Viral RNA Is Required for the Association of APOBEC3G with Human Immunodeficiency Virus Type 1 Nucleoprotein Complexes

Abstract

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...

Enzymatically Active APOBEC3G Is Required for Efficient Inhibition of Human Immunodeficiency Virus Type 1

“…APO3G is packaged into viruses or virus-like particles in a process that involves nucleocapsid protein and viral genomic RNA (4,5,7,17,21,32,39,44). Moreover, we previously reported that APO3G packaged in the presence of viral genomic RNA stably associates with viral nucleoprotein complexes and is resistant to detergent treatment while APO3G packaged in the absence of viral genomic RNA is detergent sensitive and presumably not core associated (17). It is conceivable that the lack of antiviral activity of APO3G C288S/C291A when expressed from stably transfected HeLa cells is due to aberrant packaging of the protein into HIV-1 particles.…”

“…One fraction was left untreated; the other fraction was adjusted to 0.1% Triton X-100. Untreated and detergent-treated samples were layered on top of a 20% to 60% sucrose step gradient and subjected to ultracentrifugation as described previously (16,17). Four fractions (1.1 ml each) were collected from the top.…”

APOBEC proteins and intrinsic resistance to HIV-1 infection

“…Accordingly, A3G is not encapsidated by either HIV-1 virus-like particles that lack NC domains (Schafer et al 2004;Zennou et al 2004) or other retroviruses whose Gag proteins do not form complexes with A3G (Doehle et al 2006). The identity of the RNA(s) that promote A3G packaging in HIV-1 infected cells remains somewhat unresolved, though HIV-1 genomic RNA and 7SL RNA (a small pol III transcript that is found in the signal recognition particle, SRP) have each been assigned stimulatory roles in the process (Luo et al 2004;Khan et al 2005Khan et al , 2007Wang et al 2007). The fact that A3G is an avid RNA-binding protein ( Navarro et al 2005;Iwatani et al 2006) raises an important question concerning packaging: specifically, since A3G is associated with an array of cellular ribonucleoprotein (RNP) complexes in an RNA-dependent manner (Chiu et al 2005(Chiu et al , 2006Chelico et al 2006;Kozak et al 2006;Wichroski et al 2006;Gallois-Montbrun et al 2007), how can its RNA-binding site(s) be liberated to allow engagement with the RNA that facilitates packaging?…”

“…The current consensus view is that A3G packaging is determined by specific interactions between the N-terminal CDA domain of A3G and the nucleocapsid (NC) region of Gag that are also dependent upon the binding of A3G to RNA (Luo et al 2004;Schafer et al 2004;Svarovskaia et al 2004;Zennou et al 2004;Khan et al 2005;Navarro et al 2005;Burnett & Spearman 2007;Bogerd & Cullen 2008). Accordingly, A3G is not encapsidated by either HIV-1 virus-like particles that lack NC domains (Schafer et al 2004;Zennou et al 2004) or other retroviruses whose Gag proteins do not form complexes with A3G (Doehle et al 2006).…”

APOBEC3G: an intracellular centurion

“…This interaction is further strengthened by A3G's propensity to bind single-stranded nucleic acids, particularly viral RNA at the plasma membrane site of virion budding (Schafer et al 2004;Svarovskaia et al 2004;Zennou et al 2004;Khan et al 2005;Burnett & Spearman 2007;Khan et al 2007;Soros et al 2007). The incorporation of only seven molecules of A3G into Dvif HIV virions produced by human peripheral blood mononuclear cells is sufficient to greatly impair HIV-1 replication (Xu et al 2007).…”