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 [(35)S]
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 [(35)S]
methionine-labeled products from
Met-tRNA(M) (Met) analyzed by
anion-exchange chromatography yielded more than 30
peptides, as compared to about 15 [(35)S]
methionine-labeled
peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-
tRNA(F) (Met) yielded one major (26)S-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.