Design of Vif-Derived Peptide Inhibitors with Anti-HIV-1 Activity by Interrupting Vif-CBFß Interaction.

One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFß E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFß interaction based on the sequences of Vif mutants with higher affinity for CBFß screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 µM and 55.1 µM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFß. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFß over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFß interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.

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.

HIV-1 Vif protein sequence variations in South African people living with HIV and their influence on Vif-APOBEC3G interaction.

PURPOSE: Despite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions. METHODS: Sanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity. RESULTS: The findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups. CONCLUSIONS: These results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.

Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 vif mRNA Production as Revealed by a Growth-Adaptive Mutation.

We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance vif mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances vif mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing vif mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.

APOBEC3 degradation is the primary function of HIV-1 Vif determining virion infectivity in the myeloid cell line THP-1.

HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+ T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity in A3F/A3G-null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.

Structural insights into RNA bridging between HIV-1 Vif and antiviral factor APOBEC3G.

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV's counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction.

Identification of Clinically Relevant HIV Vif Protein Motif Mutations through Machine Learning and Undersampling.

Human Immunodeficiency virus (HIV) and its clinical entity, the Acquired Immunodeficiency Syndrome (AIDS) continue to represent an important health burden worldwide. Although great advances have been made towards determining the way viral genetic diversity affects clinical outcome, genetic association studies have been hindered by the complexity of their interactions with the human host. This study provides an innovative approach for the identification and analysis of epidemiological associations between HIV Viral Infectivity Factor (Vif) protein mutations and four clinical endpoints (Viral load and CD4 T cell numbers at time of both clinical debut and on historical follow-up of patients. Furthermore, this study highlights an alternative approach to the analysis of imbalanced datasets, where patients without specific mutations outnumber those with mutations. Imbalanced datasets are still a challenge hindering the development of classification algorithms through machine learning. This research deals with Decision Trees, Naïve Bayes (NB), Support Vector Machines (SVMs), and Artificial Neural Networks (ANNs). This paper proposes a new methodology considering an undersampling approach to deal with imbalanced datasets and introduces two novel and differing approaches (MAREV-1 and MAREV-2). As theses approaches do not involve human pre-determined and hypothesis-driven combinations of motifs having functional or clinical relevance, they provide a unique opportunity to discover novel complex motif combinations of interest. Moreover, the motif combinations found can be analyzed through traditional statistical approaches avoiding statistical corrections for multiple tests.

Various strategies for developing APOBEC3G protectors to circumvent human immunodeficiency virus type 1.

Host restriction factor APOBEC3G (A3G) efficiently restricts Vif-deficient HIV-1 by being packaged with progeny virions and causing the G to A mutation during HIV-1 viral DNA synthesis as the progeny virus infects new cells. HIV-1 expresses Vif protein to resist the activity of A3G by mediating A3G degradation. This process requires the self-association of Vif in concert with A3G proteins, protein chaperones, and factors of the ubiquitination machinery, which are potential targets to discover novel anti-HIV drugs. This review will describe compounds that have been reported so far to inhibit viral replication of HIV-1 by protecting A3G from Vif-mediated degradation.

DNAJB8 facilitates autophagic-lysosomal degradation of viral Vif protein and restricts HIV-1 virion infectivity by rescuing APOBEC3G expression in host cells.

HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.

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.

Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase.

Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-ß and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.

Evolutionary Conservation of PP2A Antagonism and G2/M Cell Cycle Arrest in Maedi-Visna Virus Vif.

