Poster abstracts
Poster number 103 submitted by Christina Budding
Site-specific identification and role of m6A RNA modification in HIV-1 genomic RNA 5'UTR
Christina R. Budding (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH), Andrew Nielsen (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH), Mahfam Shariati (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
HIV-1 selects full-length genomic RNA (gRNA) from the cytoplasmic pool of cellular and viral RNA. The viral Gag polyprotein orchestrates the packaging process via specific interactions within the gRNA 5'UTR. Two N-6-methyladenosine (m6A) modifications in the HIV-1 5'UTR have previously been identified using low-resolution antibody enrichment-based m6A-seq (Tirumuru et al, eLife, 2016). N-6-methyladenosine is the most common internal mRNA modification and affects many aspects of RNA biology. Thus, m6A on viral RNA may impact viral replication through immune evasion, RNA stability, and RNA localization. The two proposed m6A 5'UTR modifications are located at A198 and A242. The first is within the tRNA primer binding site (PBS) and the second is in the viral RNA packaging signal proximal to a known site of Gag interaction. The presence of both modifications has been proposed to play a critical role in down-regulating gRNA packaging (Pereira-Montecinos et al, NAR, 2022). We hypothesize m6A modifications in the HIV-1 5'UTR influence viral assembly through modulating RNA structure, which in turn impacts tRNA primer annealing and Gag interactions. To determine the stoichiometry of m6A modification on these specific sites in the gRNA 5'UTR, we are using SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography) (Mirza et al, RNA, 2023) as well as a deoxyribozyme-RT-qPCR approach, wherein the presence of m6A at target sites inhibits deoxyribozyme cleavage (Bujnowska et al, JBC, 2020). Probed RNA was purified from Lenti-X 293T cells transfected with an NL4-3 E-R+ HIV-1 plasmid vector and from progeny viral particles. Preliminary results are consistent with a reduction of m6A in the 5'UTR of packaged genomic RNA. Current work is focused on probing m6A in gRNA produced in SupT1 T cells and performing biochemical assays to determine the effect of these modifications on tRNA primer annealing and HIV-1 Gag binding.
Keywords: m6A, HIV-1, RNA modification