Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses.     

HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation

The viral infectivity factor (Vif) encoded by HIV-1 neutralizes a potent antiviral pathway that occurs in human T lymphocytes and several leukemic T-cell lines termed nonpermissive, but not in other cells termed permissive. In the absence of Vif, this antiviral pathway efficiently inactivates HIV-1. It was recently reported that APOBEC3G (also known as CEM-15), a cytidine deaminase nucleic acid-editing enzyme, confers this antiviral phenotype on permissive cells. Here we describe evidence that Vif binds APOBEC3G and induces its rapid degradation, thus eliminating it from cells and preventing its incorporation into HIV-1 virions. Studies of Vif mutants imply that it contains two domains, one that binds APOBEC3G and another with a conserved SLQ(Y/F)LA motif that mediates APOBEC3G degradation by a proteasome-dependent pathway. These results provide promising approaches for drug discovery.     

APOBEC3G与Vif在HIV-1感染中的作用

载脂蛋白B mRNA编辑催化多肽样（apolipoprotein B mRNA-editing catalytic polypeptide-like,APOBEC）蛋白是一组胞嘧啶脱氨基酶,具有天然的抗病毒活性,对多种病毒具有抑制作用,特别是逆转录病毒. APOBEC3蛋白能够抑制人类免疫缺陷病毒(HIV-1)的感染,其中APOBEC3G和APOBEC3F的作用最强. APOBEC3G能够通过胞嘧啶脱氨基作用和非胞嘧啶脱氨基作用抑制病毒感染. HIV-1病毒感染因子(Vif) 蛋白主要经泛素-蛋白酶体途径介导APOBEC3G降解,从而拮抗其抗病毒作用. APOBEC3G和Vif之间相互作用的研究对于寻求新的抗HIV治疗靶点具有重要意义.     

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

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.     

HIV-1 Vif and APOBEC3G: Multiple roads to one goal

The viral infectivity factor, Vif, of human immunodeficiency virus type 1, HIV-1, has long been shown to promote viral replication in vivo and to serve a critical function for productive infection of non-permissive cells, like peripheral blood mononuclear cells (PBMC). Vif functions to counteract an anti-retroviral cellular factor in non-permissive cells named APOBEC3G. The current mechanism proposed for protection of the virus by HIV-1 Vif is to induce APOBEC3G degradation through a ubiquitination-dependent proteasomal pathway. However, a new study published in Retrovirology by Strebel and colleagues suggests that Vif-induced APOBEC3G destruction may not be required for Vif's virus-protective effect. Strebel and co-workers show that Vif and APOBEC3G can stably co-exist, and yet viruses produced under such conditions are fully infectious. This new result highlights the notion that depletion of APOBEC3G is not the sole protective mechanism of Vif and that additional mechanisms exerted by this protein can be envisioned which counteract APOBEC3G and enhance HIV infectivity.     

Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, or A3G) and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F) are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus (HIV)-1 requires suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5, ElonginB, and ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. Domains in Vif that mediate APOBEC3 recognition have not been fully characterized. In the present study, we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif, which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion, and functional suppression of the A3G antiviral activity. Amino acids 52 to 72 of HIV-1 Vif (including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G, and this interaction was abolished by mutations of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G, yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G, suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44, which is only important for A3G suppression, the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression. On the other hand, another motif, TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition. Both the VxIPLx_4-5LxΦx₂YWxL and TGERxW motifs are highly conserved among HIV-1, HIV-2, and various simian immunodeficiency virus Vif proteins. Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3 proteins through conserved structural features that represent attractive targets for the development of novel inhibitors.     

Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant

Human immunodeficiency virus type 1 (HIV-1) Vif counteracts the antiviral activity of the human cytidine deaminase APOBEC3G (APO3G) by inhibiting its incorporation into virions. This has been attributed to the Vif-induced degradation of APO3G by cytoplasmic proteasomes. We recently demonstrated that although APO3G has a natural tendency to form RNA-dependent homo-multimers, multimerization was not essential for encapsidation into HIV-1 virions or antiviral activity. We now demonstrate that a multimerization-defective APO3G variant (APO3G C97A) is able to assemble into RNase-sensitive high-molecular-mass (HMM) complexes, suggesting that homo-multimerization of APO3G and assembly into HMM complexes are unrelated RNA-dependent processes. Interestingly, APO3G C97A was highly resistant to Vif-induced degradation even though the two proteins were found to interact in coimmunoprecipitation experiments and exhibited partial colocalization in transfected HeLa cells. Surprisingly, encapsidation and antiviral activity of APO3G C97A were both inhibited by Vif despite resistance to degradation. These results demonstrate that targeting of APO3G to proteasome degradation and interference with viral encapsidation are distinct functional properties of Vif.     

