Han, Yingshan; Quan, Yudong; Brenner, Bluma G; Wainberg, Mark A

    2012-08-01

    The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both

  5. 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.

  6. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24522437

  8. Crystallographic Study of a Novel Sub-Nanomolar Inhibitor Provides Insight on the Binding Interactions of Alkenyldiarylmethanes with Human Immunodeficiency Virus-1 (HIV-1) Reverse Transcriptase†

    PubMed Central

    Cullen, Matthew D.; Ho, William C.; Bauman, Joseph D.; Das, Kalyan; Arnold, Eddy; Hartman, Tracy L.; Watson, Karen M.; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

    2009-01-01

    Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the backside of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC50 ≤ nM). ADAM 4 was designed to be hydrolytically stable in blood plasma, and an investigation of its hydrolysis in rat plasma demonstrated it has a significantly prolonged half-life in comparison to ADAM lead compounds 1 and 2. PMID:19775161

  9. Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

    PubMed

    Mantri, Chinmay K; Mantri, Jyoti V; Pandhare, Jui; Dash, Chandravanu

    2014-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.

  10. Methamphetamine Inhibits HIV-1 Replication in CD4+ T Cells by Modulating Anti–HIV-1 miRNA Expression

    PubMed Central

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

    2015-01-01

    Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4+ T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4+ T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti–HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4+ T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis. PMID:24434277

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

    PubMed

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

    2012-06-08

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

  12. S100A9 protein is a novel ligand for the CD85j receptor and its interaction is implicated in the control of HIV-1 replication by NK cells

    PubMed Central

    2013-01-01

    Background The reportedly broad expression of CD85j across different immune cell types suggests an importance for this molecule in the human immune system. Previous reports have shown that this receptor interacts with several HLA class-I molecules, as well as with some viral proteins. We have demonstrated that the subset of CD85j + Natural Killer (NK) cells efficiently controls human immunodeficiency virus type 1 (HIV-1) replication in monocyte-derived dendritic cells (MDDC) in vitro and this led us to hypothesize that the CD85j + NK cell-mediated anti-HIV activity in MDDC is specifically dependent on the interaction between the CD85j receptor and unknown non-HLA class-I ligand(s). Results In this study, we focused our efforts on the identification of these non-described ligands for CD85j. We found that the CD85j receptor interacts with a calcium-binding proteins of the S100 family; namely, S100A9. We further demonstrated that HIV-1 infection of MDDC induces a modulation of S100A9 expression on surface of the MDDC, which potentially influences the anti-HIV-1 activity of human NK cells through a mechanism involving CD85j ligation. Additionally, we showed that stimulation of NK cells with exogenous S100A9 enhances the control of HIV-1 infection in CD4+ T cells. Conclusions Our data show that S100A9 protein, through ligation with CD85j, can stimulate the anti-HIV-1 activity of NK cells. PMID:24156302

  13. 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

  14. Candida albicans Delays HIV-1 Replication in Macrophages

    PubMed Central

    Rodriguez Rodrigues, Christian; Remes Lenicov, Federico; Jancic, Carolina; Sabatté, Juan; Cabrini, Mercedes; Ceballos, Ana; Merlotti, Antonela; Gonzalez, Heidi; Ostrowski, Matías; Geffner, Jorge

    2013-01-01

    Macrophages are one of the most important HIV-1 target cells. Unlike CD4+ T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy. PMID:24009706

  15. The ESCRT pathway and HIV-1 budding.

    PubMed

    Usami, Yoshiko; Popov, Sergei; Popova, Elena; Inoue, Michio; Weissenhorn, Winfried; G Göttlinger, Heinrich

    2009-02-01

    HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPX(n)L (Leu-Tyr-Pro-Xaa(n)-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

  16. 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

  17. 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

  18. 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.

  19. Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

    PubMed

    Emery, Ann; Zhou, Shuntai; Pollom, Elizabeth; Swanstrom, Ronald

    2017-03-15

    Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of

  20. 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

  1. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    PubMed

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  2. HTLV-1 Tax activates HIV-1 transcription in latency models.

    PubMed

    Geddes, Victor Emmanuel Viana; José, Diego Pandeló; Leal, Fabio E; Nixon, Douglas F; Tanuri, Amilcar; Aguiar, Renato Santana

    2017-04-01

    HIV-1 latency is a major obstacle to HIV-1 eradication. Coinfection with HTLV-1 has been associated with faster progression to AIDS. HTLV-1 encodes the transactivator Tax which can activate both HTLV-1 and HIV-1 transcription. Here, we demonstrate that Tax activates HIV transcription in latent CD4(+) T cells. Tax promotes the activation of P-TEFb, releasing CDK9 and Cyclin T1 from inactive forms, promoting transcription elongation and reactivation of latent HIV-1. Tax mutants lacking interaction with the HIV-1-LTR promoter were not able to activate P-TEFb, with no subsequent activation of latent HIV. In HIV-infected primary resting CD4(+) T cells, Tax-1 reactivated HIV-1 transcription up to five fold, confirming these findings in an ex vivo latency model. Finally, our results confirms that HTLV-1/Tax hijacks cellular partners, promoting HIV-1 transcription, and this interaction should be further investigated in HIV-1 latency studies in patients with HIV/HTLV-1 co-infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Prediction of molecular properties including symmetry from quantum-based molecular structural formulas, VIF.

    PubMed

    Alia, Joseph D; Vlaisavljevich, Bess; Abbot, Matthew; Warneke, Hallie; Mastin, Tyson

    2008-10-09

    Structurally covariant valency interaction formulas, VIF, gain chemical significance by comparison with resonance structures and natural bond orbital, NBO, bonding schemes and at the same time allow for additional prediction such as symmetry of ring systems and destabilization of electron pairs with respect to reference energy of -1/2 Eh. Comparisons are based on three chemical interpretations of Sinanoğlu's theory of structural covariance: (1) sets of structurally covariant quantum structural formulas, VIF, are interpreted as the same quantum operator represented in linearly related basis frames; (2) structurally covariant VIF pictures are interpreted as sets of molecular species with similar energy; and (3) the same VIF picture can be interpreted as different quantum operators, one-electron density or Hamiltonian; for example. According to these three interpretations, bond pair, lone pair, and free radical electrons understood in terms of a localized orbital representation are recognized as having energies above, below, or equal to a predetermined reference, frequently-1/2 Eh. The probable position of electron pairs and radical electrons is predicted. The selectivity of concerted ring closures in allyl anion and cation is described. Symmetries of conjugated ring systems are predicted according to their numbers of pi-electrons and spin-multiplicity. The pi-distortivity of benzene is predicted.The 3c/2e- H-bridging bonds in diborane are derived in a natural way according to the notion that the bridging bonds will have delocalizing interactions between them consistent with results of the NBO method. Key chemical bonding motifs are described using VIF. These include 2c/1e-, 2c/2e-, 2c/3e-, 3c/2e-, 3c/3e-,3c/4e-, 4n antiaromatic, and 4n+2 aromatic bonding systems. Some common organic functional groups are represented as VIF pictures and because these pictures can be interpreted simultaneously as one-electron density and Hamiltonian operators, the valence shell

  4. Mutations in the nef and vif genes associated with progression to AIDS in elite controller and slow-progressor patients.

