Talk abstracts

Talk on Saturday 09:20-09:35am submitted by Christopher Jones

Molecular Mimicry of primer tRNA^Lys3 by HIV-1 RNA genome facilitates viral replication

Christopher P. Jones (Departments of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Jenan Saadatmand (Lady Davis Institute for Medical Research, McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada, H3T1E2), Lawrence Kleiman (Lady Davis Institute for Medical Research, McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada, H3T1E2), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Many transposable elements and viruses rely on tRNAs and tRNA-like structures during their life cycles. To initiate reverse transcription, HIV-1 anneals human tRNA^Lys3 to the complementary primer binding site (PBS), and cDNA synthesis begins from the 3´-OH of the tRNA. We have previously shown that human lysyl-tRNA synthetase (LysRS) is specifically incorporated into HIV-1 by the Gag protein and that this interaction is critical for tRNA^Lys3 packaging. Herein, we describe a novel conserved tRNA^Lys3-like element (TLE) in the 5´-UTR of the HIV-1 genome proximal to the PBS. Using electrophoretic mobility shift assays and fluorescence anisotropy binding studies, we show that LysRS interacts with the TLE, which mimics the anticodon domain of tRNA^Lys3, whereas human TrpRS and ProRS do not. The affinity of LysRS for short (23 nt) HIV-1 genome-derived RNA hairpins containing the TLE is similar to that of in vitro transcribed 76-nt tRNA^Lys3 (Kd ~ 90 nM). Human LysRS recognizes tRNA^Lys isoacceptors via a specific interaction with the anticodon (UUU in tRNA^Lys3 and CUU in tRNA^Lys1,2). Mutation of the UUU motif in the TLE to AAA or CCC greatly reduces the LysRS-TLE interaction (Kd ≥ 2.5 and 8 uM, respectively). Longer 330-nt RNAs derived from the 5´-UTR compete even more effectively for LysRS binding to a tRNA^Lys3 anticodon stem-loop mimic than tRNA^Lys3, suggesting that in addition to the U-rich motif in the TLE, other sequence elements in the 5´ UTR contribute to preferential binding of LysRS to the HIV-1 genome. 293T cells transfected with virus containing TLE mutations show no change in the level of packaged tRNA but result in a 70% decrease in the amount of tRNA^Lys3 placed onto the genome. These new findings suggest that the HIV-1 genome uses molecular mimicry of tRNA^Lys3 to facilitate primer release from LysRS and placement onto the PBS.

Keywords: tRNA-like element, HIV genome, reverse transcription