A computational exploration of global and temporal dynamics of selection pressure on HIV-1 Vif polymorphism.

Virion infectivity factor (Vif), an accessory protein of HIV-1 (human immunodeficiency virus type 1), antagonizes host APOBEC3 protein (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3) or A3 via proteasomal degradation, facilitating viral replication. HLA (Human leukocyte antigens) alleles, host restriction factors, and error-prone reverse transcription contribute to the global polymorphic dynamics of HIV, impacting effective vaccine design. Our computational analysis of over 50,000 HIV-1 M vif sequences from the Los Alamos National Laboratory (LANL) database (1998-2021) revealed positive selection pressure on the vif gene (nonsynonymous to synonymous ratio, dn/ds=1.58) and an average entropy score of 0.372 in protein level. Interestingly, over the years (1998-2021), a decreasing trend of dn/ds (1.68 to 1.47) and an increasing trend of entropy (0.309 to 0.399) was observed. The predicted mutational frequency against Vif consensus sequence decreased over time (slope = -0.00024, p < 0.0001). Sequence conservation was observed in Vif functional motifs F1, F2, F3, G, BC box, and CBF ß binding region, while variability was observed mainly in N- and C- terminal and Zinc finger region, which were dominantly under immune pressure by host HLA-I-restricted CD8+ T cell. Computational analysis of ∆∆Gstability through protein stability prediction tools suggested that missense mutation may affect Vif stability, especially in the Vif-A3 binding interface. Notably, mutations R17K and Y44F in F1 and G box were predicted to destabilize the Vif-A3 binding interface by altering bond formations with adjacent amino acids. Therefore, our analysis demonstrates Vif adaptation with host physiology by maintaining sequence conservation, especially in A3 interacting functional motifs, highlighting important therapeutic candidate regions of Vif against HIV-1 infections.

The structural basis for HIV-1 Vif antagonism of human APOBEC3G.

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1-4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene.

Characterization of an A3G-VifHIV-1-CRL5-CBFß Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host-Pathogen Complexes.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFß) to determine the structure of the (A3G-Vif-CRL5-CBFß) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFß complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Examination of the APOBEC3 Barrier to Cross Species Transmission of Primate Lentiviruses.

The transmission of viruses from animal hosts into humans have led to the emergence of several diseases. Usually these cross-species transmissions are blocked by host restriction factors, which are proteins that can block virus replication at a specific step. In the natural virus host, the restriction factor activity is usually suppressed by a viral antagonist protein, but this is not the case for restriction factors from an unnatural host. However, due to ongoing viral evolution, sometimes the viral antagonist can evolve to suppress restriction factors in a new host, enabling cross-species transmission. Here we examine the classical case of this paradigm by reviewing research on APOBEC3 restriction factors and how they can suppress human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). APOBEC3 enzymes are single-stranded DNA cytidine deaminases that can induce mutagenesis of proviral DNA by catalyzing the conversion of cytidine to promutagenic uridine on single-stranded viral (-)DNA if they escape the HIV/SIV antagonist protein, Vif. APOBEC3 degradation is induced by Vif through the proteasome pathway. SIV has been transmitted between Old World Monkeys and to hominids. Here we examine the adaptations that enabled such events and the ongoing impact of the APOBEC3-Vif interface on HIV in humans.

Maedi-visna virus Vif protein uses motifs distinct from HIV-1 Vif to bind zinc and the cofactor required for A3 degradation.

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin-RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi-visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.

Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G2/M cell cycle arrest. Vif initiates G2/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-ß/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis.IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G2/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G2/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins.

Vif (viral infectivity factor) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of host APOBEC3 (apolipoprotein B mRNA-editing enzyme catalytic subunit 3) proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and core-binding factor subunit ß, forming a four-protein complex called VCBC. The structure of VCBC-Cullin5 has recently been solved by X-ray crystallography, and, using molecular dynamics simulations, the dynamics of VCBC have been characterized. Here, we applied time-lapse high-speed atomic force microscopy to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of this complex, which we identified as the triangle, dumbbell, and globular structures. Moreover, we characterized the dynamics of each of these structures. Our data revealed the very dynamic behavior of all of them, with the triangle and dumbbell structures being the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and may support efforts to improve HIV treatment, because Vif is essential for virus survival in the cell.

Structural perspectives on HIV-1 Vif and APOBEC3 restriction factor interactions.