The canonical function of lentiviral Vif proteins is to counteract the mutagenic potential of APOBEC3 antiviral restriction factors. However, recent studies have discovered that Vif proteins from diverse HIV-1 and simian immunodeficiency virus (SIV) isolates degrade cellular B56 phosphoregulators to remodel the host phosphoproteome and induce G2/M cell cycle arrest. Here, we evaluate the conservation of this activity among non-primate lentiviral Vif proteins using fluorescence-based degradation assays and demonstrate that maedi-visna virus (MVV) Vif efficiently degrades all five B56 family members. Testing an extensive panel of single amino acid substitution mutants revealed that MVV Vif recognizes B56 proteins through a conserved network of electrostatic interactions. Furthermore, experiments using genetic and pharmacologic approaches demonstrate that degradation of B56 proteins requires the cellular cofactor cyclophilin A. Lastly, MVV Vif-mediated depletion of B56 proteins induces a potent G2/M cell cycle arrest phenotype. Therefore, remodeling of the cellular phosphoproteome and induction of G2/M cell cycle arrest are ancient and conserved functions of lentiviral Vif proteins, which suggests that they are advantageous for lentiviral pathogenesis.

[HIV Restriction Factor APOBEC3G and Prospects for Its Use in Gene Therapy for HIV].

The mechanisms for the protection of the human body from viral or bacterial agents are extremely diverse. In one such mechanism, an important role belongs to the cytidine deaminase APOBEC3 family, which is the factor of congenital immunity and protects the organism from numerous viral agents. One of the proteins of this family, APOBEC3G, is able to protect against Human Immunodeficiency Virus type 1 in the absence of viral protein Vif. In turn, Vif opposes APOBEC3G action, causing polyubiquity of the protein and degradation in the proteasome. The review describes possible ways to increase the anti-HIV activity of APOBEC3G, giving it resistance to viral protein Vif, as well as potential approaches to the use of modified APOBEC3G in gene therapy for HIV.

Aromatic disulfides as potential inhibitors against interaction between deaminase APOBEC3G and HIV infectivity factor.

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC 50 values close to or smaller than 1 µM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.

HIV-1 VIF and human APOBEC3G interaction directly observed through molecular specific labeling using a new dual promotor vector.

Over the last few decades, protein NMR isotope labeling methods using E. coli based expression have revolutionized the information accessible from biomolecular NMR experiments. Selective labeling of a protein of interest in a multi-protein complex can significantly reduce the number of cross-peaks and allow for study of large protein complexes. However, limitations still remain since some proteins are not stable independently and cannot be separately labeled in either NMR active isotope enriched or unenriched media and reconstituted into a multimeric complex. To overcome this limitation, the LEGO NMR method was previously developed using protein expression plasmids containing T7 or araBAD promoters to separately express proteins in the same E. coli after changing between labeled and unlabeled media. Building on this, we developed a method to label the Human Immunodeficiency Virus type 1 viral infectivity factor (HIV-1 Vif), a monomerically unstable protein, in complex with CBFß, it's host binding partner. We designed a dual promoter plasmid containing both T7 and araBAD promoters to independently control the expression of HIV-1 Vif in NMR active isotope enriched media and CBFß in unenriched media. Using this method, we assigned the backbone resonance and directly observed the binding of HIV-1 Vif with APOBEC3G, a host restriction factor to HIV-1.

Host AKT-mediated phosphorylation of HIV-1 accessory protein Vif potentiates infectivity via enhanced degradation of the restriction factor APOBEC3G.

HIV-1 encodes accessory proteins that neutralize antiviral restriction factors to ensure its successful replication. One accessory protein, the HIV-1 viral infectivity factor (Vif), is known to promote ubiquitination and proteasomal degradation of the antiviral restriction factor apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytosine deaminase that leads to hypermutations in the viral DNA and subsequent aberrant viral replication. We have previously demonstrated that the HIV-1 viral transcription mediator Tat activates the host progrowth PI-3-AKT pathway, which in turn promotes HIV-1 replication. Because the HIV-1 Vif protein contains the putative AKT phosphorylation motif RMRINT, here we investigated whether AKT directly phosphorylates HIV-1 Vif to regulate its function. Coimmunoprecipitation experiments showed that AKT and Vif interact with each other, supporting this hypothesis. Using in vitro kinase assays, we further showed that AKT phosphorylates Vif at threonine 20, which promotes its stability, as Vif becomes destabilized after this residue is mutated to alanine. Moreover, expression of dominant-negative kinase-deficient AKT as well as treatment with a chemical inhibitor of AKT increased K48-ubiquitination and proteasomal degradation of HIV-1 Vif. In contrast, constitutively active AKT (Myr-AKT) reduced K48-ubiquitination of Vif to promote its stability. Finally, inhibition of AKT function restored APOBEC3G levels, which subsequently reduced HIV-1 infectivity. Thus, our results establish a novel mechanism of HIV-1 Vif stabilization through AKT-mediated phosphorylation at threonine 20, which reduces APOBEC3G levels and potentiates HIV-1 infectivity.

