The goal of this project is to define the molecular mechanisms involved in the replication of enveloped RNA viruses and in particular, to understand the factors which influence the regulation and expression of viral genetic information. Studies are being carried out with the murine leukemia virus system. Current interest is focused on the organization of the MuLV pol gene and on correlation of genetic structure with pol-associated enzymatic functions. Molecular clones containing MuLV reverse transcriptase and/or endonuclease gene segments have been expressed in E. coli and specific antisera hav been generated against the expressed bacterial proteins. These antisera are useful reagents for characterizing viral pol proteins, and serve as probes for the protein structure of the virus. Recently, studies with an antiserum directed against endonuclease alone have led to the observation that the viral reverse transcriptase and endonuclease can be associated as a complete involving both non-covalent and disulfide bonds. The exact nature of this association within the virion is under investigation. The sera are also being used to define the defect in a viral mutant with an in-frame deletion in the endonuclease coding region. in addition, a clone expressing only reverse transcriptase sequences has been used to make a monospecific anti-polymerase serum and to provide a source of highly active enzyme. Large scale preparations of bacterial extracts of this clone have now been extensively purified by standard biochemical procedures and characterization of the purified bacterially expressed reverse transcriptase is in progress.
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