Human Immunodeficiency Virus Type 1 Vif Research Articles

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 β 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.

Human Immunodeficiency Virus Type 1 Vif Up-Regulates the Expression of Tat via AKT Signaling Pathway: Role of Ubiquitin Specific Protease 17.

Human immunodeficiency virus type 1 (HIV-1) has RNA genome and depends on host cellular machinery for most of its activities. Host cellular proteins modulate the expression and activity of viral proteins to combat the virus. HIV-1 proteins are known to regulate each other for the benefit of virus by exploiting these modulations. Here, we report that HIV-1 Vif increases the levels of Tat via AKT signaling pathway. We show that HIV-1 Vif activates AKT signaling pathway by inducing phosphorylation of AKT. Mdm2, downstream target of AKT signaling, increases the levels of Tat protein in ubiquitin-dependent manner by inducing Ubiquitin Specific Protease 17 (USP17), which is a deubiquitinase and stabilizes Tat protein. Thus, HIV-1 proteins exploit AKT signaling pathway to promote viral replication.

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

HIV-1 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 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 its cellular cofactor CBFβ, at 3.9 Å resolution. The structure shows that Vif and CBFβ form a platform to recruit A3F, revealing a direct A3F-recruiting role of CBFβ 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.

Simian Immunodeficiency Virus Vif and Human APOBEC3B Interactions Resemble Those between HIV-1 Vif and Human APOBEC3G.

Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient human immunodeficiency virus type 1 (HIV-1) by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire, and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, simian immunodeficiency virus SIVmac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include degradation of several human APOBEC3s and even human APOBEC3B, a non-HIV-1-restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIVmac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIVmac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap HIV-1 Vif surface residues that interact with human APOBEC3G and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIVmac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to the growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution.IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIVmac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire, including APOBEC3B. Understanding the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIVmac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes.

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.

FoxO4 negatively controls Tat-mediated HIV-1 transcription through the post-transcriptional suppression of Tat encoding mRNA.

The connection between the repression of human immunodeficiency virus type 1(HIV-1) transcription and the resting CD4+ T cell state suggests that the host transcription factors involved in the active maintenance of lymphocyte quiescence are likely to repress the viral transactivator, Tat, thereby restricting HIV-1 transcription. In this study, we analysed the interplay between Tat and the forkhead box transcription factors, FoxO1 and FoxO4. We show that FoxO1 and FoxO4 antagonize Tat-mediated transactivation of HIV-1 promoter through the repression of Tat protein expression. No effect was observed on the expression of two HIV-1 accessory proteins, Vif and Vpr. Unexpectedly, we found that FoxO1 and FoxO4 expression causes a strong dose-dependent post-transcriptional suppression of Tat mRNA, indicating that FoxO should effectively inhibit HIV-1 replication by destabilizing Tat mRNA and suppressing Tat-mediated HIV-1 transcription. In accordance with this, we observed that the Tat mRNA half-life is reduced by FoxO4 expression. The physiological relevance of our findings was validated using the J-Lat 10.6 model of latently infected cells. We demonstrated that the overexpression of a constitutively active FoxO4-TM mutant antagonized HIV-1 transcription reactivation in response to T cell activators, such as TNF-α or PMA. Altogether, our findings demonstrate that FoxO factors can control HIV-1 transcription and provide new insights into their potential role during the establishment of HIV-1 latency.

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

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

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

The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C.

The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

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

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

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

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

Requirement of HIV-1 Vif C-terminus for Vif-CBF-β interaction and assembly of CUL5-containing E3 ligase.

BackgroundHuman immunodeficiency virus type 1 (HIV-1) Vif hijacks an E3 ligase to suppress natural APOBEC3 restriction factors, and core binding factor β (CBF-β) is required for this process. Although an extensive region of Vif spanning most of its N-terminus is known to be critical for binding with CBF-β, involvement of the Vif C-terminus in the interaction with CBF-β has not been fully investigated.ResultsHere, through immunoprecipitation analysis of Vif C-terminal truncated mutants of various lengths, we identified that CBF-β binding requires not only certain amino acids (G126A, E134A, Y135A and G138A) in the HCCH region but also the HCCH motif itself, which also affects the Vif-mediated suppression of APOBEC3G/APOBEC3F (A3G/A3F). These mutants still maintained interactions with substrate A3G or A3F as well as other cellular factors ElonginB/C (ELOB/C), indicating that their structures were not functionally affected. Moreover, by determining that the BC box also is necessary for CBF-β interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-β and consequent interaction with CUL5.ConclusionsThese results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase. Identification of the new binding interface with CBF-β at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors.

