HIV reverse transcriptase (RT) remains one of the most important targets for therapeutic treatment of AIDS. It is a fascinating enzyme, in that it can carry out the complete synthesis of a double-stranded DNA copy of the single-stranded RNA genome of HIV. X-ray crystallography will be employed for the determination of the structure of the wild-type and Met184Val HIV-1 RT complexes with a monoclonal antibody Fab and an RNA:DNA template-primer, in the presence and absence of a dNTP analog. Specifically, recombinant HIV-1 RTs prepared and partially purified at Dr. Stephen Hughes's laboratory will be purified to crystallographic grade by successive chromatography steps. Crystals will be formed in the presence of the stabilizing antibody Fab. X-ray data collection will take place at CABM/Rutgers (5-6 Angstrom for pilot studies) and at the Cornell High Energy Synchrotron Source (2.8-3.0 A resolution). Depending on their degree of similarity to the solved HIV- 1 RT/Fab/DNA:DNA structure available in Dr. Arnold's laboratory, the proposed structures will be solved either by averaging the electron density for multiple closely related crystal forms of RT or by simple difference in Fourier analysis. Solution of the RT/Fab/RNA:DNA structure, with and without dNTP analog, should provide useful information regarding the mechanisms of DNA polymerization and degradation of RNA by RNase H. In addition, the solution of the structure containing the drug resistant Met184Val may provide insight into the mechanism of RT inhibition and development of drug resistance. Detailed stereochemical description of the active site in a pretransition state mode should be valuable in the design of new or in the improvement of existing inhibitors.
| Gao, H Q; Sarafianos, S G; Arnold, E et al. (1999) Similarities and differences in the RNase H activities of human immunodeficiency virus type 1 reverse transcriptase and Moloney murine leukemia virus reverse transcriptase. J Mol Biol 294:1097-113 |