Human immunodeficiency virus (HIV) is a retroviral pathogen that targets human immune cells such as CD4+ T cells, macrophages, and dendritic cells. The human apolipoprotein B mRNA- editing catalytic polypeptide 3 (APOBEC3 or A3) cytidine deaminases are a key class of intrinsic restriction factors that inhibit replication of HIV. When HIV-1 enters the cell, the immune system responds by inducing the activation of the A3 family proteins, which convert cytosines to uracils in single-stranded DNA replication intermediates, neutralizing the virus. HIV counteracts this intrinsic immune response by encoding a protein termed viral infectivity factor (Vif). Vif targets A3 to an E3 ubiquitin ligase complex for poly-ubiquitination and proteasomal degradation. Vif is unique in that it can recognize and counteract multiple A3 restriction factor substrates. Structural biology studies have provided significant insights into the overall architectures and functions of Vif and A3 proteins; however, a structure of the Vif-A3 complex has remained elusive. In this review, we summarize and reanalyze experimental data from recent structural, biochemical, and functional studies to provide key perspectives on the residues involved in Vif-A3 protein-protein interactions.

Structural basis of antagonism of human APOBEC3F by HIV-1 Vif.

HIV-1 virion infectivity factor (Vif) promotes degradation of the antiviral APOBEC3 (A3) proteins through the host ubiquitin-proteasome pathway to enable viral immune evasion. Disrupting Vif-A3 interactions to reinstate the A3-catalyzed suppression of human immunodeficiency virus type 1 (HIV-1) replication is a potential approach for antiviral therapeutics. However, the molecular mechanisms by which Vif recognizes A3 proteins remain elusive. Here we report a cryo-EM structure of the Vif-targeted C-terminal domain of human A3F in complex with HIV-1 Vif and the cellular cofactor core-binding factor beta (CBFß) at 3.9-Å resolution. The structure shows that Vif and CBFß form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFß beyond Vif stabilization, and captures multiple independent A3F-Vif interfaces. Together with our biochemical and cellular studies, our structural findings establish the molecular determinants that are critical for Vif-mediated neutralization of A3F and provide a comprehensive framework of how HIV-1 Vif hijacks the host protein degradation machinery to counteract viral restriction by A3F.

Structural Basis for a Species-Specific Determinant of an SIV Vif Protein toward Hominid APOBEC3G Antagonism.

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94 Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface on Vif that enables adaptation while maintaining protein interactions required for potent A3 antagonism.

The Role of RNA in HIV-1 Vif-Mediated Degradation of APOBEC3H.

As many as five members of the APOBEC3 family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which forms a hybrid ubiquitin ligase complex that directly binds APOBEC3 enzymes and targets them for proteasomal degradation. APOBEC3H (A3H) is unique among family members by dimerization through cellular and viral duplex RNA species. RNA binding is required for localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles and ultimately for effective virus restriction activity. Here we compared wild-type human A3H and RNA binding-defective mutants to ask whether RNA may be a factor in the functional interaction with HIV-1 Vif. We used structural modeling, immunoblotting, live cell imaging, and split green fluorescence protein (GFP) reconstitution approaches to assess the capability of HIV-1 Vif to promote the degradation of wild-type A3H in comparison to RNA binding-defective mutants. The results combined to show that RNA is not strictly required for Vif-mediated degradation of A3H, and that RNA and Vif are likely to bind this single-domain DNA cytosine deaminase on physically distinct surfaces. However, a subset of the results also indicated that the A3H degradation process may be affected by A3H protein structure, subcellular localization, and differences in the constellation of A3H interaction partners, suggesting additional factors may also influence the fate and functionality of this host-pathogen interaction.

Conformational Dynamics of the HIV-Vif Protein Complex.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-ß. A five-protein complex containing the substrate receptor (Vif, CBF-ß, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the µs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Functional proteomic atlas of HIV infection in primary human CD4+ T cells.

Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell.

HIV-1 Escape from Small-Molecule Antagonism of Vif.

The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus.IMPORTANCE Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation.

Identification of small molecule compounds targeting the interaction of HIV-1 Vif and human APOBEC3G by virtual screening and biological evaluation.

Human APOBEC3G (hA3G) is a restriction factor that inhibits human immunodeficiency 1 virus (HIV-1) replication. The virally encoded protein Vif binds to hA3G and induces its degradation, thereby counteracting the antiviral activity of hA3G. Vif-mediated hA3G degradation clearly represents a potential target for anti-HIV drug development. Herein, we have performed virtual screening to discover small molecule inhibitors that target the binding interface of the Vif/hA3G complex. Subsequent biochemical studies have led to the identification of a small molecule inhibitor, IMB-301 that binds to hA3G, interrupts the hA3G-Vif interaction and inhibits Vif-mediated degradation of hA3G. As a result, IMB-301 strongly inhibits HIV-1 replication in a hA3G-dependent manner. Our study further demonstrates the feasibility of inhibiting HIV replication by abrogating the Vif-hA3G interaction with small molecules.

Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors.

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

An improved method for purification and refolding of recombinant HIV Vif expressed in Escherichia coli.

Virion infectivity factor (Vif) is a 23 kDa protein that protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the elongin B and C subunits, Cullin 5, and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21(DE3) Escherichia coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively.

Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5.

Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif.IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

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

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 ß-sheet (ß2-ß5). 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 ß-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-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

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

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.