Poster abstracts
Poster number 19 submitted by Jonathan Kitzrow
Intrinsic conformational dynamics of the HIV-1 genomic RNA 5′ UTR
Jonathan P. Kitzrow (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Benjamin S. Brigham (Department of Molecular Biology and Microbiology, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, MA 02111), Joshua-Paolo C. Reyes (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), James B. Munro (Department of Molecular Biology and Microbiology, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, MA 02111)
Abstract:
The highly conserved 5′ untranslated region (5′ UTR) of the HIV-1 RNA genome (gRNA) is central to the regulation of virus replication. Biochemical and NMR experiments support a model in which the 5′UTR can transition between at least two conformational states. In one state, the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed and the genome is competent for dimerization and packaging into assembling virions. According to this model the conformation of the 5′UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and ensemble FRET assays. We find that the monomeric 5′UTR can spontaneously transition between two conformations, which have distinct intra-molecular base pairing. One of the observed conformations is competent for dimerization with a second 5′UTR molecule. Our results are consistent with the previously proposed model in which dimerization initiates by way of localized inter-molecular kissing-loop base pairing. We also show that the inter-molecular interface can extend beyond the kissing-loop interaction, giving rise to an extended dimer conformation. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer, but not the extended dimer. In contrast, the HIV-1 nucleocapsid protein promotes formation of the extended dimer both in the absence and presence of tRNALys3. Thus, the HIV-1 5′UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.
Keywords: RNA biology, HIV-1, FRET