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Translation of moloney murine leukemia virus and moloney murine sarcoma virus rna in nuclease treated reticulocyte extracts enhancement of the gag pol poly peptide with yeast suppressor transfer rna

Translation of moloney murine leukemia virus and moloney murine sarcoma virus rna in nuclease treated reticulocyte extracts enhancement of the gag pol poly peptide with yeast suppressor transfer rna

Cell 13(1): 189-200

ISSN/ISBN: 0092-8674

The virion RNA from Moloney murine leukemia virus (MuLV) and Moloney murine sarcoma virus (MSV) were translated in a micrococcal nuclease-treated [rabbit reticulocyte] cell-free system. The predominant polypeptides formed from 35S MuLV RNA were 78,000 and 65,000 daltons in MW, and minor components with MW of 180,000, 110,000, 52,000 and 40,000 daltons were also observed. The 30S MSV RNA yielded 2 predominant polypeptides of 62,000 and 43,000 daltons, and minor components about 72,000, 40,000 and 18,000 daltons in MW. The predominant polypeptides generated by both MuLV and MSV RNA were precursors of the core proteins by immunoprecipitation with specific antisera. The 180,000 dalton MW polypeptide encoded by MuLV RNA was immunoprecipitated both by antisera to the core protein (p30) and reverse transcriptase. The major products therefore appear to be Pr65gag and Pr78gag; an important minor product is Pr180gag-pol. Most of the products of < 65,000 daltons synthesized in either system contained sequences from the core protein precursor, but the 43,000 and 18,000 dalton MW polypeptides generated by MSV RNA did not precipitate with antisera to MULV proteins. When purified yeast suppressor tRNA was added to the translation mixture directed by 35S MuLV RNA, the amount of the Pr78gag was reduced, while the yield of the Pr180gag-pol was enhanced. Amber suppressor tRNA was about 3 times as effective as ochre suppressor tRNA and nonsuppressor tRNA. This pattern of suppression was also seen for an established amber mutation (UAG) in the synthetase gene of [phage] Q.beta. (Q.beta. aml), suggesting that it is a UAG codon which terminates synthesis of Pr78gag. In the MSV system, the amber suppressor tRNA, and to a lesser extent the ochre suppressor tRNA, markedly increased the synthesis of the 72,000 dalton MW polypeptide with a slight reduction of the 62,000 dalton protein. Since read-through between the core protein and reverse transcriptase genes occurs to a low level both in vivo and in vitro and can be enhanced in vitro by amber suppressor tRNA, these results suggest that a suppression mechanism may control the relative amounts of core protein and reverse transcriptase synthesized from 35S mRNA. Such a mechanism might be used more generally by mRNA from mammalian cells.

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