Poster abstracts

Poster number 26 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 ), Erik D. Olson, William A. Cantara, 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 three tRNALys isoacceptors are selectively packaged into virions through specific interactions between human lysyl-tRNA synthetase (hLysRS) and the viral Gag protein. tRNALys3 must be released from hLysRS prior to 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 proposed that this release is facilitated, in part, by the interaction of hLysRS with a tRNA-like element (TLE) in the HIV-1 genome (1). In the HIV-1 NL4-3 isolate the TLE is located proximal to the PBS and contains a U-rich anticodon-like loop similar to that of tRNALys3. Small angle X-ray scattering (SAXS) analysis of the NL4-3 PBS-TLE domain has revealed that this region mimics the 3D structure of tRNA (2). The goal of this project is to establish whether the TLE/tRNA mimicry is conserved across various subtypes of HIV-1. Here, we study the PBS region of the HIV-1 MAL isolate, which contains a 23-nt insertion that is known to result in an alternative secondary structure of the 5´-UTR (3). Fluorescence anisotropy assays used to investigate the binding of hLysRS to MAL-derived RNA constructs revealed moderate to high-affinity binding of hLysRS to MAL 123-218 (Kd = 499 ± 119 nM) and MAL 123-349 (Kd < 50 nM). To locate the sequence elements responsible for high-affinity binding, truncated RNAs and mutants of MAL 123-218 were tested. The experiments revealed decreased binding of hLysRS to the truncated RNAs, while more subtle mutations of MAL 123-218 did not significantly affect binding. The tertiary structure of MAL 123-218 was also analyzed using SAXS, revealing that this RNA shows remarkable structural similarity to the NL4-3 PBS-TLE region. The results obtained to date suggest that specific recognition of the TLE is based on overall 3D topology.

References:
1. Jones, C. P., Saadatmand, J., Kleiman, L., and Musier-Forsyth, K. (2013) Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing, RNA 19, 219-229.
2. Jones, C. P., Cantara, W. A., Olson, E. D., and Musier-Forsyth, K. (2014) Small-angle X-ray scattering-derived structure of the HIV-1 5' UTR reveals 3D tRNA mimicry, Proc Natl Acad Sci U S A 111, 3395-3400.
3. Goldschmidt, V., Paillart, J. C., Rigourd, M., Ehresmann, B., Aubertin, A. M., Ehresmann, C., and Marquet, R. (2004) Structural variability of the initiation complex of HIV-1 reverse transcription, J Biol Chem 279, 35923-35931.

Keywords: HIV-1, TLE, MAL