Identification of HIV-1 Vif regions required for CBF-β interaction and APOBEC3 suppression.

Human immunodeficiency virus type 1 (HIV-1) Vif requires core binding factor β (CBF-β) to degrade the host APOBEC3 restriction factors. Although a minimum domain and certain amino acids of HIV-1 Vif, including hydrophobic residues at the N-terminal, have been identified as critical sites for binding with CBF-β, other regions that potentially mediate this interaction need to be further investigated. Here, we mapped two new regions of HIV-1 Vif that are required for interaction with CBF-β by generating a series of single-site or multiple-site Vif mutants and testing their effect on the suppression of APOBEC3G (A3G) and APOBEC3F (A3F). A number of the mutants, including G84A/SIEW86-89AAAA (84/86–89), E88A/W89A (88/89), G84A, W89A, L106S and I107S in the 84GxSIEW89 and L102ADQLI107 regions, affected Vif function by disrupting CBF-β binding. These Vif mutants also had altered interactions with CUL5, since CBF-β is known to facilitate the binding of Vif to CUL5. We further showed that this effect was not due to misfolding or conformational changes in Vif, as the mutants still maintained their interactions with other factors such as ElonginB, A3G and A3F. Notably, G84D and D104A had stronger effects on the Vif-CUL5 interaction than on the Vif-CBF-β interaction, indicating that they mainly influenced the CUL5 interaction and implying that the interaction of Vif with CUL5 contributes to the binding of Vif to CBF-β. These new binding interfaces with CBF-β in HIV-1 Vif provide novel targets for the development of HIV-1 inhibitors.

Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-β promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-β is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-β from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-β interfaces. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-β) is a novel regulator of Vif-CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-β can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-β from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-β in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1.

Dispersed and Conserved Hydrophobic Residues of HIV-1 Vif Are Essential for CBFβ Recruitment and A3G Suppression

CBFβ was recently found to be a key regulator of the ability of human immunodeficiency virus type 1 (HIV-1) Vif to overcome host antiviral APOBEC3 proteins. However, the detailed molecular requirements for the Vif-CBFβ interaction are still not clear. Here, we mapped the minimum Vif domain required for CBFβ binding. In terms of CBFβ binding, the Vif N terminus was very sensitive to deletions. We determined that the Vif fragment from residues 5 to 126 was sufficient to form a stable complex with CBFβ in vitro. We also observed that ionic interactions were not the main contributor to the interaction between Vif and CBFβ. Instead, hydrophobic interactions were important for maintaining the Vif-CBFβ complex, since it could be disrupted by nonionic detergent. Site-directed mutagenesis of conserved hydrophobic amino acids revealed novel residues in Vif that were important for CBFβ binding and APOBEC3 inactivation. At least part of the well-characterized HCCH domain (residues 108 to 139) was required to form a stable Vif-CBFβ complex. Thus, the HCCH motif may have a dual role in binding both Cul5 and CBFβ. Considering the importance of Vif in HIV-1 infection, this unique Vif-CBFβ interaction represents an attractive pharmacological intervention target against HIV-1. Vif-induced APOBEC3 protein degradation was the first host antiviral mechanism against HIV-1/simian immunodeficiency virus to be revealed, yet details regarding which proteins are degraded are not fully demonstrated. Recently, host cellular factor CBFβ was found to be essential for Vif to function and promote viral infectivity. In this study, we present more critical information on the Vif-CBFβ interaction by revealing that hydrophobicity contributes the most to the Vif-CBFβ interaction and locating several novel hydrophobic sites (tryptophans and phenylalanines) that are conserved among Vif proteins from different lentiviruses and essential for Vif binding to CBFβ. Mutations on these sites result in a reduced/abolished Vif-CBFβ interaction, leading to the attenuated potency of Vif on both inducing the degradation of antiviral factors like APOBEC3G and promoting HIV-1 infectivity. Therefore, information from this study will help people to further understand how Vif acts against host antiviral mechanism, which is important for novel anti-HIV-1 drug development.

