Poster abstracts
Poster number 35 submitted by Chathuri Pathirage
Phosphomimetic S207D lysyl-tRNA synthetase binds the tRNA-like element and packaging signal of dimeric HIV-1 genomic RNA for proper primer placement
Chathuri Pathirage (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210, USA ), William A. Cantara (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210, USA ), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210, USA ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus, OH, 43210, USA )
Abstract:
Selective packaging of the HIV-1 reverse transcription primer, tRNALys3, is facilitated by interactions between lysyl-tRNA synthetase (LysRS) and HIV-1 Gag. Proper targeting of tRNALys3 to the primer binding site (PBS) in the 5´UTR of the HIV-1 genomic RNA (gRNA) has been proposed to be facilitated by LysRS binding to a PBS-adjacent tRNA-like element (TLE). During HIV-1 infection, LysRS is phosphorylated at S207 and released from a multi-aminoacyl-tRNA synthetase complex. Here, we investigated the interaction of phosphomimetic S207D LysRS with the HIV-1 5´UTR. Fluorescence anisotropy binding studies showed that S207D LysRS binds to the PBS/TLE RNA with similar affinity as WT LysRS; binding affinity increased by 2-3 fold for both proteins when the downstream packaging signal (psi) sequences capable of 5´UTR dimerization were included. RNA-protein UV crosslinking results showed that both proteins bound directly to the TLE and stem loop 1 (SL1) of psi, while additional binding sites were found in the PBS region for S207D LysRS. Selective 2´-hydroxyl acylation analyzed by primer extension (SHAPE) probing showed that S207D- but not WT-LysRS binding caused increased RNA nucleotide (nt) flexibility in the PBS and psi regions. SAXS analysis of the S207D LysRS-PBS/TLE complex revealed an elongated SAXS envelope compared to the WT LysRS complex, suggesting that S207D LysRS binds the RNA in the more “open conformation” previously observed for the protein alone. SAXS analysis of the 240-nt 5´UTR RNA monomer revealed a cruciform-like global RNA fold with the TLE and SL1 hairpins oriented in opposite directions, which may explain the lower binding affinity of LysRS variants for monomeric 5´UTR. SAXS-derived structures of primer-annealed PBS/TLE constructs revealed a conformational change in the TLE stem upon primer extension, and binding studies showed decreasing affinity for both LysRS variants in extended primer-annealed PBS/TLE constructs. These data suggest a mechanism for LysRS dissociation from the RNA during initial steps of reverse transcription. Overall, these data support a model wherein S207D LysRS binds to the 5´UTR and directs tRNA placement preferentially on dimeric gRNA, while destabilizing the PBS structure to facilitate reverse transcription initiation.
Keywords: phosphorylated LysRS, tRNA annealing, HIV-1 RNA