Poster abstracts
Poster number 86 submitted by Shuohui Liu
Capsid and SP1 domains of HIV-1 Gag contribute to genomic RNA binding selectivity
Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Erik D. Olson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Ioulia Rouzina (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH)
Abstract:
HIV-1 Gag is a polyprotein consisting of matrix (MA), capsid (CA), nulceocapsid (NC), and p6 domains, in addition to two short spacer peptides, SP1 and SP2. Both NC and MA domains are highly basic nucleic acid binding domains. How Gag selectively packages its cognate genomic RNA (gRNA) over the vast excess of cellular RNAs is not understood. Here, we study the effect of Gag mutations on its in vitro ability to distinguish between various specific and non-specific fragments of gRNA. We have previously shown that WT HIV-1 Gag binds the Psi RNA (packaging signal) and a non-Psi RNA with similar affinity at physiological salt (~150 mM NaCl), but that the salt dependence of these two binding events differs dramatically (Webb, JA, et al, RNA 2013). Gag binds Psi RNA with a strong non-electrostatic binding component and a small effective charge (Zeff~5), which we hypothesize is due to an NC-only binding mode. In contrast, HIV-1 Gag binds non-Psi RNA with a very weak non-electrostatic component and a larger effective charge (Zeff~9) that is consistent with a contribution from both NC and MA domains. In new work, a WM to AA mutation in the CA C-terminal domain, which eliminates Gag dimerization, reduces Psi RNA binding selectivity while retaining the NC-only binding mode, similar to a Gag variant lacking the MA domain. Interestingly, a Gag deletion mutant in which the CA domain is replaced by a 16-residue flexible linker, retains partial selectivity for Psi. Another linker variant, which additionally lacks SP1, almost completely loses selectivity. SP1 has been shown to be capable of undergoing a concentration-dependent random coil to helix transition, and is helical in the immature virus particle. Taken together, our observations suggest that the selectivity of Gag for gRNA may be regulated by a global Gag conformational change triggered by specific Psi RNA binding, and additionally requires CA domain protein-protein contacts, as well as a local SP1 conformational switch.
References:
Webb, Joseph A., et al. "Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging." RNA 19.8 (2013): 1078-1088.
Keywords: HIV-1, Gag, genomic RNA