Poster abstracts

Poster number 24 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 ), 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. N-6-methyladenosine (m6A) modifications in the HIV-1 genome have previously been identified using low resolution m6A-sequencing-based techniques. m6A is the most common internal mRNA modification and affects all aspects of cellular RNA biology. Thus, m6A on viral RNA may impact viral replication through multiple processes including immune evasion, RNA stability, and viral RNA localization. Two m6A motifs are located at A198 and A242 within an identified m6A-seq peak in the 5'UTR.1 The first of the modifications is within the primer binding site (PBS) and the second is located in the viral RNA packaging signal proximal to a known site of Gag interaction. The presence of both modifications has recently been proposed to play a critical role in down-regulating gRNA packaging.2 Thus, we hypothesize m6A modifications in the HIV-1 5'UTR influence viral replication and assembly through modulating RNA structure, which in turn impacts tRNA primer annealing and Gag interactions. To determine the stoichiometry of m6A on these specific sites in the gRNA 5'UTR, we are using an approach related to SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography)3 as well as a deoxyribozyme-RT-qPCR approach, wherein the presence of m6A at target sites inhibits deoxyribozyme cleavage.4 We purified RNA from Lenti-X 293T cells transfected with a plasmid encoding HIV-1 proviral DNA as well as 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, a relevant T cell line, and performing biochemical assays to determine the effect of these modifications on tRNA primer annealing and HIV-1 Gag binding.

References:
[1] Tirumuru et al (2016) N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression, Elife 5, e15528.
[2] Pereira-Montecinos et al (2022) Epitranscriptomic regulation of HIV-1 full-length RNA packaging, Nucleic Acids Res 50, 2302-2318.
[3] Liu, N. and Pan, T. (2016) Probing N 6-methyladenosine (m6A) RNA Modification in Total RNA with SCARLET, Methods Mol. Biol. 1358:285-292
[4] Bujnowska et al (2020) Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA, J. Biol. Chem. 295, 6992-7000.

Keywords: m6A, HIV-1, RNA modification