Human Immunodeficiency Virus Type 1 Vif Inhibits Packaging and Antiviral Activity of a Degradation-Resistant APOBEC3G Variant

Abstract

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...

APOBEC proteins and intrinsic resistance to HIV-1 infection

“…Perhaps the simplest way to prevent A3G action would, therefore, be to block virion encapsidation-and this is what Vif does. Though there have been reports of Vif directly impeding packaging (Mariani et al 2003;Opi et al 2007), Vif's principal function is to induce the polyubiquitylation and subsequent proteasomal degradation of A3G, thereby depleting the pool of cytosolic A3G available for incorporation into assembling virus particles (Conticello et al 2003;Marin et al 2003;Sheehy et al 2003;Stopak et al 2003;Yu et al 2003;Mehle et al 2004b;Kobayashi et al 2005). Vif accomplishes this by simultaneously binding to both A3G and the cullin5-elongin B/C-Rbx ubiquitin ligase, thus serving as an adaptor to recruit the ligase complex to its substrate ( Yu et al 2003; figure 2).…”

Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

“…Moreover, several APOBEC3 proteins have been shown to also function as inhibitors of endogenous retroviruses and LTR-retrotransposons, including the yeast retrotransposon Ty1 (Dutko et al 2005;Esnault et al 2005;Schumacher et al 2005;Bogerd et al 2006;Chen et al 2006). In all cases where this has been examined, inhibition has been shown to correlate with the specific packaging of the inhibitory APOBEC3 protein into retroviral virions or retrotransposon virus-like particles (VLPs) (Kao et al 2003;Doehle et al 2005Doehle et al , 2006Dutko et al 2005;Derse et al 2007;Goila-Gaur et al 2007;Huthoff and Malim 2007;Opi et al 2007;Aguiar et al 2008). However, given the lack of any obvious protein sequence similarity across these very diverse retroelement families, the identity of the common target that is recognized by the APOBEC3 proteins, leading to virion or VLP incorporation, has remained unclear.…”

Analysis of Human APOBEC3H Haplotypes and Anti-Human Immunodeficiency Virus Type 1 Activity

“…Vif binds both A3G and a Cul5/EloB/EloC E3 ubiquitin ligase (44), causing A3G to be degraded by proteasomes (22,35,37). Vif may also inhibit A3G activity via other proteasome-independent mechanisms (30,33,37). Vif also neutralizes A3DE and A3F, but not A3B and A3H.…”