Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

HIV-1 and other retroviruses occasionally undergo hypermutation, characterized by a high rate of G-to-A substitution. Recently, the human apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), first identified as CEM15, was shown to be packaged into retroviral virions and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minusstrand DNA, thereby inducing G-to-A hypermutation. This innate mechanism of resistance to retroviral infection is counteracted by the HIV-1 viral infectivity factor (Vif), which protects the virus by preventing the incorporation of APOBEC3G into virions by rapidly inducing its ubiquitination and proteasomal degradation. To gain insights into the mechanism by which Vif protects HIV-1 from APOBEC3G, we substituted several amino acids in human APOBEC3G with equivalent residues in simian APOBEC3Gs that are resistant to HIV-1 Vif and determined the effects of the mutations on HIV-1 replication in the presence and absence of Vif. We found that a single amino acid substitution mutant of human APOBEC3G (D128K) can interact with HIV-1 Vif but is not depleted from cells; thus, it inhibits HIV-1 replication in an HIV-1 Vif-resistant manner. Interestingly, rhesus macaque simian immunodeficiency virus 239 or HIV-2 Vif coexpression depleted the intracellular steady state levels of the D128K mutant and abrogated its antiviral activity, indicating that it can be a substrate for the proteasomal pathway. The HIV-1 Vif-resistant mutant APOBEC3G could provide a gene therapy approach to combat HIV-1 infection.

APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.Replication of human immunodeficiency virus type 1 (HIV-1) in primary cells is dependent on the expression of Vif protein, which counteracts the activity of the host cytidine deaminases APOBEC3G (APO3G) and APOBEC3F (4,25,29,32). In the absence of Vif, APO3G is incorporated into virus particles (11,16,19,20,26,27,30), resulting in hypermutation of the viral genome (19) or degradation of mutated cDNA (14, 18, 31) via a DNA repair mechanism (reviewed in references 3 and 12). Interestingly, human APO3G is not only packaged into human immunodeficiency viruses but also incorporated into simian immunodeficiency viruses and murine leukemia virus (9,18,19). Packaging of APO3G into such diverse viruses suggests that it either is a relatively nonspecific process or involves signals shared by these viruses. APO3G can bind RNA in vitro (10). Indeed, several reports have noted that the presence of viral RNA enhanced APO3G encapsidation (28); however, others (17, 23) suggested that viral RNA was not essential for APO3G packaging (2,5,8,17,23,28).To further study the role of viral RNA in the packaging of APO3G into HIV-1 virions, we first compared the packaging of APO3G into either the wild-type infectious NL4-3 virus or a helper virus (C-Help) whose RNA genome is not packaged due to a 33-base deletion in the putative RNA packaging signal (21). Virus stocks were prepared by transient cotransfection of HeLa cells with either the pNL4-3 plasmid (1) or the C-Help vector DNA and the APO3G-expressing plasmid pcDNA-APO3G (11). Viruses were collected 48 h after transfection and purified by two rounds of sucrose gradient centrifugation. Cell lysates and concentrated virus preparations were analyzed by immunoblotting (Fig. 1A). We found that packaging of APO3G into helper virus was reduced by Ͼ3.5-fold compared to packaging into NL4-3 virus (Fig. 1B). Thus, viral RNA contributes to the specific packaging of APO3G into HIV-1 virions, consistent with data reported by Svarovskaia et al. (28).If encapsidation of APO3G and viral RNA are linked, the APO3G packaging defect observed with the helper virus ( Fig. 1A and B) should be overcome by the coexpression of packaging-competent vector-derived RNA. To test this hypothesis, several packaging vectors were constructed based on the lentiviral packaging vector pHRЈCMVGFP (15). This vector contains both HIV-1 long terminal repeats (LTRs), the 5Ј-untranslated region, 35...

