STRUCTURES OF HIV REVERSE TRANSCRIPTASE WITH SUBSTRATES

Rutgers, The State Univ Of N.J., New Brunswick NJ

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Abstract

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