Talk abstracts

Talk on Saturday 08:30-08:45am submitted by William Cantara

SAXS-derived structure of HIV-1 genome 5´UTR reveals independently folded functional domains and three-dimensional tRNA mimicry

William A. Cantara (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus OH 43210), Christopher P. Jones, Erik D. Olson (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus OH 43210), William C. Ho, Steve Tuske, Eddy Arnold (Center for Advanced Biotechnology and Medicine, and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus OH 43210)

Abstract:
In all retroviruses, reverse transcription is initiated from the 3´ end of a cellular tRNA primer. In the case of HIV-1, the 3´ 18 nucleotides of human tRNA^Lys3 are perfectly complementary to the primer binding site (PBS) within the 5´UTR. After annealing, the 3´ end of tRNA^Lys3 is extended by reverse transcriptase (RT). Only a single copy of tRNA^Lys3 is required to initiate reverse transcription, but all three human tRNA^Lys isoacceptors (~20-25 molecules) are selectively packaged into virions, along with stoichiometric amounts of human lysyl-tRNA synthetase (LysRS). LysRS is specifically packaged into virions via its interaction with the viral Gag protein, thus leading to the enrichment of tRNA^Lys in virions. Recently, we have shown that LysRS binds with high affinity to the viral RNA, in part, due to the presence of a tRNA-like element (TLE) in the U-rich stem loop proximal to the PBS, which mimics the anticodon of tRNA^Lys3 (Jones et al, RNA 2013). However, whether the 5´UTR possesses more extensive tRNA structural mimicry is unknown. In addition, it remains unclear precisely how tRNA^Lys3 annealing affects the conformation of the PBS domain of the HIV-1 genome. Using small-angle X-ray scattering and molecular dynamics simulations, we report the solution structure of the first 223 nucleotides of the 5′UTR comprising the transactivation response (TAR) element, polyadenylation (PolyA) stem loop, and PBS region. These data show that the TAR and PolyA domains adopt long co-axially stacked helices. Analysis of the apo and 18-nt DNA (anti-PBS) primer-annealed PBS domain reveal a tRNA-like tertiary fold. The conformations of the individual domains do not change significantly in the context of the full 223-nt RNA, suggesting the absence of long-range tertiary contacts. SAXS studies of the Psi packaging signal and of complexes between the genomic RNA, anti-PBS, and HIV-1 RT will also be presented.

Keywords: SAXS, HIV, 5UTR