    PubMed

    Cruz, Nadia V G; Amorim, Raquel; Oliveira, Fátima E; Speranza, Francisco A C; Costa, Luciana J

    2013-04-01

    Progression towards AIDS can vary from 5 to 10 years from the establishment of the primary infection by HIV-1 to more than 10 years in the complete absence of antiretroviral therapy. Several factors can contribute to the outcome of HIV infection, including host genetic and viral replicating characteristics. Historically, nef-deleted viral genomes have been associated with disease progression. Therefore, the lentiviral Nef protein is regarded as a progression factor. The objective of this work was to characterize the nef gene from a group of treatment naive patients infected with HIV-1 for more than 10 years. These patients were classified as long-term non-progressors, elite controller, and slow-progressors according to clinical and laboratorial data. A premature stop codon within the nef gene leading to the expression of a truncated peptide was observed on samples from the elite controller patient. For the slow-progressor patients, several degrees of deletions at the C-terminal of Nef were observed predicting a loss of function of this protein. The vif gene was characterized for these patients and a rare mutation that predicts a miss localization of the Vif protein to the nucleus of infected cells that could prevent its function as an APOBEC neutralization factor was also observed. These data indicate the importance of the HIV accessory proteins as factors that contribute to the outcome of AIDS.

  5. Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination.

    PubMed

    Herate, Cecile; Vigne, Clarisse; Guenzel, Carolin A; Lambele, Marie; Rouyez, Marie-Christine; Benichou, Serge

    2016-04-12

    Through incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism. We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.

  6. Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

    PubMed Central

    Nomaguchi, Masako; Yokoyama, Masaru; Kono, Ken; Nakayama, Emi E.; Shioda, Tatsuo; Doi, Naoya; Fujiwara, Sachi; Saito, Akatsuki; Akari, Hirofumi; Miyakawa, Kei; Ryo, Akihide; Ode, Hirotaka; Iwatani, Yasumasa; Miura, Tomoyuki; Igarashi, Tatsuhiko

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells. PMID:23966385

  7. Hyperthermia stimulates HIV-1 replication.

    PubMed

    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.

  8. 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

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

    PubMed

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

    2016-01-01

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

  10. Tobacco smoking effect on HIV-1 pathogenesis: role of cytochrome P450 isozymes

    PubMed Central

    Ande, Anusha; McArthur, Carole; Kumar, Anil; Kumar, Santosh

    2014-01-01

    Introduction Tobacco smoking is highly prevalent among the HIV-1-infected population. In addition to diminished immune response, smoking has been shown to increase HIV-1 replication and decrease response to antiretroviral therapy, perhaps through drug–drug interaction. However, the mechanism by which tobacco/nicotine increases HIV-1 replication and mediates drug–drug interaction is poorly understood. Areas covered In this review, the authors discuss the effects of smoking on HIV-1 pathogenesis. Since they propose a role for the cytochrome P450 (CYP) pathway in smoking-mediated HIV-1 pathogenesis, the authors briefly converse the role of CYP enzymes in tobacco-mediated oxidative stress and toxicity. Finally, the authors focus on the role of CYP enzymes, especially CYP2A6, in tobacco/nicotine metabolism and oxidative stress in HIV-1 model systems monocytes/macrophages, lymphocytes, astrocytes and neurons, which may be responsible for HIV-1 pathogenesis. Expert opinion Recent findings suggest that CYP-mediated oxidative stress is a novel pathway that may be involved in smoking-mediated HIV-1 pathogenesis, including HIV-1 replication and drug–drug interaction. Thus, CYP and CYP-associated oxidative stress pathways may be potential targets to develop novel pharmaceuticals for HIV-1-infected smokers. Since HIV-1/TB co-infections are common, future study involving interactions between antiretroviral and antituberculosis drugs that involve CYP pathways would also help treat HIV-1/TB co-infected smokers effectively. PMID:23822755

  11. Interaction between the cytoplasmic domains of HIV-1 Vpu and CD4: role of Vpu residues involved in CD4 interaction and in vitro CD4 degradation.

    PubMed

    Margottin, F; Benichou, S; Durand, H; Richard, V; Liu, L X; Gomas, E; Benarous, R

    1996-09-15

    The Vpu and CD4 cytoplasmic domains were found, by using a two-hybrid assay in yeast, to interact in the absence of their membrane anchor domains. Studies on several deletion and point mutants revealed that the overall structure of the Vpu cytoplasmic domain is required for this interaction. The Vpu amino acid residues involved in the interaction with CD4 were identified. Deletion of the C-terminal residues of Vpu, required for CD4 degradation, as well as the double mutation on the casein kinase II phosphorylation sites S52N-S56N, also involved in CD4 degradation, resulted in the loss of interaction with CD4 and in the inability to induce CD4 degradation. These results suggest that the ability of Vpu to mediate the degradation of CD4 is linked to its capacity to physically interact with CD4. However, additional mutagenesis on the S52 site revealed that the interaction between the cytoplasmic domains of Vpu and CD4 is not sufficient for in vitro Vpu-mediated CD4 degradation.

  12. Analysis of structural water and CH···π interactions in HIV-1 protease and PTP1B complexes using a hydrogen bond prediction tool, HBPredicT.

    PubMed

    Yesudas, Joshy P; Sayyed, Fareed Bhasha; Suresh, Cherumuttathu H

    2011-02-01

    A hydrogen bond prediction tool HBPredicT is developed for detecting structural water molecules and CH···π interactions in PDB files of protein-ligand complexes. The program adds the missing hydrogen atoms to the protein, ligands, and oxygen atoms of water molecules and subsequently all the hydrogen bonds in the complex are located using specific geometrical criteria. Hydrogen bonds are classified into various types based on (i) donor and acceptor atoms, and interactions such as (ii) protein-protein, (iii) protein-ligand, (iv) protein-water, (v) ligand-water, (vi) water-water, and (vii) protein-water-ligand. Using the information in category (vii), the water molecules which form hydrogen bonds with the ligand and the protein simultaneously-the structural water-is identified and retrieved along with the associated ligand and protein residues. For CH···π interactions, the relevant portions of the corresponding structures are also extracted in the output. The application potential of this program is tested using 19 HIV-1 protease and 11 PTP1B inhibitor complexes. All the systems showed presence of structural water molecules and in several cases, the CH···π interaction between ligand and protein are detected. A rare occurrence of CH···π interactions emanating from both faces of a phenyl ring of the inhibitor is identified in HIV-1 protease 1D4L.

