Talk abstracts
Talk on Monday 10:35-10:50am submitted by Kaylee Grabarkewitz
Role of transcriptional start-site differences in the HIV-1 genomic RNA 5´UTR on Gag binding and RNA dimerization
Kaylee Grabarkewitz (Chemistry and Biochemistry Graduate Program), 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), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research The Ohio State University, Columbus, OH)
Abstract:
Human Immunodeficiency Virus type 1 (HIV-1) has become one of the world’s deadliest pathogens. During the HIV-1 retroviral lifecycle, reverse transcription converts the genomic RNA (gRNA) into double-stranded DNA, which is incorporated into the host genome. Following transcription, the full-length viral RNA has two fates: it serves as mRNA for translation of viral polyproteins Gag and Gag-Pol, and as gRNA, which is packaged as a dimer into new virions. The latter is orchestrated by the HIV-1 Gag protein through interactions with the 5´UTR. Due to transcription start-site heterogeneity, multiple full-length viral RNA species with a variable number of guanines at the 5´end of the gRNA (1G, 2G, and 3G) are produced. Surprisingly, several studies have shown that the number of 5´ G residues affects the localization of the gRNA: 1G RNA is selectively packaged into 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. Gag specifically recognizes and binds to a packaging signal (Psi) within the 5´UTR; previous work suggested that the packaging selectivity can’t be explained by differences in binding affinity alone. To understand other factors that may contribute to selective gRNA packaging, we used mass photometry (MP) and native mass spectrometry (nMS) to investigate Gag-RNA interactions. Using MP, we observed higher order binding to Psi RNA relative to a non-Psi region of the 5´UTR. MP and nMS were used 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, strength of dimer interactions, and in the interaction between the 2G vs. 4G 5´UTR and the full-length Gag protein.
Keywords: HIV-1 RNA, Native Mass Spectrometry , HIV-1 Gag