Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays

Abstract

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral t sequences almost as well as to the HIV-1 W riboprobe.

Cooperation of an RNA Packaging Signal and a Viral Envelope Protein in Coronavirus RNA Packaging

“…For example, in alphaviruses, the capsid protein specifically recognizes the packaging signal and is the basis for the specific encapsidation of viral genomic RNA (30,63,80). In retrovirus human immunodeficiency virus type 1, the NCp7 domain of Gag polyprotein has been shown elsewhere to specifically recognize the human immunodeficiency virus packaging signal and is principally responsible for the specific encapsidation of the unspliced genomic RNA into the virus particles (8,10,20,29). In the case of hepatitis B virus, viral RNA packaging occurs through the specific binding of P protein to the encapsidation signal, followed by addition of multiple C proteins to viral RNA to form the nucleocapsid (5,16,34).…”

Preclinical evaluation of a mercaptobenzamide and its prodrug for NCp7-targeted inhibition of human immunodeficiency virus

“…The alteration of any one of the zinc-chelating amino acids or surrounding amino acids results in the production of noninfectious virus. NCp7 functions at multiple stages of HIV replication, including: reverse transcription, integration, Tat-promoted transcription, auto-catalysis of Gag-Pol precursors to form functional protease, promotion of Gag assembly and interaction with viral genomic RNA, and protection of double-stranded HIV genomic RNA from degradation (Buckman et al, 2003;Guo et al, 2000;Hargittai et al, 2004;Turpin et al, 1996;Zybarth and Carter, 1995;Berkowitz et al, 1993;Dawson and Yu, 1998;Shubsda et al, 2002;Lapadat-Tapolsky et al, 1993). The mechanistic activities of NCp7 suggest a potentially high genetic barrier to the selection of resistance and suggest that any resistant strains that may emerge might suffer significant loss of fitness.…”

APOBEC3G cytidine deaminase association with coronavirus nucleocapsid protein

“…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(Berkowitz et al, , 1995Poon et al, 1996;Zhang and Barklis, 1997), NC contains an I domain that is responsible for Gag-Gag interactions (Bennett et al, 1993;Bowzard et al, 1998). Heterologous polypeptides capable of self-association have been shown to confer the ability to efficiently 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).…”