Amino-Terminal Region of the Human Immunodeficiency Virus Type 1 Nucleocapsid Is Required for Human APOBEC3G Packaging

Abstract

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.The Vif protein, which modulates viral infectivity (15, 21-23, 29, 38, 45, 59, 62, 78-80, 82, 84, 88) and pathogenicity (13, 28, 29, 33, 34,52), is present in nearly all lentiviruses, excluding equine infectious anemia virus. Recently, it has been shown that APOBEC3G has antiviral activity and that its activity is suppressed by Vif (73). APOBEC3G belongs to a family of proteins that have cytidine deaminase activity (35,73,89), and its cellular function is still unknown (35,73,89). Vif mutant, but not wild-type, human immunodeficiency virus type 1 (HIV-1) produced in the presence of APOBEC3G undergoes hypermutations in newly synthesized viral DNA (30, 44,53,55,94), presumably due to C-to-U modification during minus-strand viral DNA synthesis (30, 44,53,55,94).HIV-1 Vif can be coimmunoprecipitated with human APOBEC3G from virus-infected or -transfected cells, suggesting that Vif and APOBEC3G may form a complex (39,56,74,81,93). However, whether Vif directly interacts with APOBEC3G is still unclear (57). Amino acids 54 to 124 of human APOBEC3G have been shown to mediate interaction with HIV-1 Vif (9). Recent observations indicate that a single amino acid variation (at position 128) between human and monkey APOBEC3G determines the species-specific sensitivity to Vif of primate immunodeficiency viruses (4, 54, 71). However, whether this position is involved in APOBEC3G interaction with Vif (4, 54, 71, 91) or whether it is involved in direct interaction between these ...

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).…”

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).…”

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

“…As a result, it creates stop codons or G-A transitions in the newly synthesized viral cDNA which is then subjective to elimination by host DNA repair machinery [74,76]. APOBEC3G confers its antiviral effect by encapsidating into the virus particles through interaction with viral Gag protein [77][78][79][80][81]. Thus, APOBEC3G represents an innate host defense mechanism against HIV infection.…”