Specific Deubiquitinating Enzymes Promote Host Restriction Factors Against HIV/SIV Viruses.

Hijacking host ubiquitin pathways is essential for the replication of diverse viruses. However, the role of deubiquitinating enzymes (DUBs) in the interplay between viruses and the host is poorly characterized. Here, we demonstrate that specific DUBs are potent inhibitors of viral proteins from HIVs/simian immunodeficiency viruses (SIVs) that are involved in viral evasion of host restriction factors and viral replication. In particular, we discovered that T cell-functioning ubiquitin-specific protease 8 (USP8) is a potent and specific inhibitor of HIV-1 virion infectivity factor (Vif)-mediated apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3)G (A3G) degradation. Ectopic expression of USP8 inhibited Vif-induced A3G degradation and suppressed wild-type HIV-1 infectivity even in the presence of Vif. In addition, specific DUBs repressed Vpr-, Vpu-, and Vpx-triggered host restriction factor degradation. Our study has revealed a previously unrecognized interplay between the host's DUBs and viral replication. Enhancing the antiviral activity of DUBs therefore represents an attractive strategy against HIVs/SIVs.

The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process.

HIV-1 Vif plays an essential role in viral replication by antagonizing anti-viral cellular restriction factors, a family of APOBEC3 proteins. We have previously shown that naturally-occurring single-nucleotide mutations in the SA1D2prox region, which surrounds the splicing acceptor 1 and splicing donor 2 sites of the HIV-1 genome, dramatically alter the Vif expression level, resulting in variants with low or excessive Vif expression. In this study, we investigated how these HIV-1 variants with poor replication ability adapt and evolve under the pressure of APOBEC3 proteins. Adapted clones obtained through adaptation experiments exhibited an altered replication ability and Vif expression level compared to each parental clone. While various mutations were present throughout the viral genome, all replication-competent adapted clones with altered Vif expression levels were found to bear them within SA1D2prox, without exception. Indeed, the mutations identified within SA1D2prox were responsible for changes in the Vif expression levels and altered the splicing pattern. Moreover, for samples collected from HIV-1-infected patients, we showed that the nucleotide sequences of SA1D2prox can be chronologically changed and concomitantly affect the Vif expression levels. Taken together, these results demonstrated the importance of the SA1D2prox nucleotide sequence for modulating the Vif expression level during HIV-1 replication and adaptation.

APOBEC3B Potently Restricts HIV-2 but Not HIV-1 in a Vif-Dependent Manner.

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

STUB1/CHIP promotes ubiquitination and degradation of HIV-1 Vif to restore the cellular level of APOBEC3G protein.

HIV-1 accessory protein Vif is required for neutralization of cellular restriction factor APOBEC3G through its ubiquitination and proteasomal degradation which allows replication of HIV-1 in non-permissive cells. This function of Vif is required for maintaining the genomic integrity of HIV-1. We here report that the Vif interacts with the cellular E3 ubiquitin ligase CHIP and the level of Vif protein gets reduced by the expression of CHIP. Reduction of Vif by CHIP expression is due to its increased rate of degradation as shown by cycloheximide (CHX) chase assay. CHIP expression also resulted in the ubiquitination of Vif protein in a dose dependent manner. The role of CHIP in the ubiquitination and degradation was confirmed by the endogenous knockdown of CHIP using CRISPR Cas9 method. Loss of endogenous CHIP protein showed the stabilization of Vif with concomitant destabilization of APOBEC3G. As expected Vif mediated ubiquitination of APOBEC3G was also reduced in CHIP knockdown cells. These results established that CHIP functions as a negative regulator of Vif protein which in-turn stabilizes APOBEC3G.