Cell-free synthesis of rauscher murine leukemia virus “gag” and “gag-pol” precursor polyproteins from virion 35 S RNA in a mRNA-dependent translation system derived from mouse tissue culture cells

Abstract

Using a mRNA-dependent cell-free protein synthesis sytem derived from mouse tissue culture cell extracts by treatment with micrococcal nuclease, we have examined the capacity of 35S genomic RNA from Rauscher leukemia virus (RLV) to code for the synthesis of viral proteins. Analysis of the polypeptide product by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that RLV 358 RNA directed the synthesis of RLV-specific polypeptides of 55,000, 65,000, 75,000, and 200,000 apparent molecular weights, identical in size to the known intracellular precursors of the RLV mature polypeptides. None of the individual mature virus proteins appeared to be synthesized. If canavanine, an arginine analogue, was substituted for arginine under conditions for cell-free protein synthesis, a RLV-specific polypeptide with a molecular weight of approximately 80,000 was synthesized at the expense of the 65,000 and 75,000 MW polypeptides. Monospecific antisera directed against p30, p15, p12, and p10 recognized the 65,000, 75,000, 80,000 and 200,000 MW polypeptides, indicating that each shared antigenic determinants with all of these “gag” proteins. Using the appropriate authentic RLV precursor polypeptides as standards, comparative tryptic maps were performed with the [ 3H]tyrosine or [ 35Snine-labeled in vitro-synthesized 65,000 and 200,000 MW polypeptides. The 65,000 MW polypeptide was found to be identical to Pr658 gag, 2 a 65,000 MW RLV gag protein precursor obtained from infected cells by immunoprecipitation and gel electrophoresis. The 200,000 MW in vitro-synthesized polypeptide was found to contain methionine-labeled tryptic peptides characteristic of the RLV reverse transcriptase (“pol”) and p30.