Poster abstracts

Poster number 7 submitted by Alice Duchon

Retroviral RNA and Protein Interactions with Host Cell Aminoacyl-tRNA Synthetases

Alice Duchon, Tiffany Rye-McCurdy (Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Rebecca Kaddis (Penn State College of Medicine, Medicine, Hershey, PA 17033), Christopher Jones (Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Jenan Saadatmand, Lawrence Kleiman (Lady Davis Institute for Medical Research, McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada, H3T1E2), Leslie Parent (Penn State College of Medicine, Medicine, Hershey, PA 17033), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
During the retroviral lifecycle, specific host cell tRNAs are used as primers for reverse transcriptase (RT). In the case of HIV-1 and Rous sarcoma virus (RSV), tRNA^Lys,3 and tRNA^Trp, respectively, serve this role. These tRNAs are selectively packaged along with their cognate lysyl- and tryptophanyl-tRNA synthetases (LysRS and TrpRS). Retroviral assembly is tightly regulated through interactions involving the Gag protein, which directs the assembly process, viral genomic RNA, and cellular factors. HIV-1 Gag is required for packaging of LysRS, and the RT domain of Gag-Pol is implicated in selective tRNA^Lys packaging. The RT region of RSV Gag-Pol is crucial for tRNA^Trp recruitment, but the interactions that dictate TrpRS packaging are unknown. In this work, using antibodies against chicken TrpRS we confirmed the packaging of TrpRS into RSV particles. Moreover, our preliminary immunofluorescence confocal imaging studies suggested that a subpopulation of TrpRS was co-localized with Gag and the viral RNA in the nucleus. We hypothesize that RSV Gag-TrpRS interactions may be initiated in the nucleus, and studies to probe both Gag-TrpRS and genome-TrpRS co-localization are underway.
In HIV-1, the 3’-18 nt of tRNA^Lys,3 anneal to a complementary primer binding site (PBS) located near the 5’ end of the genome. A region upstream of the PBS strongly resembles the tRNA^Lys anticodon loop, which is the major binding/recognition element of the synthetase. This “tRNA‐Like Element” (TLE), binds LysRS with a similar affinity as tRNA^Lys. Moreover, cell-based assays suggest that LysRS-TLE interaction facilitates placement of tRNA^Lys onto the PBS and contributes to viral infectivity. By analogy to this work, a putative tRNA^Trp anticodon domain mimic has been identified in the RSV genome. Fluorescence anisotropy binding assays suggest a short, 60 nt, and longer, 419 nt, segment of the genome containing the TLE region, are able to bind to and compete with tRNA^Trp as a substrate for TrpRS. Future studies will test the role of TrpRS/genomic RNA interaction on tRNA^Trp priming and viral infectivity.

Keywords: RSV, TrpRS, TLE