A flexible system has been developed for studying the structures of complexes of HIV-1 reverse transcriptase (RT) with nucleic acid template- primers and inhibitors at 2.8 Angstrom resolution. We have thus far determined the structure of a ternary complex of HIV-1 RT with a monoclonal antibody Fab fragment (Fab28) and a 19 base/18 base dsDNA (19/18 dsDNA) template-primer at 3.5 Angstrom resolution and are refining the structure at 2.8 Angstrom resolution. The crystal form that we are using is particularly advantageous because 1) it is providing the first atomic structure of any polymerase bound to nucleic acid in a mode relevant for polymerization, and 2) complexes containing antiviral compounds, different template-primers, and HIV-1 RT mutants can be studied by the straightforward difference Fourier method. Since only the altered portion of the structure needs to be solved, all of the proposed studies can be done with relative facility. Specifically, using the HIV-1 RT/Fab28 crystals, we will solve and refine the structures of variants that contain three different nucleic acid template-primers (19/18 dsDNA, 30/18 dsDNA, and 30/18 RNA/DNA). These RT/Fab/template-primer complexes will be the reference structures for all of the other determinations. We will determine the structures of the RT/Fab/DNA crystals containing triphosphate forms of the nucleoside inhibitors AZT and ddI, using both wild-type and nucleoside-resistant HIV- 1 RT mutants. The structure of an HIV-1 /HIV-2 chimera in which the HIV-2 polymerase active site has been transplanted onto HIV-1 RT will be determined, thus permitting examination of the structure of the HIV-2 RT active site. The structural information gained from this work will be helpful in understanding the molecular mechanisms of DNA polymerization, RT inhibition, and development of resistance to nucleoside inhibitors. The detailed stereochemical description of nucleoside triphosphate inhibitor complexes with HIV-1 RT should be valuable in the design and refinement of antiviral agents.
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