Poster abstracts

Poster number 123 submitted by Petra Wallenmeyer

XL-SHAPE Analysis of Human Lysyl-tRNA Synthetase Interaction with HIV-1 Genomic RNA

Petra C. Wallenmeyer (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Kevin A. Garayalde Batista (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Joshua Hatterschide (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH)

Abstract:
Reverse transcription of HIV-1 genomic RNA (gRNA) is initiated from the 3' end of human tRNALys3, which is annealed to the primer binding site (PBS) in the 5'UTR. All tRNALys isoacceptors are specifically packaged into HIV-1 particles, along with human lysyl-tRNA synthetase (LysRS). Moreover, specific interactions between a tRNA-like element (TLE) in the 5'UTR and LysRS are proposed to facilitate targeting of tRNALys3 to the PBS.1 The TLE mimics the U-rich anticodon loop of tRNALys3 and is located proximal to the PBS. A previous study showed that high-affinity LysRS binding to the 5'UTR required additional stem-loop (SL) elements within the gRNA Psi packaging signal.1 Previous work also showed that the isoform of LysRS that is packaged into HIV-1 particles is phosphorylated on S207.2 To further characterize the interaction between the HIV-1 5’UTR and LysRS and to understand the effect of LysRS phosphorylation on this interaction, cross-linking and selective 2'-hydroxyl acylation analyzed by primer extension (XL-SHAPE) studies were performed using WT LysRS and the phosphomimetic S207D LysRS variant. SHAPE studies revealed that both proteins confer moderate RNA flexibility changes in the TLE of both WT dimeric and monomeric RNA constructs. Both proteins also confer decreases in flexibility in a bulge in SL1 of Psi, consistent with studies showing that SL1 contributes to high-affinity binding of LysRS to gRNA.1 Thus, a specific SL1:LysRS interaction may contribute to LysRS binding to the 5′UTR and cross-linking studies currently underway will address this question.

References:
1. Jones CP, Saadatmand J, Kleiman L, Musier-Forsyth K. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing. RNA. 2013;19(2):219-229.

2. Duchon AA, St-Gelais C, Titkemeier N, Hatterschide J, Musier-Forsyth K. HIV-1 exploits dynamic multi-aminoacyl-tRNA synthetase complex to enhance viral replication. J. Virol. 2017, in press.

Keywords: HIV, XL-SHAPE, LysRS