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

    PubMed

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

    2011-08-15

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  16. HIV-1 subtypes in Yugoslavia.

    PubMed

    Stanojevic, Maja; Papa, Anna; Papadimitriou, Evagelia; Zerjav, Sonja; Jevtovic, Djordje; Salemovic, Dubravka; Jovanovic, Tanja; Antoniadis, Antonis

    2002-05-01

    To gain insight concerning the genetic diversity of HIV-1 viruses associated with the HIV-1 epidemic in Yugoslavia, 45 specimens from HIV-1-infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty-one of 45 specimens (91.2%) were identified as pol subtype B, 2 of 45 as subtype C (4.4%), 1 of 45 as CRF01_AE (2.2%), and 1 as CRF02_AG recombinant (2.2%). Nucleotide divergence among subtype B sequences was 4.8%. Results of this study show that among HIV-1-infected patients in Yugoslavia subtype B predominates (91.5%), whereas non-B subtypes are present at a low percentage, mostly related to travel abroad.

  17. The Vpr protein from HIV-1: distinct roles along the viral life cycle

    PubMed Central

    Le Rouzic, Erwann; Benichou, Serge

    2005-01-01

    The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle. PMID:15725353

  18. Tat is required for efficient HIV-1 reverse transcription.

    PubMed Central

    Harrich, D; Ulich, C; García-Martínez, L F; Gaynor, R B

    1997-01-01

    The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription. PMID:9135139

  19. Flexible Use of Nuclear Import Pathways by HIV-1

    PubMed Central

    Lee, KyeongEun; Ambrose, Zandrea; Martin, Thomas D.; Oztop, Ilker; Mulky, Alok; Julias, John G.; Vandegraaff, Nick; Baumann, Joerg G.; Wang, Rui; Yuen, Wendy; Takemura, Taichiro; Shelton, Kenneth; Taniuchi, Ichiro; Li, Yuan; Sodroski, Joseph; Littman, Dan R.; Coffin, John M.; Hughes, Stephen H.; Unutmaz, Derya; Engelman, Alan; KewalRamani, Vineet N.

    2010-01-01

    Summary The cellular and viral determinants required for HIV-1 infection of nondividing cells have been a subject of intense scrutiny. Here we identify the 68 kDa subunit of cleavage factor Im, CPSF6, as an inhibitor of HIV-1 infection. When enriched in the cytoplasm by high level expression or mutation, CPSF6 prevents nuclear entry of the virus. Similar to TRIM5 and Fv1 type restrictions, CPSF6 targets the viral capsid (CA). N74D mutation of the HIV-1 CA leads to a loss of interaction with CPSF6 and evasion of the nuclear import restriction. Interestingly, N74D mutation of CA changes HIV-1 nucleoporin (NUP) requirements. Whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics the NUP requirements of feline immunodeficiency virus (FIV) and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs. PMID:20227665

  20. 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

  1. Elucidation of the time course of adenosine deaminase APOBEC3G and viral infectivity factor vif in HIV-2287-infected infant macaques

    PubMed Central

    Endsley, Aaron N.; Ho, Rodney J.Y.

    2012-01-01

    Background Although the interactions of cellular cytidine deaminase A3G and viral infection factor (vif) of human immunodeficiency virus (HIV) were reported, regulation of A3G after in vivo HIV infection and disease progression is not known. Methods Time courses of plasma virus, CD4+ T lymphocyte Macaca levels, and concentrations of A3G and vif transcripts were determined in infant macaques infected with HIV-2287. These in vivo results were compared with those collected in vitro in HIV-2-infected T cells. Results Human immunodeficiency virus-infected macaques exhibited plasma viremia (≥108 copies/ml) followed by a precipitous CD4+ T-cell (from 40–70 to ≤5%) decline. An initial increase in A3G transcripts coincides with early increases in virus and vif RNA. As virus load continues to increase, A3G RNA decreases but recovers at a later phase as virus level stabilizes. Pearson correlation analysis revealed strong interactions of A3G–CD4, vif–CD4, and A3G–vif. Conclusions There is a time-dependent A3G and vif RNA interaction throughout the course of HIV infection. PMID:22017399

  2. 1,4-Bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene, a small molecule, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular Lens epithelium-derived growth factor.

    PubMed

    Gu, Wan-gang; Ip, Denis Tsz-Ming; Liu, Si-jie; Chan, Joseph H; Wang, Yan; Zhang, Xuan; Zheng, Yong-tang; Wan, David Chi-Cheong

    2014-04-25

    Translocation of viral integrase (IN) into the nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). As the first discovered cellular factor to interact with IN, Lens epithelium-derived growth factor (LEDGF/p75) plays an important role in the process of integration. Disruption of the LEDGF/p75-IN interaction has provided a great interest for anti-HIV agent discovery. In this work, we reported that one small molecular compound, 1,4-bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene(Compound 15), potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution at 1 μM. The putative binding mode of Compound 15 was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Compound 15 suppressed viral replication by measuring p24 antigen production in HIV-1IIIB acute infected C8166 cells with EC50 value of 11.19 μM. Compound 15 might supply useful structural information for further anti-HIV agent discovery. Copyright © 2014. Published by Elsevier Ireland Ltd.

  3. Preexposure prophylaxis is efficacious for HIV-1 prevention among women using depot medroxyprogesterone acetate for contraception.

