The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3, referred to as A3) proteins are cellular cytidine deaminases that potently restrict retrovirus replication. However, HIV-1 viral infectivity factor (Vif) counteracts the antiviral activity of most A3 proteins by targeting them for proteasomal degradation. To date, the structure of an A3 protein containing a Vif-binding interface has not been solved. Here, we report a high-resolution crystal structure of APOBEC3C and identify the HIV-1 Vif-interaction interface. Extensive structure-guided mutagenesis revealed the role of a shallow cavity composed of hydrophobic or negatively charged residues between the α2 and α3 helices. This region is distant from the DPD motif (residues 128-130) of APOBEC3G that participates in HIV-1 Vif interaction. These findings provide insight into Vif-A3 interactions and could lead to the development of new pharmacologic anti-HIV-1 compounds.
Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant (p. I231V, with allele (V) frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) (RH = 0.53, 95% CI: 0.37-0.76). A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression.
Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV), Hepatitis B virus (HBV), Human Papilloma virus (HPV), and Human T Cell Leukemia virus (HTLV). The best characterized members of this family are APOBEC3G (A3G) and APOBEC3F (A3F) and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif). Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.
The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminate how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.
Human DNA cytosine-to-uracil deaminases catalyze mutations in both pathogen and cellular genomes. APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H restrict human immunodeficiency virus 1 (HIV-1) infection in cells deficient in the viral infectivity factor (Vif), and have the potential to catalyze sublethal levels of mutation in viral genomes in Vif-proficient cells. At least two APOBEC3 enzymes, and in particular APOBEC3B, are sources of somatic mutagenesis in cancer cells that drive tumor evolution and may manifest clinically as recurrence, metastasis, and/or therapy resistance. Consequently, APOBEC3 enzymes are tantalizing targets for developing chemical probes and therapeutic molecules to harness mutational processes in human disease. This review highlights recent efforts to chemically manipulate APOBEC3 activities.
Viral infectivity factor (Vif) is protective against APOBEC3G (A3G)-mediated viral cDNA hypermutations, and development of molecules that inhibit Vif mediated A3G degradation is a novel strategy for blocking HIV-1 replication. Through optimizations of the central ring of N-(2-methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (RN-18), we found a potent compound 12c with EC50 value of 1.54 μM, enhancing the antiviral activity more than 150-fold compared with RN-18 in nonpermissive H9 cells. 12c protected A3G from degradation by inhibiting Vif function. Besides, 12c suppressed different HIV-1 clinical strains (HIV-1KM018, HIV-1TC-1 and HIV-1WAN) and drug-resistant strains (NRTI, NNRTI, PI, and FI) with relatively high activities. Amidation of 12c with glycine gave a prodrug 13a, improving the water solubility about 2600-fold compared with 12c. Moreover, 13a inhibited the virus replication efficiently with an EC50 value of 0.228 μM. These results suggested that the prodrug 13a is a promising candidate agent for the treatment of AIDS.
The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5'-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.
The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4+ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine to adenine hypermutation through deamination of cytosine to form uracil in (-)DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity than A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 alone, A3G and A3F when combined had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer, in the absence of RNA, which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded (-)DNA exposed for longer periods of time enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties when compared to each individual counterpart.
HIV type 1 overcomes the host restriction factor apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing steady-state level of Vif protein, however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have previously reported that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2, and that over-expression of CBFβ disrupted the interaction between MDM2 and Vif. Knockdown of CBFβ reduced steady-state level of Vif in MDM2-proficient cells, but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ , degraded faster than wild-type Vif in MDM2-proficient cells, but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ .