Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that is the causative agent of AIDS. Human tRNALys,3, is selected as the natural primer for HIV-1 reverse transcriptase (RT). Prior to reverse transcription, the 18 nucleotides at the 3' end of this tRNA are annealed to a complementary sequence on the viral genome called the primer binding site (PBS). Although specific molecular interactions occur between the tRNALys,3 primer and various HIV components, the details of these interactions are not understood at the molecular level. Moreover, the complexes formed by human tRNALys,3, RT, and other HIV components such as the HIV-1 nucleocapsid protein (NC) and the RNA genome are attractive targets for new therapeutic agents. Therefore, we propose: 1. To elucidate the molecular interactions responsible for initiation of tRNA-primed DNA synthesis from complementary and non-complementary PBS sequences, (a) Specifically designed chimeric tRNALys,3 variants containing tRNAPro-specific domains, as well as HIV-RNA templates containing altered PBSs will be prepared and tested in in vitro primer/template annealing and reverse transcription assays. (b) Primer tRNAs containing randomized anticodon- and D-stem-loop sequences will be generated and employed in in vitro selection (SELEX) experiments. This study will delineate specific primer tRNA nucleotide bases that are critical for the initiation process. (c) Deoxy-phosphorothioate modification interference experiments will be carried out to identify backbone 2'-hydroxyl groups and phosphate oxygens that are crucial to tRNA primer annealing and extension. For (a)-(c) the results of assays using complementary versus non-complementary PBSs and heat-annealed versus NC-annealed primer/template complexes will be compared. 2. To probe the mechanism of primer tRNALys,3 unwinding and RNA-RNA annealing by HIV-1 NC, (a) Nuclease digestion experiments will be used to probe NC-induced conformational changes in tRNALys,3. (b) Fluorescence resonance energy transfer (FRET) measurements using steady-state and time-resolved techniques will be carried out to investigate NC unwinding of tRNALys,3 in the absence and presence of template. The formation of NC-annealed versus heat-annealed primer/template binary complexes will also be compared. (c) Stopped-flow fluorescence techniques will be used to investigate the kinetic mechanism of NC unwinding and annealing of tRNALys,3.
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