Poster abstracts
Poster number 62 submitted by Jonathan Kitzrow
Probing the structure of the HIV-1 5´UTR with varying transcription start sites
Jonathan Kitzrow (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Luke Lamorelle (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)
Abstract:
The full-length HIV-1 transcript can act as either mRNA, encoding the Gag and Gag-Pol polyproteins, or as genomic RNA (gRNA) that is selected by Gag during virion assembly. The HIV-1 5′UTR, which is part of all full-length HIV-1 transcripts, is known to regulate many important processes during the viral lifecycle, including RNA splicing, translation, dimerization, and packaging. These processes are regulated, in part, by interactions between host/viral proteins and conserved structural elements within the 5′UTR. Recently, the number of guanosines at the 5′ end of the HIV-1 RNA transcript has been shown to vary due to heterogeneous transcriptional start-site (TSS) selection.1 5´-cap-1G transcripts are primarily dimeric and enriched in virions, whereas 5´-cap-2G/3G transcripts are primarily monomeric and enriched on polysomes.1,2 Mutations designed to destabilize the transactivation response element (TAR) and polyadenylation (PolyA) stem loops of the 5′UTR impair gRNA dimerization.2,3 Taken together, these results suggest that different TSSs in HIV-1 transcripts influence downstream structures within the 5´UTR, as well as 5´UTR function. To establish the effects of 5´ end heterogeneity on 5´UTR structure, we performed differential selective 2′-hydroxylation analyzed by primer extension (SHAPE) on 5´UTR RNAs with varying numbers of 5´G nucleotides. Results to date suggest nucleotides within TAR/PolyA are more reactive to SHAPE reagents (i.e., less structured) in G3 constructs, whereas nucleotides within the HIV-1 psi packaging element are more reactive in G1 constructs. Thus, preliminary probing studies are consistent with the hypothesis that 5’UTR structure varies depending on TSS selection.
References:
1. Masuda T, Sato Y, Huang YL, Koi S, Takahata T, Hasegawa A, Kawai G, Kannagi M. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. Sci Rep. 2015;5:17680.
2. Kharytonchyk S, Monti S, Smaldino PJ, Van V, Bolden NC, Brown JD, Russo E, Swanson C, Shuey A, Telesnitsky A, Summers MF. Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome. Proc Natl Acad Sci U S A. 2016;113(47):13378-83.
3. Das AT, Vrolijk MM, Harwig A, Berkhout B. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging. Retrovirology. 2012;9:59.
Keywords: HIV-1, SHAPE, RNA Structure