Cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by Rauscher murine leukemia virus 35S RNA

Abstract

Translation of Rauscher murine leukemia virus (RLV) 35S RNA in a mRNA-dependent protein-synthesizing system results in the synthesis of polypeptides with apparent molecular weights of 200,000, 75,000 and 65,000 daltons. Each of these polypeptides was immunoprecipitable with anti-p30 serum, while only the 200,000 dalton size class was specifically recognized by antiserum prepared against purified reverse transcriptase. intracellular Pr200 gag-pol has been shown to contain p30 tryptic peptide sequences (Arcement et al., 1976) and to share antigenic determinants with the gag proteins p30, p15, p12 and p10 and the reverse transcriptase (Jamjoom, Naso and Arlinghaus, 1977). These results and others (Jamjoom et al., 1977) led us to the conclusion that Pr200 gag-pol is the initial translation product that leads to the formation of mature reverse transcriptase. In the present study, nine of eleven methionine-containing tryptic peptides found in a anti-reverse transcriptase-precipitable 80,000 dalton molecular weight virion polypeptide (p80 pol) were contained in Pr200 gag-pol. In addition, virion p80 pol co-migrates in SDS-polyacrylamide gels with the major polypeptide found in partially purified preparations of active reverse transcriptase. The in vitro synthesized 200,000 dalton polypeptide was identical to intracellular Pr200 gag-pol as determined by comparing ion-exchange profiles of methionine-labeled tryptic peptides. The frequency of synthesis of the in vitro synthesized Pr200 gag-pol was 1 25 to 1 20 that of the combined synthesis of the 65,000 and 75,000 dalton gag precursors. A similar ratio of Pr200 gag-pol to Pr65 gag and Pr80 gag has been observed in viral infected cells. Thus 35S viral genomic RNA is an mRNA for both gag and pol gene products. Experiments with the arginine analogue canavanine suggest that the usual termination signal occurs after the translation of Pr80 gag. We propose that the Pr200 gag-pol results from an occasional read-through (5% frequency) of a single termination codon at the end of the gag gene.