APOBEC3F Determinants of HIV-1 Vif Sensitivity

HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus-human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the α3 and α4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.     

The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif

The human protein apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), also known as CEM-15, mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates. Because DNA deamination takes place after virus entry into target cells, APOBEC3G function is dependent on its association with the viral nucleoprotein complexes that synthesize cDNA and must therefore be incorporated into virions as they assemble in infected cells. Here we show that the HIV-1 virion infectivity factor (Vif) protein protects the virus from APOBEC3G-mediated inactivation by preventing its incorporation into progeny virions, thus allowing the ensuing infection to proceed without DNA deamination. In addition to helping exclude APOBEC3G from nascent virions, Vif also removes APOBEC3G from virus-producing cells by inducing its ubiquitination and subsequent degradation by the proteasome. Our findings indicate that pharmacologic strategies aimed at stabilizing APOBEC3G in HIV-1 infected cells should be explored as potential HIV/AIDS therapeutics.     

Status of APOBEC3G/F in cells and progeny virions modulated by Vif determines HIV-1 infectivity

We examined various HIV-1 Vif mutants for interaction with APOBEC3 proteins (A3G/A3F). All replication-defective proviral mutants were found to carry A3G/A3F in virions, and of these, a replication-defective mutant with Vif that binds to A3G in cells but not in virions was noted. Furthermore, a mutant Vif protein that suppresses A3F activity but does not exclude A3F from virions was identified. We also showed that incorporation of Vif into virions is dependent on its interaction with A3G/A3F. Taken together, we concluded that functional binding of Vif to A3G/A3F in cells and/or virions is critical for viral infectivity.     

Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function

The human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif) overcomes the antiviral activity of APOBEC3G to protect HIV-1 DNA from G-to-A hypermutation. Vif targets APOBEC3G for ubiquitination and proteasomal degradation by forming an SCF-like E3 ubiquitin ligase complex composed of Cullin5, Elongin B, and Elongin C (Vif-BC-Cul5) through a novel SOCS-box motif. In this paper, we have established an in vitro ubiquitin conjugation assay with purified Vif-BC-Cul5 complex and reported that the Vif-BC-Cul5 complex could function as an E3 ligase for APOBEC3G in vitro. A Vif-BC-Cul5 complex promotes the in vitro ubiquitination of the wild type, APOBEC3G but not that of D128K mutant, which does not interact with Vif. We have also investigated several loss-of-function Vif mutants. One mutant, SLQ144/146AAA, lost its activity on APOBEC3G because it could not form a complex due to mutations in SOCS-box motif. Other mutants, C114S and C133S, also lost their activity because of loss of the E3 ligase activity of a Vif-BC-Cul5 complex, although these mutants retained the ability to bind to APOBEC3G as well as Cul5 complex. These findings suggest that the E3 ubiquitin ligase activity of the Vif-BC-Cul5 complex is essential for Vif function against APOBEC3G.     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.     

The Roles of APOBEC3G Complexes in the Incorporation of APOBEC3G into HIV-1

Background

The incorporation of human APOBEC3G (hA3G) into HIV is required for exerting its antiviral activity, therefore the mechanism underlying hA3G virion encapsidation has been investigated extensively. hA3G was shown to form low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. The function of different forms of hA3G in its viral incorporation remains unclear.

Methodology/Principal Findings

In this study, we investigated the subcellular distribution and lipid raft association of hA3G using subcellular fractionation, membrane floatation assay and pulse-chase radiolabeling experiments respectively, and studied the correlation between the ability of hA3G to form the different complex and its viral incorporation. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.

Conclusions/Significance

Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.