    PubMed

    Heffron, Renee; Mugo, Nelly; Were, Edwin; Kiarie, James; Bukusi, Elizabeth A; Mujugira, Andrew; Frenkel, Lisa M; Donnell, Deborah; Ronald, Allan; Celum, Connie; Baeten, Jared M

    2014-11-28

    To evaluate preexposure prophylaxis (PrEP) efficacy for HIV-1 prevention among women using depot medroxyprogesterone acetate (DMPA) for contraception and men whose HIV-1-infected partners use DMPA. Secondary analysis of data from a randomized placebo-controlled trial of daily oral tenofovir and emtricitabine/tenofovir PrEP among heterosexual Kenyan and Ugandan HIV-1 serodiscordant couples. PrEP efficacy for HIV-1 prevention was compared among HIV-1-uninfected women using DMPA versus no hormonal contraception and among HIV-1 uninfected men whose HIV-1-infected female partners used DMPA versus no hormonal contraception. Of 4747 HIV-1 serodiscordant couples, 901 HIV-1-uninfected women used DMPA at some point during follow-up, 1422 HIV-1-uninfected women used no hormonal contraception, 1568 HIV-1-uninfected men had female partners who used DMPA, and 2626 men had female partners who used no hormonal contraception. PrEP efficacy estimates for HIV-1 prevention, compared with placebo, were similar among women using DMPA and those using no hormonal contraception (64.7 and 75.5%, adjusted interaction P = 0.65). Similarly, for men whose female partners used DMPA, PrEP efficacy did not differ from men whose partners used no hormonal contraception (90.0 versus 81.7%, adjusted interaction P = 0.52). PrEP is efficacious for HIV-1 prevention among women using DMPA and men whose partners use DMPA, suggesting PrEP could mitigate the potential increased HIV-1 acquisition and transmission risks that have been associated with DMPA use. Women at risk for HIV-1 choosing DMPA could maintain this contraceptive method and add PrEP to achieve prevention of unintended pregnancy and HIV-1.

  4. A novel enzyme-linked immunosorbent assay for screening HIV-1 fusion inhibitors targeting HIV-1 Gp41 core structure.

    PubMed

    Pang, Wei; Wang, Rui-Rui; Gao, Yue-Dong; Yang, Liu-Meng; Sun, Yi; Huang, Jing-Fei; Tien, Po; Zheng, Yong-Tang

    2011-02-01

    The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein mediates the fusion of viral and host cell membranes. As the HIV-1 enters the host cells, the 2 helical regions, HR1 and HR2, in the ectodomain of gp41 can form a 6-helix bundle, which brings the viral and target cell membranes to close proximity and serves as an attractive target for developing HIV-1 fusion inhibitors. Now, there are several cell- and molecule-based assays to identify potential HIV-1 fusion inhibitors targeting gp41. However, these assays cannot be used universally because they are time-consuming, inconvenient, and expensive. In the present study, the authors expressed and purified GST-HR121 and C43-30a proteins that were derived from the HIV-1 gp41 ectodomain region. GST-HR121 has a function similar to the HR1 peptide of gp41, whereas C43-30a is an HR2-derived peptide that added 50 amino acid residues (aa) in the N-terminal of C43. Further research found they could interact with each other, and a potential HIV-1 fusion inhibitor could inhibit this interaction. On the basis of this fact, a novel, rapid, and economic enzyme-linked immunosorbent assay was established, which can be developed for high-throughput screening of HIV-1 fusion inhibitors.

  5. HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles.

    PubMed

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

    2004-08-27

    The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.

  6. Plausibility of HIV-1 Infection of Oral Mucosal Epithelial Cells

    PubMed Central

    Herzberg, M.C.; Vacharaksa, A.; Gebhard, K.H.; Giacaman, R.A.; Ross, K.F.

    2011-01-01

    The AIDS pandemic continues. Little is understood about how HIV gains access to permissive cells across mucosal surfaces, yet such knowledge is crucial to the development of successful topical anti-HIV-1 agents and mucosal vaccines. HIV-1 rapidly internalizes and integrates into the mucosal keratinocyte genome, and integrated copies of HIV-1 persist upon cell passage. The virus does not appear to replicate, and the infection may become latent. Interactions between HIV-1 and oral keratinocytes have been modeled in the context of key environmental factors, including putative copathogens and saliva. In keratinocytes, HIV-1 internalizes within minutes; in saliva, an infectious fraction escapes inactivation and is harbored and transferable to permissive target cells for up to 48 hours. When incubated with the common oral pathogen Porphyromonas gingivalis, CCR5− oral keratinocytes signal through protease-activated receptors and Toll-like receptors to induce expression of CCR5, which increases selective uptake of infectious R5-tropic HIV-1 into oral keratinocytes and transfer to permissive cells. Hence, oral keratinocytes—like squamous keratinocytes of other tissues—may be targets for low-level HIV-1 internalization and subsequent dissemination by transfer to permissive cells. PMID:21441479

  7. Nuclear Exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding.

    PubMed

    Bennett, Ryan P; Presnyak, Vladimir; Wedekind, Joseph E; Smith, Harold C

    2008-03-21

    Human APOBEC3G (hA3G) is a host factor that defends against HIV-1 as well as other exogenous retroviruses and endogenous retroelements. To this end, hA3G is restricted to the cytoplasm of T lymphocytes where it interacts with viral RNA and proteins to assemble with viral particles causing a post-entry block during reverse transcription. hA3G also exhibits a mechanism to inhibit the reverse transcription of retroelements by RNA binding and sequestration into mRNA processing centers in the cytoplasm. We have determined that the molecular basis for this specialized property of hA3G is a novel cytoplasmic retention signal (CRS) that is necessary and sufficient to restrict wild-type hA3G and chimeric constructs to the cytoplasm. The CRS resides within amino acids 113-128 and is embedded within a basic flanking sequence and does not require RNA binding to retain hA3G in the cytoplasm. Paralogs of hA3G that have nuclear or cytoplasmic distributions differ from hA3G within the region encompassing the CRS motif with respect to charge and amino acid composition. We propose that the CRS enables hA3G to interact with cytoplasmic factors, and thereby enables hA3G to serve in host cell defense by restricting an antiviral sentinel to the cytoplasm. The CRS lies in a region involved in both Gag and Vif interactions; therefore, identification of this motif has important implications for the design of therapeutics that target HIV-1 while maintaining antiviral and cellular functions.

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

    PubMed

    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.

  9. Identification of dominant negative human immunodeficiency virus type 1 Vif mutants that interfere with the functional inactivation of APOBEC3G by virus-encoded Vif.

    PubMed

    Walker, Robert C; Khan, Mohammad A; Kao, Sandra; Goila-Gaur, Ritu; Miyagi, Eri; Strebel, Klaus

    2010-05-01

    APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.

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

    PubMed

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

    2014-05-01

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

  11. Population genomics of intrapatient HIV-1 evolution

    PubMed Central

    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 100bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. DOI: http://dx.doi.org/10.7554/eLife.11282.001 PMID:26652000

  12. Authentic HIV-1 integrase inhibitors

    PubMed Central

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

    2010-01-01

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

  13. 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

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

    PubMed

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

    2008-01-15

    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 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. 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 mug/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.

  15. 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

  16. Developing strategies for HIV-1 eradication

    PubMed Central

    Durand, Christine M.; Blankson, Joel N.; Siliciano, Robert F.

