Poster abstracts
Poster number 53 submitted by Chathuri Pathirage
Human lysyl-tRNA synthetase binds to packaging-competent dimeric HIV-1 5’UTR RNA to facilitate tRNA 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), William A. Cantara (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), 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)
Abstract:
Selective packaging of the HIV-1 reverse transcription primer, human 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. Recent studies have shown that 5’ transcriptional start site (TSS) heterogeneity leads to gRNAs starting with 1G or 3Gs, which differ in structure and function. 1G RNAs adopt a conformation that favors dimerization and are preferably packaged to virions, while 3G RNAs fold into dimer-incompetent structures and serve as viral mRNAs for translation. We previously showed that gRNA 5´UTR binding affinity of both WT and phosphomimetic S207D LysRS proteins increased when the downstream packaging signal (psi) sequences capable of 5´UTR dimerization were included. UV crosslinking (XL)-based mapping of protein binding sites revealed that both WT and S207D LysRS proteins bind to TLE and psi regions of the 5´UTR. The impact of TSS heterogeneity on LysRS binding and the role of phosphorylation in tRNA primer placement are unknown. Using electromobility shift assays, we showed that both LysRS variants bind preferentially to dimeric 1G RNA, suggesting that LysRS selects for packaging competent gRNA for tRNA placement in a phosphorylation-independent manner. Although LysRS alone does not show any chaperone activity for tRNA annealing to viral gRNA, the HIV-1 Gag protein chaperones the annealing of tRNALys3 pre-bound to LysRS. This data support a model wherein LysRS recruits tRNA to packaging-competent 1G RNA, thereby facilitating Gag-chaperoned annealing of tRNA to the PBS. Studies aimed at characterizing binding stoichiometries of LysRS variants to 1G/3G RNAs using mass photometry are currently underway.
Keywords: HIV-1 RNA, LysRS, tRNA primer annealing