Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Abstract

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short ($10 nucleotides) singlestranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.

Equine Infectious Anemia Virus Resists the Antiretroviral Activity of Equine APOBEC3 Proteins through a Packaging-Independent Mechanism

“…This EIAV sequence was cloned in frame into the BamHI/XhoI sites of pGEX4T. The pGEX4T-HIV NC plasmid, which encodes HIV-1 NC fused to GST, has been previously described (5).…”

Analysis of the human immunodeficiency virus-1 RNA packageome

“…Encapsidation of the host cell restriction factors APOBEC3F and APOBEC3G into HIV-1 virions requires RNA, and it was shown recently that many cellular RNAs are capable of mediating packaging (Apolonia et al 2015). Since, in vitro, APOBEC3G requires at least 10 nt of G-rich single-stranded RNA to form ternary complexes with NC (Bogerd and Cullen 2008), newly made ncRNAs could be the RNAs that have sufficient stretches of protein-free single-stranded RNA to confer packaging of APOBEC3 proteins in vivo. Packaging of newly synthesized RNAs could also potentially impact infectivity.…”

Structural Model for Deoxycytidine Deamination Mechanisms of the HIV-1 Inactivation Enzyme APOBEC3G

“…Virus infectivity factor facilitates polyubiquitination of Apo3G, resulting in proteasomal degradation (2)(3)(4)(5)(6). When virus infectivity factor is absent, however, Apo3G is encapsulated in the HIV virus particle, most likely as a multimer (7), through interactions with HIV RNA or 7SL RNA and the nucleocapsid portion of Gag (7)(8)(9)(10)(11)(12)(13)(14)(15). Following release from the HIV virion into T cells, Apo3G generates large numbers of deaminations, typically 5Ј-CCC3 CCU, in viral (Ϫ) single-stranded cDNA during reverse transcription (16 -18).…”