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), 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), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University)
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. This release is partly facilitated 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. 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 tRNA mimicry and hLysRS binding is conserved across two prototypical HIV-1 subtypes. Here, we study the 5`-UTR region of the MAL isolate, which contains a 23-nt insertion that is known to result in an alternative secondary structure relative to the NL4-3 isolate(3). Fluorescence anisotropy assays used to investigate the binding of hLysRS to MAL-derived genomic RNA constructs revealed high-affinity binding to a 229-nt MAL construct (Kd=47±13 nM). Truncation of this construct at the 3` end yielded a 98-nt construct with ~10-fold reduced binding (Kd=485±133 nM). No binding was observed to smaller stem-loop elements derived from the 98-nt construct and point mutations of this construct did not significantly affect binding. The tertiary structure of both the 229-nt and the 98-nt MAL RNAs was analyzed using SAXS, revealing that these RNAs show remarkable structural similarity to analogous NL4-3-derived RNAs. The results suggest that specific recognition of the HIV-1 PBS-TLE domain by hLysRS is largely based on overall 3D topology rather than sequence-specific recognition.
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 USA 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