Poster abstracts
Poster number 26 submitted by Samantha Sarni
Unlocking the mechanism of HIV-1 viral assembly nucleation with native mass spectrometry
Samantha H. Sarni (OSBP, Chemistry and Biochemistry, Center for RNA Biology, Resource for Native Mass Spectrometry Guided Structural Biology), Erik D. Olson (OSBP, Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research), Shuohui Liu (Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research), Karin Musier-Forsyth (OSBP, Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research), Vicki H. Wysocki (OSBP, Chemistry and Biochemistry, Center for RNA Biology, Resource for Native Mass Spectrometry Guided Structural Biology)
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 are aimed at disrupting immature viral particle assembly. This work aims to unlock the mechanism by which the virus nucleates immature particle assembly around a viral dimeric RNA genome (gRNA) using native mass spectrometry (nMS). 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 widely 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. This system has defied traditional structural biology techniques (cryo-EM, X-ray, NMR) due to its size, heterogeneity, and dynamic nature. Here, MS was used to determine the stoichiometry of Gag:RNA interactions. Gag bound to Psi RNA (100-nucleotide construct) with 1:1 and 2:1 stoichiometry but bound to non-Psi RNA of similar size with exclusively 1:1 stoichiometry. These data suggest the possibility of two different mechanisms: (1) Two independent binding sites are present on Psi, or (2) Psi can induce higher order assembly under conditions where non-Psi RNA does not. To distinguish between these two binding mechanisms, a strictly monomeric double mutant, W184A/M185A Gag (WM-Gag), was purified and used for nMS binding assays. In the presence of Psi, WM-Gag exclusively forms a 1:1 WM-Gag:Psi complex, suggesting that higher order assembly is stimulated by Psi.
Keywords: Native Mass Spectrometry, RNA, Structural Biology