Friday, February 17th, 2017
3:30 p.m. / Olin 04
Erik D. Olson ’12, The Ohio State University, presents “Multiple roles of HIV-1 Gag and Psi RNA in ensuring specific genomic RNA recognition and assembly”.
In cells infected by HIV-1, host RNA is present in vast excess to viral RNA, yet the virus specifically packages full-length, dimeric genomic RNA (gRNA). The “Psi element” within the 5´-untranslated region (5´UTR) is critical for gRNA packaging through interaction with the HIV-1 Gag polyprotein. Gag is made up of 3 major domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Under physiological conditions, Gag binding affinity for Psi versus non-Psi RNAs is not significantly different; thus, the mechanism for specific gRNA packaging is unclear. To investigate the mechanism of Gag selectivity for gRNA, salt-titration binding assays were previously performed (Webb, JA et al., RNA 2013), allowing measurement of the strength of the specific component of binding and the number of charges that mediate the protein-RNA interaction. We found that in the context of Gag, both the MA and NC domains interact with non-Psi RNAs, while only the NC domain interacts with Psi RNAs. We investigated the elements of Psi that contribute to specific HIV-1 Gag interaction and found three specific sites that were critical for promoting specific Gag binding. Interestingly, the MA domain of Gag is also responsible for recognizing phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a plasma membrane-specific lipid, and targeting Gag-gRNA assemblies to the plasma membrane to initiate virion assembly. Previous liposome binding assays found that RNAs inhibit Gag binding to membranes that lack PI(4,5)P2. In an effort to identify the specific RNA species that confer this inhibition of Gag membrane binding, we tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P2. We found that specific tRNAs and Psi RNAs inhibit Gag binding to membranes lacking PI(4,5)P2. This result suggests that in addition to its role in gRNA packaging, the Psi element may also play a role in ensuring virion assembly at correct sites on the plasma membrane.