    2014-01-01

    Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication, transforming the outlook for infected patients. However, reservoirs of replication-competent forms of the virus persist during HAART, and when treatment is stopped, high rates of HIV-1 replication return. Recent insights into HIV-1 latency, as well as a report that HIV-1 infection was eradicated in one individual, have renewed interest in finding a cure for HIV-1 infection. Strategies for HIV-1 eradication include gene therapy and hematopoietic stem cell transplantation, stimulating host immunity to control HIV-1 replication, and targeting latent HIV-1 in resting memory CD4+ T cells. Future efforts should aim to provide better understanding of how to reconstitute the CD4+ T cell compartment with genetically engineered cells, exert immune control over HIV-1 replication, and identify and eliminate all viral reservoirs. PMID:22867874

  17. Cell activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting exosomes from cells expressing defective HIV-1.

    PubMed

    Arenaccio, Claudia; Chiozzini, Chiara; Columba-Cabezas, Sandra; Manfredi, Francesco; Federico, Maurizio

    2014-06-12

    A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4+ T lymphocytes. We observed that unstimulated human primary CD4+ T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-α in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4+ T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4+ T lymphocytes. TNF-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4+ T lymphocytes. Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4+ T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis

  18. 4'-Thio-oligo-beta-D-ribonucleotides: synthesis of beta-4'-thio-oligouridylates, nuclease resistance, base pairing properties, and interaction with HIV-1 reverse transcriptase.

    PubMed Central

    Bellon, L; Barascut, J L; Maury, G; Divita, G; Goody, R; Imbach, J L

    1993-01-01

    We present the synthesis and the study of properties of a new series of modified oligonucleotides, namely 4'-thio-oligo-beta-D-ribonucleotides (4'-S-RNA). Homo-oligonucleotides of this class (4'-SU6 and 4'-SU12) were prepared from the previously known thionucleosides using the phosphoramidite methodology. The comparison of the substrate properties of 4'-SU6 and its natural analog U6 with respect to four nucleases indicates that the former is much more resistant than the latter. Such resistance to nucleases in addition to relatively high Tm values for 4'-SU12 hybridized with Poly(A) show that these new 4'-S-RNA are good candidates for potential antisense effects. The oligonucleotides 4'-SU6 and 4'-SU12 have been also evaluated as non sequence specific inhibitors of HIV-1 reverse transcriptase. All available evidences, based primarily on fluorescence measurements, are consistent with the binding of 4'-SU6 and 4'-SU12 to RT at a site which is different from the polymerase site of the enzyme. PMID:7683133

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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.

  1. Caveolin-1 mediated uptake via langerin restricts HIV-1 infection in human Langerhans cells.

    PubMed

    van den Berg, Linda M; Ribeiro, Carla M S; Zijlstra-Willems, Esther M; de Witte, Lot; Fluitsma, Donna; Tigchelaar, Wikky; Everts, Vincent; Geijtenbeek, Teunis B H

    2014-12-31

    Human Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown. Here, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin(+) caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs. Thus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.

  2. Impairment of B-cell functions during HIV-1 infection.

    PubMed

    Amu, Sylvie; Ruffin, Nicolas; Rethi, Bence; Chiodi, Francesca

    2013-09-24

    A variety of B-cell dysfunctions are manifested during HIV-1 infection, as reported early during the HIV-1 epidemic. It is not unusual that the pathogenic mechanisms presented to elucidate impairment of B-cell responses during HIV-1 infection focus on the impact of reduced T-cell numbers and functions, and lack of germinal center formation in lymphoid tissues. To our understanding, however, perturbation of B-cell phenotype and function during HIV-1 infection may begin at several different B-cell developmental stages. These impairments can be mediated by intrinsic B-cell defects as well as by the lack of proper T-cell help. In this review, we will highlight some of the pathways and molecular interactions leading to B-cell impairment prior to germinal center formation and B-cell activation mediated through the B-cell receptor in response to HIV-1 antigens. Recent studies indicate a regulatory role for B cells on T-cell biology and immune responses. We will discuss some of these novel findings and how these regulatory mechanisms could potentially be affected by the intrinsic defects of B cells taking place during HIV-1 infection.

  3. 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

  4. Characterization of a New HIV-1 CRF01_AE/CRF07_BC Recombinant Virus in Guangxi, China.

    PubMed

    Kong, Desheng; Wang, Yan; Wang, Chen; Liang, Shujia; Feng, Yi; Ruan, Yuhua; Shao, Yiming; Ma, Liying

    2017-07-10

    Currently, more and more kinds of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified in China. A novel second-generation CCR5-tropic HIV-1 recombinant virus (GX2016EU09) was identified here, which was isolated from a HIV-1-infected man who had sex with men (MSM) in Guangxi, China. Phylogenetic analysis of near full-length genome (NFLG) showed that the novel HIV-1 recombinant GX2016EU09 clustered with CRF01_AE reference sequences, but set up a distinct branch. Recombinant analysis showed that the NFLG of GX2016EU09 composed of CRF01_AE (as the backbone) and CRF07_BC, with nine recombination break points observed in the gag, pol, vif, vpr, tat, rev, vpu, env, and nef regions. To our knowledge, this HIV-1 URF differs from previously documented CRF01_AE/CRF07_BC forms. The emergence of a novel CRF01_AE/CRF07_BC recombinant strain indicates the increasing complexity of the HIV-1 epidemic among the MSM group in Guangxi, China.

  5. Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

    PubMed Central

    Tamamis, Phanourios; Floudas, Christodoulos A.

    2013-01-01

    HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity. PMID:24048002

  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. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

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

    1993-12-31

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

  8. Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe.

    PubMed

    Nkeze, Joseph; Li, Lin; Benko, Zsigmond; Li, Ge; Zhao, Richard Y

    2015-01-01

    The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe). Each one of the HIV-1 genes was cloned and expressed individually in fission yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between fission yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in fission yeast and mammalian cells. Consistent with the observation in fission yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress. Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

  9. 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.

  10. HIV-1 Vpu Accessory Protein Induces Caspase-mediated Cleavage of IRF3 Transcription Factor*

    PubMed Central

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

    2014-01-01

    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

  11. Role of semen in HIV-1 transmission: inhibitor or facilitator?

    PubMed

    Doncel, Gustavo F; Joseph, Theresa; Thurman, Andrea R

    2011-03-01

    Sexual transmission of human immunodeficiency virus type 1 (HIV-1) accounts for 60-90% of new infections, especially in developing countries. During male-to-female transmission, the virus is typically deposited in the vagina as cell-free and cell-associated virions carried by semen. But semen is more than just a carrier for HIV-1. Evidence from in vitro and in vivo studies supports both inhibitory and enhancing effects. Intrinsic antiviral activity mediated by cationic antimicrobial peptides, cytotoxicity, and blockage of HIV-dendritic cell interactions are seminal plasma properties that inhibit HIV-1 infection. On the contrary, neutralization of vaginal acidic pH, enhanced virus-target cell attachment by seminal amyloid fibrils, opsonization by complement fragments, and electrostatic interactions are factors that facilitate HIV-1 infection. The end result, i.e., inhibition or enhancement of HIV mucosal infection, in vivo, likely depends on the summation of all these biological effects. More research is needed, especially in animal models, to dissect the role of these factors and establish their relevance in HIV-1 transmission. © 2010 John Wiley & Sons A/S.

