Initiation of Picornavirus Protein Synthesis in Ascites Cell Extracts

Abstract

The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [³⁵S]methionine from rabbit liver Met-tRNA_M ^Met and fMet-tRNA_F ^Met into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNA_M ^Met continued for 20 min. Tryptic digests of [³⁵S]methionine-labeled products from Met-tRNA_M ^Met analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [³⁵S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNA_F ^Met yielded one major ²⁶S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNA_F ^Met when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.

• translation in vitro
• Met-tRNA
• tryptic mapping
• encephalomyocarditis RNA