Poster abstracts
Poster number 18 submitted by Kaylee Grabarkewitz
Investigating the role of transcriptional start-site differences in the HIV-1 genomic RNA 5´UTR on Gag binding
Kaylee Grabarkewitz (Department of Chemistry and Biochemistry, Center for RNA Biology, Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University, Columbus, OH), Vicki Wysocki (Department of Chemistry and Biochemistry, Center for RNA Biology, Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH)
Abstract:
Since the AIDS pandemic began in the early 1980s, Human Immunodeficiency Virus type 1 (HIV-1) has become one of the world’s deadliest pathogens. During the HIV-1 retroviral lifecycle, the process of reverse transcription converts the genomic RNA (gRNA) into double-stranded DNA, which is incorporated into the host genome. Following transcription, the full-length gRNA has two fates: it serves as mRNA for translation of viral polyproteins Gag and Gag-Pol, or as gRNA, which is packaged as a dimer into new virions. The latter is orchestrated by the HIV-1 Gag protein through specific interactions with the 5´UTR. Transcription start-site heterogeneity results in the synthesis of full-length viral RNA species with a variable number of guanines at the 5´ end of the gRNA (1G, 2G, and 3G). Surprisingly, several studies have shown that the number of 5´ G residues affects the localization of the gRNA: 1G RNA was found to be selectively packaged in the virion even though 3G RNA is the most abundant transcript in the cell. RNA structural differences between 1G and 3G 5´UTR RNAs were recently reported. The 1G RNA has an exposed dimerization initiation site and exposed G-rich Gag binding sites; these motifs are sequestered in the 3G RNA. We use native mass spectrometry and mass photometry to investigate RNA dimerization and Gag-RNA binding stoichiometry using 2G and 4G 5´UTR RNAs designed to mimic 1G/3G RNA plus an m7G cap. Preliminary data are consistent with differences in the dimerization efficiencies and interactions between the 2G vs. 4G 5´UTR and the full-length Gag protein. Understanding how these critical processes are regulated at the level of RNA structure may inform new RNA-based therapeutic strategies.
Keywords: HIV-1 RNA, Native Mass Spectrometry , HIV-1 Gag