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.

Stability of APOBEC3F in the Presence of the APOBEC3 Antagonist HIV-1 Vif Increases at the Expense of Co-Expressed APOBEC3H Haplotype I.

The seven human APOBEC3 enzymes (APOBEC3A through H, excluding E) are host restriction factors. Most of the APOBEC3 enzymes can restrict HIV-1 replication with different efficiencies. The HIV-1 Vif protein combats APOBEC3-mediated restriction by inducing ubiquitination and degradation in the proteasome. APOBEC3F and APOBEC3G can hetero-oligomerize, which increases their restriction capacity and resistance to Vif. Here we determined if APOBEC3C, APOBEC3F, or APOBEC3G could hetero-oligomerize with APOBEC3H haplotype I. APOBEC3H haplotype I has a short half-life in cells due to ubiquitination and degradation by host proteins, but is also resistant to Vif. We hypothesized that hetero-oligomerization with APOBEC3H haplotype I may result in less Vif-mediated degradation of the interacting APOBEC3 and stabilize APOBEC3H haplotype I, resulting in more efficient HIV-1 restriction. Although we found that all three APOBEC3s could interact with APOBEC3H haplotype I, only APOBEC3F affected APOBEC3H haplotype I by surprisingly accelerating its proteasomal degradation. However, this increased APOBEC3F levels in cells and virions in the absence or presence of Vif and enabled APOBEC3F-mediated restriction of HIV-1 in the presence of Vif. Altogether, the data suggest that APOBEC3 enzymes can co-regulate each other at the protein level and that they cooperate to ensure HIV-1 inactivation rather than evolution.

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.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

[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.

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.

HIV-1 Vif Gained Breadth in APOBEC3G Specificity after Cross-Species Transmission of Its Precursors.

APOBEC3G (A3G) is a host-encoded cytidine deaminase that potently restricts retroviruses such as HIV-1 and depends on its ability to package into virions. As a consequence of this, HIV-1 protein Vif has evolved to antagonize human A3G by targeting it for ubiquitination and subsequent degradation. There is an ancient arms race between Vif and A3G highlighted by amino acids 128 and 130 in A3G that have evolved under positive selection due to Vif-mediated selective pressure in Old World primates. Nonetheless, not all possible amino acid combinations at these sites have been sampled by nature, and the evolutionary potential of species to resist Vif antagonism is not clear. To explore the evolutionary space of positively selected sites in the Vif-binding region of A3G, we designed a combinatorial mutagenesis screen to introduce all 20 amino acids at sites 128 and 130. Our screen uncovered mutants of A3G with several interesting phenotypes, including loss of antiviral activity and resistance of Vif antagonism. However, HIV-1 Vif exhibited remarkable flexibility in antagonizing A3G 128 and 130 mutants, which significantly reduces viable Vif resistance strategies for hominid primates. Importantly, we find that broadened Vif specificity was conferred through loop 5 adaptations that were required for cross-species adaptation from Old World monkey A3G to hominid A3G. Our evidence suggests that Vif adaptation to novel A3G interfaces during cross-species transmission may train Vif toward broadened specificity that can further facilitate cross-species transmissions and raise the barrier to host resistance. IMPORTANCE APOBEC3G (A3G) is an antiviral protein that potently restricts retroviruses like HIV. In turn, the HIV-1 protein Vif has evolved to antagonize A3G through degradation. Two rapidly evolving sites in A3G confer resistance to unadapted Vif and act as a barrier to cross-species transmission of retroviruses. We recently identified a single amino acid mutation in a simian immunodeficiency virus (SIV) Vif that contributed to the cross-species origins of SIV infecting chimpanzee and, ultimately, the HIV-1 pandemic. This mutation broadened specificity of this Vif to both antagonize the A3G of its host while simultaneously overcoming the A3G barrier in the great apes. In this work, we explore the evolutionary space of human A3G at these rapidly evolving sites to understand if the broadened Vif specificity gained during cross-species transmission confers an advantage to HIV-1 Vif in its host-virus arms race with A3G.

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.

Degradation-Independent Inhibition of APOBEC3G by the HIV-1 Vif Protein.

The ubiquitin-proteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.

Demystifying Cell Cycle Arrest by HIV-1 Vif.

Although APOBEC3 degradation is the canonical function of HIV-1 Vif, this viral protein also induces potent cell cycle arrest through a newly defined mechanism. Here, we review recent advances in this area and propose that the scope of this activity may go beyond subversion of the host cell cycle.

Multifaceted HIV-1 Vif interactions with human E3 ubiquitin ligase and APOBEC3s.

APOBEC3 (A3) proteins are a family of host antiviral restriction factors that potently inhibit various retroviral infections, including human immunodeficiency virus (HIV)-1. To overcome this restriction, HIV-1 virion infectivity factor (Vif) recruits the cellular cofactor CBFß to assist in targeting A3 proteins to a host E3 ligase complex for polyubiquitination and subsequent proteasomal degradation. Intervention of the Vif-A3 interactions could be a promising therapeutic strategy to facilitate A3-mediated suppression of HIV-1 in patients. In this structural snapshot, we review the structural features of the recently determined structure of human A3F in complex with HIV-1 Vif and its cofactor CBFß, discuss insights into the molecular principles of Vif-A3 interplay during the arms race between the virus and host, and highlight the therapeutic implications.

Loop 1 of APOBEC3C Regulates its Antiviral Activity against HIV-1.

APOBEC3 deaminases (A3s) provide mammals with an anti-retroviral barrier by catalyzing dC-to-dU deamination on viral ssDNA. Within primates, A3s have undergone a complex evolution via gene duplications, fusions, arms race, and selection. Human APOBEC3C (hA3C) efficiently restricts the replication of viral infectivity factor (vif)-deficient Simian immunodeficiency virus (SIVΔvif), but for unknown reasons, it inhibits HIV-1Δvif only weakly. In catarrhines (Old World monkeys and apes), the A3C loop 1 displays the conserved amino acid pair WE, while the corresponding consensus sequence in A3F and A3D is the largely divergent pair RK, which is also the inferred ancestral sequence for the last common ancestor of A3C and of the C-terminal domains of A3D and A3F in primates. Here, we report that modifying the WE residues in hA3C loop 1 to RK leads to stronger interactions with substrate ssDNA, facilitating catalytic function, which results in a drastic increase in both deamination activity and in the ability to restrict HIV-1 and LINE-1 replication. Conversely, the modification hA3F_WE resulted only in a marginal decrease in HIV-1Δvif inhibition. We propose that the two series of ancestral gene duplications that generated A3C, A3D-CTD and A3F-CTD allowed neo/subfunctionalization: A3F-CTD maintained the ancestral RK residues in loop 1, while diversifying selection resulted in the RK â†’ WE modification in Old World anthropoids' A3C, possibly allowing for novel substrate specificity and function.

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.