The goal of this research is to define biochemical mechanisms of avian retrovirus replication through the study of the interactions of viral proteins with nucleic acids. These multidisciplinary model studies have direct application to development of anti-HIV agents.
The first aim of this proposal is to study the interaction of the viral gag nucleocapsid (NC) and matrix (MA) proteins and their Pr76gag precursor with viral RNA and to evaluate their role in virion assembly. Molecular genetic techniques will be used to make amino acid substitutions at highly conserved or biochemically important residues of the NC and MA proteins. The effect these changes have on viral replication in vivo and on the biochemical properties of the mutant proteins purified either from virus or bacteria will be determined. The role of the Pr76gag or NC and MA proteins in the recognition of specific packaging signals in viral RNA will also be studied.
The second aim of this proposal is to elucidate the mechanism of action of the DNA endonuclease associated with the viral integration (IN) protein and its role in viral DNA integration. Biochemical and molecular genetic experiments are outlined to relate the sequences at the termini of long terminal repeats (LTR) of viral DNA required for specific cleavage in vitro by the IN protein to those required for integration in vivo. This will be accomplished by analyzing the effect of small U5 LTR deletions on both cleavage and on integration of viral DNA. The requirement for inverted repeat sequences in the LTR in linear and supercoiled DNA substrates for IN protein activity and integration in vivo will be evaluated. Reconstitution of a viral DNA integration system in vitro using purified components will be attempted. In a related project, the role of secondary structure in the U5 region of viral RNA for initiation of reverse transcription will be studied. The observation that such structures potentially exist in other retrovirus RNAs including HIV suggest that this is a general phenomenon of replication.
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