2013 Rustbelt RNA Meeting
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Poster number 23 submitted by Roopa Comandur

HIV-1 subtype-specific differences in tRNALys targeting to viral RNA primer binding site

Roopa Comandur (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH 43210 ), Christopher P. Jones (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH 43210 ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH 43210 )

Abstract:
Human tRNALys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1). All tRNALys isoacceptors are selectively packaged into budding virions through specific interactions between human lysyl-tRNA synthetase (hLysRS) and the viral Gag protein. tRNALys3 must be released from hLysRS for annealing to the complementary primer binding site (PBS) in the 5’-UTR of the HIV-1 genome in order to initiate reverse transcription. We have shown that this release is facilitated, in part, by the interaction of hLysRS with a tRNA-like element (TLE) in the HIV-1 genome. In HIV-1 NL4-3, which is a clade B subtype, the TLE is located proximal to the PBS and mimics the U-rich anticodon loop of tRNALys. The goal of this project is to establish the conservation of the TLE across various subtypes of HIV-1. We were particularly interested in investigating the HIV-1 MAL isolate, which is a complex recombinant of clades A, D and I. This isolate is representative of clade A and contains a 20-nt insertion near the PBS, which is known to result in an alternative secondary structure fold of the 5’-UTR. We have used fluorescence anisotropy assays to investigate the binding of hLysRS to MAL-derived RNA constructs. The assays reveal moderate to high affinity binding of hLysRS to MAL 123-218 (a 98-mer RNA containing the PBS; Kd = 413±22 nM) and MAL 123-349 (a 229-mer RNA containing the PBS and the dimer initiation signal, DIS; Kd < 50 nM). To locate the sequence elements responsible for the high-affinity binding, truncated genomic RNAs are being designed and tested. An A-rich 23-nt stem-loop RNA previously shown by NMR to mimic the tRNA anticodon structure showed only weak binding to hLysRS (Kd > 5 uM). The results obtained to date suggest differences in the mechanism of tRNALys3 targeting to the PBS and release from hLysRS in different subtypes of HIV-1.

Keywords: HIV-1, TLE, MAL