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 process of reverse transcription in an effort to correlate genetic structure with enzymatic function. Portions of the MuLV pol gene have been expressed in E. coli. The expression system consists of (i) a vector carrying the bacteriophage gamma PL prommoter and sequences encoding the first 13 amino acids of the gamma cI protein fused (out-of-frame) to the Beta-galactosidase coding region and (ii) a lac-host with a temperature-sensitive Gamma repressor. Recombinants containing in-frame inserts of the reverse transcriptase and endonuclease coding regions have been isolated. At 42 degrees C these clones express large amounts of high molecular weight lac Z fusion proteins, which have been injected into rabbits to elicit antibody production. Analysis of wild-type and mutant virions by immunoprecipitation and Western blot techniques has demonstrated that the antisera react specifically with two proteins present only in wild-type MuLV: the 80K reverse transcriptase; and a 47K protein which we can now identify as the viral endonuclease. Proteins expressed by clones engineered to eliminate lac Z expression can be partially solubilized with high salt and detergents, and some reverse transcriptase activity has been detected in extracts of clones containing the reverse transcriptase coding sequence. In other studies, revertants of the frameshift pol mutant clone 23 have been isolated. Efforts are underway to make molecular clones of these revertants to determine the mechanism by which the mutation is repaired. Experiments involving in vitro mutagenesis of selected regions of the MuLV genome have also been initiated.
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