This proposal intends to investigate both structural and functional aspects of the ribonucleaseH (RNaseH) activity of HIV RT and evaluate its potential as a target for chemotherapy. Using a novel metal chelate affinity resin, several forms of the genetically engineered 15 kDa RNaseH domain (p15 RNaseH) will be purified and characterized. In the first instance, p15 RNaseH will be subjected to biophysical analyses by both X-ray crystallography and low angle neutron scattering, in an attempt to define the structure of the isolated domain. The nucleolytic properties of HIV-1 RT/RNaseH will be evaluated with a series of point mutants whose RNaseH capacity has been either totally or partially impaired by in vitro site- directed mutagenesis. Using high resolution gel electrophoresis, experiments discriminating between the exo- and endonucleolytic activities will be carried out on RNaseH, as either the purified domain, p66 RT/RNaseH homodimer or p66/p51 RT/RNaseH heterodimer. The interaction of recombinant p15 RNaseH with model DNA/RNA hybrids substrates is proposed, in an attempt to better understand how the enzyme interacts with each nucleic acid component of the hybrid. These studies will provide valuable information for later biophysical studies to characterize the structure of RNaseH complexed with a defined DNA/RNA hybrid. In an attempt to elucidate potential interdependence between the polymerising and Rnaseh domains of HIV RT, chimaeric RT/RNaseH molecules will be constructed between HIV-1 RT and portions of either the HIV-2 or equine infectious anaemia virus (EIAV) enzyme. These chimaerae will be analyzed for their polymerase and Rnaseh properties, as well as capacity to reconstitute into enzymatically active heterodimer with the 51 kDa HIV-1 RT subunit. Small amino acid insertions will also be introduced at the junction between the polymerase and Rnaseh domains of p66 RT to determine if these can be spatially separated without impairment of enzymatic activity. Finally, in order to evaluate the requirement of p15 Rnaseh for viral replication, mutant proviruses will be constructed which fail to mature the RNaseH domain from p66 RT, and their infectivity determined.
