Poster abstracts

Poster number 43 submitted by Samantha Sarni

Unlocking the mechanism of HIV-1 viral assembly nucleation with native mass spectrometry

Samantha H. Sarni (OSBP, Chemistry and Biochemistry, National Resource for Native Mass Spectrometry Guided Structural Biology), Erik D. Olson (OSBP, Chemistry and Biochemistry), Shuohui Liu (Chemistry and Biochemistry), Karin Musier-Forsyth (Chemistry and Biochemistry, OSBP), Vicki H. Wysocki (OSBP, Chemistry and Biochemistry, National Resource for Native Mass Spectrometry Guided Str)

Abstract:
The HIV-1 pandemic has claimed over 36 million lives, warranting investigation of the mechanism of virion assembly to guide the development of novel therapeutics. Of the numerous drugs available to treat individuals, none of are aimed at disrupting the assembly of the immature viral particle. This work aims to probe the mechanism by which the virus nucleates immature particle assembly around a single dimeric RNA genome (gRNA) using native mass spectrometry (MS). Virion assembly requires Gag, the primary viral structural protein that interacts with the 5’-untranslated region (5’UTR) of the viral gRNA. Gag specifically recognizes and binds to a packaging signal sequence (Psi) within the 5’UTR, yet promiscuously interacts with any nucleic acid. Gag is an intrinsically dynamic precursor protein with RNA binding sites at each terminus. It is postulated that Gag binds Psi using one RNA binding domain in an extended conformation, while using both ends to bind non-Psi RNA in a compact conformation. The size, heterogeneity, and dynamic nature of this system has precluded traditional structural biology techniques. Here, MS was used to determine the stoichiometry of Gag:RNA interactions. Gag bound to Psi with 1:1 and 2:1 stoichiometry but bound to non-Psi RNA with primarily 1:1 stoichiometry. These data suggest either that two independent binding sites are present on Psi or that Psi can induce Gag dimerization under conditions where non-Psi RNA does not. These data are consistent with Gag conformational differences that depend on the identity of the bound RNA.

Keywords: Native Mass Spectrometry, RNA, Structural Biology