HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme consisting of a 66-kDa subunit (termed p66) and a p66- derived 51-kDa subunit (p51). The DNA polymerase and ribonuclease H (RNase H) activities of RT are entirely dependent on the quaternary structure of the enzyme. Recent studies have shown that nonnucleoside RT inhibitors (NNRTI) can modulate the inter-subunit interactions between the p66 and p51 polypeptides of RT. In this regard, NNRTI can be classified into 3 distinct groups. The first group, which includes derivatives of 2',5'-Bis-O-(tert-butyldimethylsilyi)- beta-D-ribofuranosyl]-3'spiro-5""""""""-(4""""""""-amino-1''2""""""""-oxathiole-2"""""""",2""""""""-dioxide) thymine (TSAO-T) and N-acyl hydrazones, destabilize the inter-subunit interactions in HIV-1 RT. The second group, which includes nevirapine and efavirenz, enhance the inter-subunit interactions in HIV-1 RT. The third group, which includes delavirdine, elicits no effect. The molecular mechanisms by which NNRTI binding to RT can modulate the inter-subunit interactions of the enzyme, and the impact that this modulation has on RT enzymatic functioning is not known. To this end, the project described in this proposal comprises two Specific Aims. (1) To determine the mechanism by which NNRTI modulate HIV-1 RT intersubunit interactions and intra-subunit conformational changes. HIV-1 RT p66/p51 heterodimer formation is a complex process that involves inter-subunit interactions and intra-subunit conformational changes. Assay systems based on fluorescence resonance energy transfer (FRET) or RT tryptophan fluorescence will be developed that monitor the bimolecular protein-protein interactions and unimolecular conformational changes in RT. These assay systems will then be used to generate quantitative thermodynamic and kinetic data regarding the p66/p51 RT dimerization process and how NNRTI-binding can impact on it. (2) To define the molecular interactions in the HIV-1 RT dimer interface and to evaluate the consequences of altering the intrinsic dimeric stability on enzymatic activity. The NNRTI-BP is small compared to the RT dimer interface and it is not understood how NNRTI binding can globally affect the inter-subunit interactions in RT. It is also not certain as to whether the NNRTI-mediated inhibition of RT is a direct result of modulating the enzyme's inter-subunit interactions. To address these issues, this aim seeks to generate high-resolution functional data regarding the importance of amino acid side chains in the RT dimer interface near to, and removed from, the NNRTI-BP. Furthermore, RT containing mutations that either stabilize or destabilize the dimeric stability of the enzyme will be assessed for their capacity to carry out both DNA polymerase and ribonuclease H (RNase H) activities. The results of our studies should provide significant insight into the nature of the protein-protein interactions in the p66/p51 RT heterodimer and the mechanisms of NNRTI action.
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