Dataset Information

The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G, APOBEC3F, and APOBEC3H.

ABSTRACT: The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48. The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-APOBEC3 interface.

SUBMITTER: Binka M

PROVIDER: S-EPMC3255910 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G, APOBEC3F, and APOBEC3H.

Binka Mawuena M Ooms Marcel M Steward Myeika M Simon Viviana V

Journal of virology 20111019 1

The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A ...[more]

PMID: 22013041

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Project description:UNLABELLED:The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCE:HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

Project description:Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H ?-helixes ?3 and ?4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes ?3 and ?4 interact with the Vif ?-sheet (?2-?5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif ?-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

Project description:UnlabelledHIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. The HIV-1 accessory protein Vif (virion infectivity factor) enhances viral infectivity through anti-retroviral factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) degradation; however, little is known regarding Vif function in EC. Here, the anti-APOBEC3G activities of clonal, plasma HIV RNA-derived Vif sequences from 46 EC, 46 noncontrollers (NC), and 44 individuals with acute infection (AI) were compared. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viruses were generated by cotransfecting 293T cells with expression plasmids encoding patient-derived Vif, human APOBEC3G, VSV-G, and a vif/env-deficient luciferase-reporter HIV-1 proviral DNA clone. Viral stocks were used to infect 293T cells, and Vif anti-APOBEC3G activity was quantified in terms of luciferase signal. On average, the anti-APOBEC3G activities of EC-derived Vif sequences (median log10 relative light units [RLU], 4.54 [interquartile range {IQR}, 4.30 to 4.66]) were significantly lower than those of sequences derived from NC (4.75 [4.60 to 4.92], P < 0.0001) and AI (4.74 [4.62 to 4.94], P < 0.0001). Reduced Vif activities were not associated with particular HLA class I alleles expressed by the host. Vif functional motifs were highly conserved in all patient groups. No single viral polymorphism could explain the reduced anti-APOBEC3G activity of EC-derived Vif, suggesting that various combinations of minor polymorphisms may underlie these effects. These results further support the idea of relative attenuation of viral protein function in EC-derived HIV sequences.ImportanceHIV-1 elite controllers (EC) are rare individuals who are able to control plasma viremia to undetectable levels without antiretroviral therapy. Understanding the pathogenesis and mechanisms underpinning this rare phenotype may provide important insights for HIV vaccine design. The EC phenotype is associated with beneficial host immunogenetic factors (such as HLA-B*57) as well as with functions of attenuated viral proteins (e.g., Gag, Pol, and Nef). In this study, we demonstrated that HIV-1 Vif sequences isolated from EC display relative impairments in their ability to counteract the APOBEC3G host restriction factor compared to Vif sequences from normal progressors and acutely infected individuals. This result extends the growing body of evidence demonstrating attenuated HIV-1 protein function in EC and, in particular, supports the idea of the relevance of viral factors in contributing to this rare HIV-1 phenotype.

Project description:UnlabelledThe multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF(JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA.ImportanceVif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly, fixation of mutations that replaced H42/43D or W79S in viral RNA lagged behind the appearance of high viral loads. In one exceptional JRCSFvifH42/43D infection, vif was unchanged but replication proceeded after a long delay. These results suggest that Vif binds and inhibits the non-cytosine deaminase activities of intact A3G and intact A3F, allowing JRCSFvifH42/43D and JRCSFvifW79S to replicate with reduced fitness. Subsequently, enhanced Vif function is acquired by enforced mutations. In infected cells, JRCSFΔvif and JRCSFvifH42/43DW79S are exposed to active A3F and A3G and fail to replicate. JRCSFvifH42/43D Vif degrades A3F and, in some cases, overcomes A3G mutagenic activity to replicate. Vif may have evolved to inhibit A3F and A3G by stoichiometric binding and subsequently acquired the ability to target these proteins to proteasomes.

Dataset Information

The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G, APOBEC3F, and APOBEC3H.

Publications

The activity spectrum of Vif from multiple HIV-1 subtypes against APOBEC3G, APOBEC3F, and APOBEC3H.

Similar Datasets

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