Degradation-Independent Inhibition of APOBEC3G by the HIV-1 Vif Protein.
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ABSTRACT:
SUBMITTER: Stupfler B
PROVIDER: S-EPMC8066197 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The human APOBEC3G (A3G) enzyme restricts HIV-1 in the absence of the viral accessory protein viral infectivity factor (Vif) by deaminating viral cDNA cytosines to uracils. These uracil lesions base-pair with adenines during the completion of reverse transcription and result in A3G signature G-to-A mutations in the viral genome. Vif protects HIV-1 from A3G-mediated restriction by forming an E3-ubiquitin ligase complex to polyubiquitinate A3G and trigger its degradation. Prior studies indicated that Vif may also directly block the enzymatic activity of A3G and, provocatively, that Vif-derived peptides, Vif 25-39 and Vif 105-119, are similarly inhibitory. Here, we show that Vif 25-39 does not inhibit A3G enzymatic activity and that the inhibitory effect of Vif 105-119 and that of a shorter derivative Vif 107-115, although recapitulated, are non-specific. We also elaborate a simple method for assaying DNA cytosine deaminase activity that eliminates potential polymerase chain reaction-induced biases. Our results show that these Vif-derived peptides are unlikely to be useful as tools to study A3G function or as leads for the development of future therapeutics.
| S-EPMC5186364 | biostudies-literature
Project description:APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-) T cells and had an IC50 as low as 8.4 ?M and a TC50 of >100 ?M when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 ?M). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.
| S-EPMC4400358 | biostudies-literature
Project description:Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) is a host restriction factor that impedes HIV-1 replication. Viral integrity is salvaged by HIV-1 virion infectivity factor (Vif), which mediates A3G polyubiquitination and subsequent cellular depletion. Previous studies have implied that A3G polyubiquitination is essential for Vif-induced degradation. However, the contribution of polyubiquitination to the rate of A3G degradation remains unclear. Here, we show that A3G polyubiquitination is essential for degradation. Inhibition of ubiquitin-activating enzyme E1 by PYR-41 or blocking the formation of ubiquitin chains by over-expressing the lysine to arginine mutation of ubiquitin K48 (K48R) inhibited A3G degradation. Our A3G mutagenesis study showed that lysine residues 297, 301, 303, and 334 were not sufficient to render lysine-free A3G sensitive to Vif-mediated degradation. Our data also confirm that Vif could induce ubiquitin chain formation on lysine residues interspersed throughout A3G. Notably, A3G degradation relied on the lysine residues involved in polyubiquitination. Although A3G and the A3G C-terminal mutant interacted with Vif and were modified by ubiquitin chains, the latter remained more resistant to Vif-induced degradation. Furthermore, the A3G C-terminal mutant, but not the N-terminal mutant, maintained potent antiviral activity in the presence of Vif. Taken together, our results suggest that the location of A3G ubiquitin modification is a determinant for Vif-mediated degradation, implying that in addition to polyubiquitination, other factors may play a key role in the rate of A3G degradation.
| S-EPMC5010486 | biostudies-other
Project description:UnlabelledVif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G.ImportanceIn this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.
| S-EPMC3993832 | biostudies-literature
Project description:The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.
| S-EPMC7153695 | biostudies-literature
Project description:Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged ?4-?4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.
| S-EPMC4670588 | biostudies-literature
Project description:Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.
| S-EPMC5125868 | biostudies-literature
Project description:The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
| S-EPMC3648125 | biostudies-literature
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.
| S-EPMC4403497 | biostudies-literature
Project description: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 ?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.
| S-EPMC3139112 | biostudies-literature