Talk Abstracts

Talk on Friday 02:00-02:15pm submitted by Chathuri Pathirage

SHAPE probing of the HIV-1 Reverse Transcriptase Initiation Complex reveals RNA flexibility changes adjacent to the Primer-Binding Site

Chathuri Pathirage (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University ), William Cantara (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University), Steven Tuske (Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ), Eddy Arnold (Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ), Karin Musier-Forsyth (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
HIV-1 uses host tRNA^Lys3 as the primer for reverse transcription (RT). The 18 nucleotides (nt) at the 3’ end of tRNA^Lys3 base pair with a complementary primer-binding site (PBS) sequence in the HIV-1 5’ UTR to initiate RT. Extended base-pairing interactions are necessary for efficient RT initiation. Based on recent cryo-EM data using a truncated PBS construct and crosslinking approach to stabilize the complex, tRNA^Lys3 is restructured into an extended helix with a base paired anticodon loop in the reverse transcriptase initiation complex (RTIC). Here, we performed UV crosslinking coupled with selective 2’- hydroxyl acylation analyzed by primer extension (XL-SHAPE) on the complete 5’UTR to probe conformational changes upon tRNA annealing and reverse transcriptase (RTase) binding. Nt flexibility was decreased in the 4 nt downstream of the PBS upon unmodified tRNA annealing to the 5’UTR, suggesting extended primer-template base pairing. We also observed increased flexibility in the primer activation signal (PAS) sequence of the 5’UTR upon tRNA annealing, suggesting dynamics in this region. SHAPE probing of tRNA^Lys3 heat-annealed to the 5’UTR showed that the anticodon loop of the tRNA has high SHAPE reactivity, suggesting that it maintains canonical structural features. UV crosslinking of RTase to the primer-template complex identified crosslinking sites in the PBS and SL2 regions of the 5’UTR packaging signal. The SHAPE reactivity profile of the 5’UTR when RTase is bound to the heat-annealed primer-template complex showed increased nt flexibility in the region upstream of the PBS, suggesting a destabilizing effect. Overall, our results indicate that 5’UTR regions adjacent to the PBS undergo significant nt flexibility changes upon both primer annealing and RTase binding, while tRNA^Lys3 maintains canonical structural features in the anticodon and D domains. Studies aimed at establishing the role of tRNA modifications in RT initiation, are underway.

Keywords: HIV-1 RNA, reverse transcription initiation, tRNA primer annealing