The long term objective is to provide a structural basis for understanding the function of HIV proteins that interact with RNA-DNA. This will be achieved through biochemical studies and the determination of crystal structures of proteins and their complexes with the nucleic acids with which they interact; proteins to be pursued include reverse transcriptase (RT), tat fragments and tat fusion proteins, rev and integrase. The high resolution crystal structures that result from these studies will form the basis for designing inhibitors which may function as anti-AIDS drugs. The present alpha-carbon backbone model of reverse transcriptase, built to fit a 3.5 alpha electron density map, will be extended to include full backbone and side chains and refined at the highest resolution possible (3.0 to 2.8 alpha). Using this refined structure it will be possible to design modifications to the non-nucleotide inhibitor, nevirapine, which has been bound to these crystals. Further, regions of this enzyme whose sequence is conserved among homologous RT molecules can be located in 3-D and used as potential targets for design of new inhibitors. Towards the goal of understanding how the nucleic acid substrates bind to this enzyme, the structure of present crystals of RT complexed with tRNA will be solved and attempts to grow crystals of RT complexed with DNA undertaken. To test hypotheses concerning the structural basis for fidelity of this enzyme, we shall make mutant enzymes with reduced fidelity. Studies of the transactivator, tat, will focus on determining the structure of crystals of a 24 residue peptide complexed with 27 nucleotide TAR RNA as well as biochemical and structural studies of tat fusion proteins with CAP. Attempts will be made to crystallize rev and integrase along with appropriate complexes with nucleic acids.
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