Influence of primate lentiviral Vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of APOBEC3G

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral evasion of the host antiviral protein APOBEC3G, also known as CEM15. Vif mutant but not wild-type HIV-1 viruses produced in the presence of APOBEC3G have been shown to undergo hypermutations in newly synthesized viral DNA upon infection of target cells, presumably resulting from C-to-U modification during minus-strand viral DNA synthesis. We now report that HIV-1 Vif could induce rapid degradation of human APOBEC3G that was blocked by the proteasome inhibitor MG132. The efficiency of Vif-induced downregulation of APOBEC3G expression depended on the level of Vif expression. A single amino acid substitution in the conserved SLQXLA motif reduced Vif function. Vif proteins from distantly related primate lentiviruses such as SIVagm were unable to suppress the antiviral activity of human APOBEC3G or the packaging of APOBEC3G into HIV-1 Vif mutant virions, due to a lack of interaction with human APOBEC3G. In the presence of the proteasome inhibitor MG132, virion-associated Vif increased dramatically. However, increased virion packaging of Vif did not prevent virion packaging of APOBEC3G when proteasome function was impaired, and the infectivity of these virions was significantly reduced. These results suggest that Vif function is required during virus assembly to remove APOBEC3G from packaging into released virions. Once packaged, virion-associated Vif could not efficiently block the antiviral activity of APOBEC3G.     

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.     

HIV-1 Viral Infectivity Factor (Vif) Alters Processive Single-stranded DNA Scanning of the Retroviral Restriction Factor APOBEC3G

APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (−)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of Vif_IIIB- and Vif_HXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, Vif_HXB2 inhibited the jumping and Vif_IIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.     

Differential Sensitivity of “Old” versus “New” APOBEC3G to Human Immunodeficiency Virus Type 1 Vif

HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inhibiting its encapsidation into virions. Here, we compared the relative sensitivity to Vif of APOBEC3G in stable HeLa cells containing APOBEC3G (HeLa-A3G cells) versus that of newly synthesized APOBEC3G. We observed that newly synthesized APOBEC3G was more sensitive to degradation than preexisting APOBEC3G. Nevertheless, preexisting and transiently expressed APOBEC3G were packaged with similar efficiencies into vif-deficient human immunodeficiency virus type 1 (HIV-1) virions, and Vif inhibited the encapsidation of both forms of APOBEC3G into HIV particles equally well. Our results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of “old” and “new” APOBEC3G.     

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.     

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.     

HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability

The human immunodeficiency virus type 1 (HIV-1) relies on Vif (viral infectivity factor) to overcome the potent antiviral function of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, also known as CEM15). Using an APOBEC3G-specific antiserum, we now show that Vif prevents virion incorporation of endogenous APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T cells. Vif achieves this depletion by both impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the APOBEC3G protein by the 26S proteasome. Vif physically interacts with APOBEC3G, and expression of Vif alone in the absence of other HIV-1 proteins is sufficient to cause depletion of APOBEC3G. These findings highlight how the bimodal translational and posttranslational inhibitory effects of Vif on APOBEC3G combine to markedly suppress the expression of this potent antiviral enzyme in virally infected cells, thereby effectively curtailing the incorporation of APOBEC3G into newly formed HIV-1 virions.     

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.     

Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging

APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.     

Production of infectious SIVagm from human cells requires functional inactivation but not viral exclusion of human APOBEC3G

The virus infectivity factor (Vif) is a protein encoded by most primate lentiviruses. Recent evidence suggests that HIV-1 Vif reduces the intracellular levels of the host cytidine deaminase APOBEC3G (Apo3G) and inhibits its packaging into virions. These functions of Vif are thought to be species-specific. Accordingly, HIV-1 Vif can target only human Apo3G (hApo3G), whereas, African green monkey simian immunodeficiency virus (SIVagm) Vif can inhibit African green monkey but not human Apo3G. Consistent with this, we found that SIVagm Vif does not affect the stability of exogenously and endogenously expressed hApo3G and does not prevent packaging of exogenous and endogenous hApo3G into SIVagm virions. Nevertheless, SIVagm Vif supported spreading infection of SIVagm virus in the hApo3G-positive human A3.01 T cell line and rescued infectivity of viruses produced from Apo3G-expressing HeLa cells. Sequence analysis verified that SIVagm Vif inhibited the accumulation of hApo3G-induced mutations, suggesting that SIVagm Vif is indeed active in human cells. Our data suggest that SIVagm Vif can inhibit hApo3G activity without inducing its intracellular degradation or preventing its packaging into virions.     

The tyrosine kinases Fyn and Hck favor the recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles

The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.     

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.     

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.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between Vif and ElonginC

The HIV-1 viral infectivity factor (Vif) protein is essential for viral replication. Vif recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3G is packaged into budding HIV-1 virions and introduces multiple mutations in the newly synthesized minus-strand viral DNA to restrict virus replication. Thus, the A3G-Vif-E3 complex represents an attractive target for development of novel anti-HIV drugs. In this study, we identified a potent small molecular compound (VEC-5) by virtual screening and validated its anti-Vif activity through biochemical analysis. We show that VEC-5 inhibits virus replication only in A3G-positive cells. Treatment with VEC-5 increased cellular A3G levels when Vif was coexpressed and enhanced A3G incorporation into HIV-1 virions to reduce viral infectivity. Coimmunoprecipitation and computational analysis further attributed the anti-Vif activity of VEC-5 to the inhibition of Vif from direct binding to the ElonginC protein. These findings support the notion that suppressing Vif function can liberate A3G to carry out its antiviral activity and demonstrate that regulation of the Vif-ElonginC interaction is a novel target for small-molecule inhibitors of HIV-1.