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif plays an important role in regulating virus infectivity (8,38). It is now well established that HIV-1 Vif can counteract the human cytidine deaminase APOBEC3G (APO3G). The inhibition of APO3G's antiviral effects has been attributed to a reduction in cellular expression of APO3G protein, which is due to Vif-mediated degradation of APO3G by cytoplasmic proteasomes (6,20,25,27,34,37,43). On the other hand, we recently found that Vif could prevent encapsidation of a degradation-resistant APO3G variant, suggesting that Vif can inhibit the APO3G antiviral activity through multiple independent mechanisms (29). In the absence of Vif, APO3G is efficiently packaged into HIV virions and inhibits virus replication. A number of studies reported that the presence of APO3G in the virus can result in hypermutation of the viral minus-strand cDNA during reverse transcription (11,18,23,24,42,45), inhibition of reverse transcription (9), tRNA annealing or tRNA processing (10, 26), DNA strand transfer (19, 26), or integration (22, 26).Some of these effects do not require catalytically active APO3G (19,22), and several reports suggested that deaminase-defective APO3G and APO3F have antiviral activity when transiently coexpressed with HIV-1 in 293T cells (3,12,28,35). Our own data concerning the antiviral properties of the deaminase-defective APO3G C288S/C291A mutant supported these conclusions (30). However, in our previous study we found that comparable inhibition of viral infectivity required higher levels of deaminase-defective APO3G protein than that of wild type (wt) (30). The purpose of the current study was to characterize in more detail the antiviral properties of deaminase-defective APO3G. We p...

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.APOBEC3G (APO3G) belongs to a family of cytidine deaminases that in humans includes APOBEC1, APOBEC2, eight APOBEC3 variants designated APOBEC3A through -3H, and APOBEC4, as well as activation-induced deaminase (7,12,30,40). The activity of APO3G interferes with the replication of a broad range of retroviruses and can also block hepatitis B virus (39), whose replication cycle involves the reverse transcription of a pregenomic RNA intermediate (33). Interestingly, human immunodeficiency virus type 1 (HIV-1) Vif can counteract the antiviral activity of APO3G by inhibiting its incorporation into virions (13,24,34,36). This inhibition is at least in part due to a reduction in cellular APO3G expression, which has been attributed primarily to Vif-mediated degradation of APO3G by cytoplasmic proteasomes (6,19,24,25,34,36,44).It is now well established that packaging of APO3G into virus particles can result in hypermutation of the viral minus-strand cDNA during reverse transcription (10,17,22,23,43,45). Interestingly, human APO3G is not only packaged into human immunodeficiency viruses but is also incorporated into simian immunodeficiency viruses and murine leukemia virus (10,22,23). The efficient packaging of APO3G into such diverse viruses could suggest that the mechanism of APO3G packaging is either relatively nonspecific or requires signals shared by these viruses. The observation that deletions in various regions of APO3G can prevent packaging into virus particles (4, 18) argues in favor of a specific packaging mechanism. APO3G binds to RNA in vitro, a property that it shares with other members of the...

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.The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif plays an important role in regulating virus infectivity (10, 42). It is now well established that HIV-1 Vif can counteract the human cytidine deaminase APOBEC3G (APO3G). The inhibition of APO3G has been attributed to a reduction in cellular expression levels, which is due to Vifmediated degradation of APO3G by cytoplasmic proteasomes (8,24,29,30,39,41,50). In the absence of Vif, APO3G is efficiency packaged into HIV virions. The presence of APO3G in the virus can result in hypermutation of the viral minusstrand cDNA during reverse transcription (RT) (12,22,26,28,49,51), inhibition of RT (11a), or deamination-independent inactivation of the virus (2,32,34,40).APO3G has the propensity to form multimers, a property that it shares with other members of the APOBEC family of proteins (13,21,31,34,40,47). In our previous study, we demonstrated that human APO3G is capable of forming multimeric complexes in transfected HeLa cells (34). The multimerization of APO3G was sensitive to RNase treatment, suggesting an involvement of viral or cellular RNA in this process. Interestingly, multimerization was also exquisitely sensitive to mutation of a single cysteine residue (C97) that is part of an N-terminal zinc finger motif. However, the C97A mutation abolished neither catalytic nor antiviral activity of APO3G (34). Mutation of C288 and C291, on the other hand, did not impair APO3G oligomerization but completely abrogated APO3G deaminase activity. Nevertheless, this deaminase-deficient APO3G mutant was packaged into vif-defective HIV-1 virions and retained partial antiviral activity when transiently expressed in HeLa cells (34). APO3G assembles into intracellular high-molecular-mass (HMM) ribonucleoprotein complexes in acti...

Background: Efficient incorporation of the cellular cytidine deaminase APOBEC3G (APO3G) into HIV-1 virions is necessary for its antiviral activity. Even though cellular RNAs are known to be nonspecifically incorporated into virus particles, we have previously found that encapsidation of APO3G into HIV-1 virions is specifically enhanced by viral genomic RNA. Intracellularly, APO3G was found to form large RNA-protein complexes involving a variety of cellular RNAs. The goal of this study was to investigate the possible contribution of host RNAs recently identified in intracellular APO3G ribonucleoprotein complexes to APO3G's encapsidation into HIV-1 virions.