  12. Cyclophilin B enhances HIV-1 infection

    SciTech Connect

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

    2016-02-15

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

  13. 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

  14. Antiretroviral treatment effect on immune activation reduces cerebrospinal fluid HIV-1 infection.

    PubMed

    Sinclair, Elizabeth; Ronquillo, Rollie; Lollo, Nicole; Deeks, Steven G; Hunt, Peter; Yiannoutsos, Constantin T; Spudich, Serena; Price, Richard W

    2008-04-15

    To define the effect of antiretroviral therapy (ART) on activation of T cells in cerebrospinal fluid (CSF) and blood, and interactions of this activation with CSF HIV-1 RNA concentrations. Cross-sectional analysis of 14 HIV-negative subjects and 123 neuroasymptomatic HIV-1-infected subjects divided into 3 groups: not on ART (termed "offs"), on ART with plasma HIV-1 RNA >500 copies/mL ("failures"), and on ART with plasma HIV-1 RNA HIV-1, it maintained a coincident relation to CSF HIV-1 in both viremic groups. In addition to correlation with CSF HIV-1 concentrations, CD8 activation in blood and CSF correlated with CSF WBCs and CSF neopterin. Multivariate analysis confirmed the association of blood CD8 T-cell activation, along with plasma HIV-1 RNA and CSF neopterin, with CSF HIV-1 RNA levels. The similarity of CD8 T-cell activation in blood and CSF suggests these cells move from blood to CSF with only minor changes in CD38/HLA-DR expression. Differences in the relation of CD8 activation to HIV-1 concentrations in the blood and CSF in the 2 viremic groups suggest that changes in immune activation not only modulate CSF HIV-1 replication but also contribute to CSF treatment effects. The magnitude of systemic HIV-1 infection and intrathecal macrophage activation are also important determinants of CSF HIV-1 RNA levels.

  15. Antiretroviral prophylaxis of perinatal HIV-1 transmission and the potential impact of antiretroviral resistance.

    PubMed

    Nolan, Monica; Fowler, Mary Glenn; Mofenson, Lynne M

    2002-06-01

    Since 1994, trials of zidovudine, zidovudine and lamivudine, and nevirapine have demonstrated that these antiretroviral drugs can substantially reduce the risk of perinatal HIV-1 transmission. With reductions in drug price, identification of simple, effective antiretroviral regimens to prevent perinatal HIV-1 transmission, and an increasing international commitment to support health care infrastructure, antiretrovirals for both perinatal HIV-1 prevention and HIV-1 treatment will likely become more widely available to HIV-1-infected persons in resource-limited countries. In the United States, widespread antiretroviral usage has been associated with increased antiretroviral drug resistance. This raises concern that drug resistance may reduce the effectiveness of perinatal antiretroviral prophylaxis as well as therapeutic intervention strategies. The purpose of this article is to review what is known about resistance and risk of perinatal HIV transmission, assess the interaction between antiretroviral resistance and the prevention of perinatal HIV-1 transmission, and discuss implications for current global prevention and treatment strategies.

  16. Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells.

    PubMed

    Kanizsai, Szilvia; Ongrádi, Joseph; Aradi, János; Nagy, Károly

    2014-12-01

    Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Cockrell, Adam S; van Praag, Henriette; Santistevan, Nicholas; Ma, Hong; Kafri, Tal

    2011-06-24

    HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation.

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

    PubMed Central

    2011-01-01

    , our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation. PMID:21702950

  1. 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

  2. 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

  3. 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

  4. 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.

  5. APOBEC3G-Mediated G-to-A Hypermutation of the HIV-1 Genome: The Missing Link in Antiviral Molecular Mechanisms

    PubMed Central

    Okada, Ayaka; Iwatani, Yasumasa

    2016-01-01

    APOBEC3G (A3G) is a member of the cellular polynucleotide cytidine deaminases, which catalyze the deamination of cytosine (dC) to uracil (dU) in single-stranded DNA. These enzymes potently inhibit the replication of a variety of retroviruses and retrotransposons, including HIV-1. A3G is incorporated into vif-deficient HIV-1 virions and targets viral reverse transcripts, particularly minus-stranded DNA products, in newly infected cells. It is well established that the enzymatic activity of A3G is closely correlated with the potential to greatly inhibit HIV-1 replication in the absence of Vif. However, the details of the underlying molecular mechanisms are not fully understood. One potential mechanism of A3G antiviral activity is that the A3G-dependent deamination may trigger degradation of the dU-containing reverse transcripts by cellular uracil DNA glycosylases (UDGs). More recently, another mechanism has been suggested, in which the virion-incorporated A3G generates lethal levels of the G-to-A hypermutation in the viral DNA genome, thus potentially driving the viruses into “error catastrophe” mode. In this mini review article, we summarize the deaminase-dependent and deaminase-independent molecular mechanisms of A3G and discuss how A3G-mediated deamination is linked to antiviral mechanisms. PMID:28066353

  6. Computational anti-AIDS drug design based on the analysis of the specific interactions between immunophilins and the HIV-1 gp120 V3 loop. Application to the FK506-binding protein.

