This proposal describes kinetic investigations of the first step of HIV retroviral reverse transcription using state-of-the-art single molecule fluorescence energy transfer (FRET) measurements and fluorescence correlation spectroscopy (FCS). The HIV retrovirus utilizes a host tRNA/Lys,3 as a fluorescence correlation spectroscopy (FCS). The HIV retrovirus utilizes a host cell tNA Lys,3 as a primer for minus strand DNA synthesis, and the tRNA unwinding and annealing to the viral RNA genome is promoted by the HIV nucleocapsid protein (NC). The kinetics and mechanism of NC's involvement in tRNA unwinding and annealing remain largely unknown and may be complex due to reversibility in stoichiometry issues. In this work, a combination of FCS and single molecule FRET measurements will be used to characterize the interaction between tRNA/Lys,3 and NC in the absence of the viral RNA template as well as the interaction of NC with the RNA template. Additionally, single molecule methods will be used to study the kinetics of the unwinding and annealing of single tRNAs. NC is a target for new antiviral agents, and increased knowledge of NC's role in unwinding and annealing will enhance these efforts. Additionally, the single molecule kinetic techniques developed in this research for RNA/protein interactions will be applicable to many other complex biochemical problems where conventional kinetics methods fail due to conformational and stoichiometric heterogeneity in the system.
| Cukalac, Tania; Kan, Wan-Ting; Dash, Pradyot et al. (2015) Paired TCR?? analysis of virus-specific CD8(+) T cells exposes diversity in a previously defined 'narrow' repertoire. Immunol Cell Biol 93:804-14 |
| Cosa, Gonzalo; Harbron, Elizabeth J; Zeng, Yining et al. (2004) Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein. Biophys J 87:2759-67 |