A Naturally Occurring Vif Mutant (I107T) Attenuates Anti-APOBEC3G Activity and HIV-1 Replication

The human immunodeficiency virus type 1 (HIV-1) Vif protein counteracts the antiviral activity of the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of proteins by targeting the proteins for degradation through the ubiquitin–proteasome pathway. Previous mutagenic studies have shown that multiple domains of Vif are required for interacting with APOBEC3G proteins and the proteasome pathway. However, very few mutagenesis and functional analyses of patient-derived Vif proteins have been conducted. In this study, we amplified and cloned the HIV-1 vif genes from the peripheral blood mononuclear cells (PBMCs) of five HIV-1-infected individuals in Nairobi and further tested the impact of the genes on anti-A3G activity and HIV-1 replication. The gene sequence analysis revealed high genetic variation of vif genes from different HIV-1-infected individuals. Interestingly, the Vif proteins derived from two of the three long-term survivors (LTSs) displayed a significantly impaired ability to mediate the degradation of A3G. In particular, a single amino acid change (I107T) in one of the non-functional LTS Vif variants, which has not been previously identified in the Los Alamos databases of vif sequences, was found to be responsible for the lack of anti-A3G activity. Further study demonstrated that HIV-1 carrying an I107T Vif mutation displayed significantly reduced fitness in A3G+ T cells and PBMCs. Moreover, co-infecting A3G+ T cells with both the wild-type and I107T Vif viruses resulted in decreased viral replication. Overall, the results of this study indicate that the HIV-1 Vif residue I107 is important for its anti-APOBEC3G activity and viral replication, which may have implications for viral fitness in vivo.

HIV Type 1 Viral Infectivity Factor and the RUNX Transcription Factors Interact with Core Binding Factor β on Genetically Distinct Surfaces

Human immunodeficiency virus type 1 (HIV-1) requires the cellular transcription factor core binding factor subunit β (CBFβ) to stabilize its viral infectivity factor (Vif) protein and neutralize the APOBEC3 restriction factors. CBFβ normally heterodimerizes with the RUNX family of transcription factors, enhancing their stability and DNA-binding affinity. To test the hypothesis that Vif may act as a RUNX mimic to bind CBFβ, we generated a series of CBFβ mutants at the RUNX/CBFβ interface and tested their ability to stabilize Vif and impact transcription at a RUNX-dependent promoter. While several CBFβ amino acid substitutions disrupted promoter activity, none of these impacted the ability of CBFβ to stabilize Vif or enhance degradation of APOBEC3G. A mutagenesis screen of CBFβ surface residues identified a single amino acid change, F68D, that disrupted Vif binding and its ability to degrade APOBEC3G. This mutant still bound RUNX and stimulated RUNX-dependent transcription. These separation-of-function mutants demonstrate that HIV-1 Vif and the RUNX transcription factors interact with cellular CBFβ on genetically distinct surfaces.

Signals in APOBEC3F N-terminal and C-terminal Deaminase Domains Each Contribute to Encapsidation in HIV-1 Virions and Are Both Required for HIV-1 Restriction

Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) inhibit human immunodeficiency virus type-1 (HIV-1) replication. In the absence of HIV-1 Vif, A3F and/or A3G are incorporated into assembling virions and exert antiviral functions in subsequently infected target cells. Encapsidation of A3F or A3G within the protease-matured virion core following their incorporation into virions is hypothesized to be important for the antiviral function of these proteins. In this report, we demonstrated that A3F was quantitatively encapsidated in the mature virion core. In distinct contrast, A3G was distributed both within and outside of the virion core. Analysis of a series of A3F-A3G chimeras comprised of exchanged N- and C-terminal deaminase domains identified a 14 amino acid segment in the A3F C-terminal deaminase domain that contributed to preferential encapsidation and anti-HIV activity. Amino acid residue L306 in this C-terminal segment was determined to be necessary, but not sufficient, for these effects. Amino acid residue W126 in the N-terminal deaminase domain was determined also to contribute to preferential encapsidation and antiviral activity of A3F. Analysis of the A3F (W126A L306A) double mutant revealed that both residues are required for full anti-HIV function. The results reported here advance our understanding of the mechanisms of A3F virion encapsidation and antiviral function and may lead to innovative strategies to inhibit HIV-1 replication.

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

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

Multifaceted Counter-APOBEC3G Mechanisms Employed by HIV-1 Vif

In the absence of human immunodeficiency virus type 1 (HIV-1) Vif protein, the host antiviral deaminase apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative single-stranded DNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here, we show that (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δ vif-virus-associated A3G, (ii) encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) purified HIV-1 Vif protein and the Vif-derived peptide Vif25–39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G antiviral activity.