    PubMed

    Andrianov, A M

    2008-08-01

    The object of the study was to model the structural complex of the FK506-binding protein (FKBP) with the CRK peptide imitating the central region of the HIV-1 V3 loop, as well as to define the FKBP stretch giving rise to the binding site for V3 the synthetic copy of which, on the assumption of preserving the spatial peptide structure in the free state, can be considered as a promising applicant for the role of antiviral drug. To this end, the following successive steps were carried out: (i) the NMR-based conformational analysis of CRK was put into practice, and, in the light of the results derived, the best energy CRK structure meeting the requirements of the input NMR data was identified; (ii) molecular docking of the CRK structure with the X-ray FKBP conformation was implemented, and energy refining the simulated structural complex was realized; (iii) the matrix of distances between amino acids of the ligand and receptor was computed to specify the FKBP stretch keeping in touch with CRK followed by analyzing the types of interactions stabilizing the over-molecular ensemble; (iv) 3D structure of this stretch in the unbound status referred to as the FKBP peptide was predicted, and its collation with the X-ray conformation of the identical FKBP site was performed; (v) the potential energy function and its constituents were studied for the structural complex generated by molecular docking of the CRK molecule with the FKBP peptide; and (vi) from all evidence, the virtual FKBP-derived peptide was submitted to be utilized as a prospective structural framework in the anti-HIV-1 drug design. Summing up the results obtained, the following principal conclusion was drawn: a high affinity of the V3 loop peptide to the FKBP is based on the principle of "mirror similarity" that implies the near resemblance of 3D structures for the two individual fragments of the receptor and ligand, which, most likely, accounts for recognizing the immunophilin by V3 and determines the

  7. Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection

    PubMed Central

    Salazar-Gonzalez, Jesus F.; Salazar, Maria G.; Keele, Brandon F.; Learn, Gerald H.; Giorgi, Elena E.; Li, Hui; Decker, Julie M.; Wang, Shuyi; Baalwa, Joshua; Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Guffey, M. Brad; Bar, Katharine J.; Davis, Katie L.; Ochsenbauer-Jambor, Christina; Kappes, John C.; Saag, Michael S.; Cohen, Myron S.; Mulenga, Joseph; Derdeyn, Cynthia A.; Allen, Susan; Hunter, Eric; Markowitz, Martin; Hraber, Peter; Perelson, Alan S.; Bhattacharya, Tanmoy; Haynes, Barton F.; Korber, Bette T.; Hahn, Beatrice H.

    2009-01-01

    Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses. PMID:19487424

  8. 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.

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

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-09

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

  10. Allosteric inhibition of HIV-1 integrase activity

    PubMed Central

    Engelman, Alan; Kessl, Jacques J.; Kvaratskhelia, Mamuka

    2013-01-01

    HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development. PMID:23647983

  11. Willingness of Kenyan HIV-1 serodiscordant couples to use antiretroviral based HIV-1 prevention strategies

    PubMed Central

    Heffron, Renee; Ngure, Kenneth; Mugo, Nelly; Celum, Connie; Kurth, Ann; Curran, Kathryn; Baeten, Jared M.

    2012-01-01

    Introduction Antiretroviral treatment (ART) and pre-exposure prophylaxis (PrEP) have demonstrated efficacy as new HIV-1 prevention approaches for HIV-1 serodiscordant couples. Methods Among Kenyan HIV-1 serodiscordant heterosexual couples participating in a clinical trial of PrEP, we conducted a cross-sectional study and used descriptive statistical methods to explore couples' willingness to use antiretrovirals for HIV-1 prevention. The study was conducted prior to July 2011, when studies among heterosexual populations reported that ART and PrEP reduced HIV-1 risk. Results For 181 couples in which the HIV-1 infected partner had a CD4 count ≥350 cells/μL and had not yet initiated ART (and thus did not qualify for ART under Kenyan guidelines), 60.2% of HIV-1 infected partners (69.4% of men and 57.9% of women) were willing to use early ART (at CD4 ≥350 cells/μL) for HIV-1 prevention. Among HIV-1 uninfected partners, 92.7% (93.8% of men and 86.1% of women) reported willingness to use PrEP. When given a hypothetical choice of early ART or PrEP for HIV-1 prevention, 52.5% of HIV-1 infected participants would prefer to initiate ART early and 56.9% of HIV-1 uninfected participants would prefer to use PrEP. Conclusions Nearly 40% of Kenyan HIV-1 infected individuals in known HIV-1 serodiscordant partnerships reported reservations about early ART initiation for HIV-1 prevention. PrEP interest in this PrEP-experienced population was high. Strategies to achieve high uptake and sustained adherence to ART and PrEP for HIV-1 prevention in HIV-1 serodiscordant couples will require responding to couples' preferences for prevention strategies. PMID:22595872

  12. 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

  13. 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

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. The HIV-1 epidemic in South Africa.

    PubMed

    Puren, A J

    2002-01-01

    The first reported cases of HIV-1 infection in South Africa occurred in 1982. Two distinct HIV-1 epidemic patterns were recognized. Initially the infection was prevalent in white males who had sex with males. The HIV-1 clade B was associated with this group. By 1989, the second epidemic was recognized primarily in the black population. Infections in this case were mainly heterosexual in origin. The HIV-1 clade involved was mainly C. The national HIV-1 sero-prevalence in antenatal attendees was less than 1% in 1990 and by 1994 this figure had risen to 7.5%. The most recent antenatal surveillance for HIV-1 sero-prevalence in 1999 revealed the following. The national prevalence rate for 1999 was 22.4% compared with the 1998 rate of 22.8%. The data highlighted the profound effect the epidemic had and will have on the disease burden in South Africa and by extension on the social and economic fronts. This view was emphasised by the impact HIV-1 infection had on tuberculosis. For example, sentinel surveys have attributed 44% of tuberculosis cases to HIV-1 infection. Moreover, the high prevalence of sexually transmitted infections will certainly exacerbate the HIV-1 epidemic.

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

    PubMed

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

    2008-09-01

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

  18. Cytopathic Mechanisms of HIV-1

    PubMed Central

    Costin, Joshua M

    2007-01-01

    The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein. PMID:17945027

  19. Cytopathic mechanisms of HIV-1.

    PubMed

    Costin, Joshua M

    2007-10-18

    The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein.

  20. Cell signaling pathways and HIV-1 therapeutics.

    PubMed

    He, Johnny J

    2011-06-01

    Host-virus interactions permeate every aspect of both virus life cycle and host response and involve host cell macromolecular machinery and viral elements. It is these intimate interactions that mandate the outcomes of the infection and pathogenesis. It is also these intimate interactions that lay the foundation for the development of pharmaceutical interventions. HIV-1 is no exception in these regards. In the first two decades, HIV/AIDS research has led to the successful development of a number of antiviral inhibitors and the landmark formulation of the suppressive therapy. It has become apparent that this therapy does not offer a complete solution to cure and eradicate the virus. Meanwhile, this therapy has changed the overall landscape of HIV-associated neurological disorders to a more common and prevalent form so-called minor cognitive motor disorder. Thus, there is an important and continued need for new anti-HIV therapeutics. We believe that this is an excellent opportunity to compile and present the latest works being done during the last few years in this exciting field of HIV-host interactions, particularly cell signaling pathways. We hope that this special issue composed of one brief report, eight thematic reviews, and two original articles will serve to foster the exchange of new scientific ideas on HIV-host interactions and anti-HIV therapy and eventually contribute to HIV/AIDS eradication.

