Poster abstracts

Poster number 13 submitted by Grace Crowe

Effects of 5′ cap modification on HIV-1 5′ UTR structure and RNA-protein interactions

Grace E. Crowe (Ohio State Biochemistry Program), Karin Musier-Forsyth (Department of Chemistry and Biochemistry)

Abstract:
During human immunodeficiency virus type 1 (HIV-1) infection, host RNA Polymerase II uses transcription start site heterogeneity to produce viral RNA transcripts with one (1G), two, or three (3G) 5′ guanosines. The 5′-untranslated region (UTR) is highly structured and folds into different conformations that favor the packaging of viral RNA into virions (1G RNA, primarily dimeric) or the translation of RNA into viral proteins (3G RNA, primarily monomeric). Like host mRNAs, HIV-1 RNA is modified with a 5′ cap and a 3′ polyA tail. Two proposed HIV-1 RNA 5′ cap modifications are m7G with a 2′-O-methyl group on the first nucleotide (i.e., Cap1; found on most eukaryotic mRNAs) and m2,2,7G (i.e., trimethylguanosine,TMG; found mainly on snRNAs and snoRNAs). The 5′ UTR structure determines RNA fate, yet the relationship between 1G or 3G RNA structure and 5′ cap modification is poorly understood. We hypothesize that the 3G 5′ UTR structure may be favorable for TMG-capping and 2′-O-methylation, which marks 3G RNAs as translation-competent. In contrast, 1G RNA structure disfavors extensive 5′-cap modification to maintain a correctly folded 5′ UTR structure for efficient packaging. Using an A-cap to mimic the loss of base pairing in the TMG-cap, native gel electrophoresis showed that 1G RNA adopted a 3G-like structure and exhibited a decrease in dimerization that was rescued using a compensatory C58U mutation. Streptavidin bead pulldowns of biotinylated 5′-UTR constructs yielded distinct interactomes. Planned studies will characterize the authentic 5′-cap identities on HIV-1 RNAs using mass spectrometry. Together, this work will aid in our understanding of the interplay between viral RNA structure and host capping mechanisms.

Keywords: RNA structure, RNA-protein interactions, HIV-1