APOBEC3G cytidine deaminase association with coronavirus nucleocapsid protein Shui-Mei Wang, Chin-Tien Wang Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan a b s t r a c ta r t i c l ei n f o Article history: Received 2 December 2008 Returned to author for revision 9 January 2009 Accepted 10 March 2009 Available online 5 April 2009 Keywords: APOBEC3G Coronavirus SARS-CoV HCoV-229E HIV-1 Nucleocapsid We previously reported that replacing HIV-1 nucleocapsid (NC) domain with SARS-CoV nucleocapsid (N) residues 2213, 215421, or 234421 results in effi cient virus-like particle (VLP) production at a level comparable to that of wild-type HIV-1. In this study we demonstrate that these chimeras are capable of packaging large amounts of human APOBEC3G (hA3G), and that an HIV-1 Gag chimera containing the carboxyl-terminal half of human coronavirus 229E (HCoV-229E) N as a substitute for NC is capable of directing VLP assembly and effi ciently packaging hA3G. When co-expressed with SARS-CoV N and M (membrane) proteins, hA3G was effi ciently incorporated into SARS-CoV VLPs. Data from GST pull-down assays suggest that the N sequence involved in NhA3G interactions is located between residues 86 and 302. Like HIV-1 NC, the SARS-CoV or HCoV-229E N-associated with hA3G depends on the presence of RNA, with the fi rst linker region essential for hA3G packaging into both HIV-1 and SARS-CoV VLPs. The results raise the possibility that hA3G is capable of associating with different species of viral structural proteins through a potentially common, RNA-mediated mechanism. 2009 Elsevier Inc. All rights reserved. Introduction Apolipoprotein B mRNA-editingenzyme-catalytic polypeptide-like 3G (APOBEC3G) is a member of the APOBEC3 family of cytidine deaminases (Cullen, 2006). Human APOBEC3G (hA3G) can be packaged into HIV-1 virions to mediate C-to-U editing on nascent proviral minus strands during reverse transcription, resulting in the inhibition of viral replication (Harris et al., 2003; Mangeat et al., 2003; Yu et al., 2004; Zhang et al., 2003). Accordingly, hA3G as an anti-viral factor confers innate immunity to HIV-1. However, HIV-1 encodes Vif, which counteracts hA3G by inducing hA3G degradation via the ubiquitinproteasome pathway, thereby blocking its incorporation into budding virions (Mehle et al., 2004; Yu et al., 2003). The ability of Vif to negate hA3G function is both dependent on physical association and partly species-specifi cin other words, Vif cannot counteract a form of A3G from a different species. This ineffi cient VifhA3G interaction means that HIV-1 Vif is capable of counteracting human and chimpanzee A3G, but not mouse A3G (mA3G) or African green monkey A3G (Mariani et al., 2003). Other researchers have shown that hA3G or other APOBEC family members can confer innate immunity to a wide range of retroviruses as well as HBV (which is similar to retroviruses in that it also goes through a reverse transcription step during genomic replication) (Cullen, 2006). The incorporation of A3G into HIV-1 virions is mediated by the Gag nucleocapsid (NC) domain (Alce and Popik, 2004; Cen et al., 2004; Khan et al., 2005; Luo et al., 2004; Schafer et al., 2004; Zennou et al., 2004). In addition to playing a key role in viral RNA packaging (Berkowitz et al., 1993, 1995; Poon et al., 1996; Zhang and Barklis, 1997), NC contains an I domain that is responsible for GagGag interactions (Bennett et al., 1993; Bowzard et al., 1998). Hetero- logous polypeptides capable of self-association have been shown to confer the ability to effi ciently produce chimeric VLPs when substituted for HIV-1 NC (Accola et al., 2000; Burniston et al., 1999; Johnson et al., 2002; Zhang et al., 1998). However, replacing NC with a leucine-zipper motif that does not encapsidate RNA abolishes hA3G packaging without signifi cantly affecting HIV-1 virion production (Zennou et al., 2004), suggesting RNA involvement in hA3G incorporation. This is consistent with the proposal that RNA is required for either hA3G viral incorporation or GaghA3G interaction (Khan et al., 2005; Schafer et al., 2004; Svarovskaia et al., 2004; Zennou et al., 2004). We previously demonstrated that HIV-1 Gag mutants containing severe acute respiratory syndrome coronavirus nucleocapsid (SARS-CoV N) coding sequences as NC substitutes can effectively assemble VLPs (Wang et al., 2008). While completely unrelated, the SARS-CoV N protein is similar to HIV-1 NC in that it contains putative proteinprotein interaction domains (He et al., 2004; Narayanan et al., 2003; Surjit et al., 2004; Yu et al., 2005) and plays a role in viral RNA packaging (Hsieh et al., 2005; Huang et al., 2004a). Given that SARS-CoV N possesses a RNA-binding property, it is likely that assembly-competent chimeras containing a replacement of HIV-1 NC by an SARS-CoV N sequence