  1. HIV-1 Proteins Accelerate HPA Axis Habituation in Female Rats

    PubMed Central

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

    2015-01-01

    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 facilitate 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

  2. Breast milk transmission of HIV-1.

    PubMed

    Nduati, R; John, G

    1995-12-01

    Breast milk provides infants and children immunologic, nutritional, and child spacing benefits. Yet it also transmits some viruses, for example, HIV-1. The World Health Organization recommends that, in conditions with poor access to breast milk substitutes, HIV-positive women should still breast feed due to the nutritional and infectious risk of artificial feeding. It appears that breast fed infants experience a slower progression of AIDS and death. Vertical transmission of HIV-1 may occur during pregnancy, at delivery, or through breast milk. The HIV-1 transmission rate via breast milk from acutely infected women is estimated to be 29-36%. A meta-analysis of case reports and small case series of women with chronic HIV-1 infection indicated a breast feeding transmission rate of 14%. Studies suggest that the likelihood of HIV-1 transmission via breast milk increases as duration of breast feeding increases. Infants with detectable HIV-1 DNA tend to have mothers whose absolute CD4 counts are less than 400 and have severe vitamin A deficiency. Breast milk has HIV-1 specific immunoglobulins (IgG, IgA, and IgM). It appears that HIV-1 elicits a local immune response. Breast milk of HIV-1 positive mothers with non-infected children tends to still have IgM and IgA until 18 months. Potential risk factors for breast milk transmission of HIV-1 include cracked nipples and mastitis in the mother; oral thrush, malnutrition, inflammation of the lips, and mucosal compromise in the infant; and vigorous suction of the neonate and use of the wrong equipment for suctioning. Inhibiting factors of HIV-1 in breast milk are bovine and human lactoferrin and a membrane associated protein that attaches to the CD4 receptor and thus prevents attachment of the HIV antigen gp120 to the CD4 receptor on T-cells.

  3. 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.

  4. A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140.

    PubMed

    Guttman, Miklos; Lee, Kelly K

    2013-11-01

    The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.

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

    PubMed Central

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

    2015-01-01

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

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

    DOE PAGES

    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

  7. Quantifying Ongoing HIV-1 Exposure in HIV-1–Serodiscordant Couples to Identify Individuals With Potential Host Resistance to HIV-1

    PubMed Central

    Mackelprang, Romel D.; Baeten, Jared M.; Donnell, Deborah; Celum, Connie; Farquhar, Carey; de Bruyn, Guy; Essex, Max; McElrath, M. Juliana; Nakku-Joloba, Edith; Lingappa, Jairam R.

    2012-01-01

    Background. Immunogenetic correlates of resistance to HIV-1 in HIV-1–exposed seronegative (HESN) individuals with consistently high exposure may inform HIV-1 prevention strategies. We developed a novel approach for quantifying HIV-1 exposure to identify individuals remaining HIV-1 uninfected despite persistent high exposure. Methods. We used longitudinal predictors of HIV-1 transmission in HIV-1 serodiscordant couples to score HIV-1 exposure and define HESN clusters with persistently high, low, and decreasing risk trajectories. The model was validated in an independent cohort of serodiscordant couples. We describe a statistical tool that can be applied to other HESN cohorts to identify individuals with high exposure to HIV-1. Results. HIV-1 exposure was best quantified by frequency of unprotected sex with, plasma HIV-1 RNA levels among, and presence of genital ulcer disease among HIV-1–infected partners and by age, pregnancy status, herpes simplex virus 2 serostatus, and male circumcision status among HESN participants. Overall, 14% of HESN individuals persistently had high HIV-1 exposure and exhibited a declining incidence of HIV-1 infection over time. Conclusions. A minority of HESN individuals from HIV-1–discordant couples had persistent high HIV-1 exposure over time. Decreasing incidence of infection in this group suggests these individuals were selected for resistance to HIV-1 and may be most appropriate for identifying biological correlates of natural host resistance to HIV-1 infection. PMID:22926009

  8. [Virtual screening of small molecular HIV-1 entry inhibitor NC-2 targeting gp120 and its action mechanism].

    PubMed

    Duan, Heng; Wang, Yuqin; Song, Deshou; Chen, Zhipeng; Qiu, Jiayin; Lu, Lu; Jiang, Shibo; Liu, Shuwen; Tan, Suiyi

    2013-06-01

    To screen the HIV-1 entry inhibitors targeting HIV-1 gp120 from the IBS natural product database by virtual screening based on the binding mode of the neutralizing antibody VRC01 with HIV-1 gp120 and investigate the anti-viral activities of the inhibitors and their action mechanisms. The binding interaction of the candidate molecules binding gp120 and changes of the binding free energy were analyzed by MM-PBSA calculation. The anti-HIV-1 activities of the tested compounds were detected by HIV-1 pseudotyped virus, laboratory-adapted HIV-1 and a cell-cell fusion assay. The cytotoxicity of the studied molecules was examined by XTT colorimetric assay. The mechanisms of the anti-viral activities of the candidate molecules were analyzed using enzyme-linked immunosorbent assay. A total of 19 molecules with distinct reduction of the binding free energy after binding with gp120 were screened from 40000 molecules. Among them, NC-2 showed anti-HIV-1 activities against HIV-1 pseudotyped virus and laboratory-adapted HIV-1, and was capable of blocking HIV-1 envelope-mediated cell-cell fusion. The IC50 of NC-2 for inhibiting HIV-1IIIB and pseudotyped HIV-1JRFL infection were 1.95∓0.44 µmol/L and 10.58∓0.13 µmol/L, respectively. The results of ELISA suggested that NC-2 could inhibit the binding of HIV-1 gp120 to CD4 without blocking the formation of gp41 six-helix bundle in vitro. This computer-based virtual screening method can be used to screen HIV-1 entry inhibitors targeting gp120. Using this virtual screening approach combined with anti-viral activity screening, we obtained a potent HIV-1 entry inhibitor NC-2 with novel structure.

  9. Novel Acylguanidine-Based Inhibitor of HIV-1

    PubMed Central

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

    2016-01-01

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

  10. 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.

  11. DDX3 RNA helicase is required for HIV-1 Tat function.

    PubMed

    Yasuda-Inoue, Mariko; Kuroki, Misao; Ariumi, Yasuo

    2013-11-22

    Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Structure based Virtual Screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75).

    PubMed

    Panwar, Umesh; Singh, Sanjeev Kumar

    2017-09-26

    HIV -1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to because of its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine- Z742267384, Maybridge- HTS02400, and Specs- AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behaviour and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.

  13. HIV-1-induced AIDS in monkeys.