Initiation of picornavirus protein synthesis in ascites cell extracts.
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.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor; D002474:Cell-Free System; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000378:metabolism; D005815:Genetic Code; D007700:Kinetics; D008574:Mengovirus / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D051379:Mice; D010442:Peptide Chain Initiation, Translational; D010455:Peptides / Q000032:analysis; D010849:Picornaviridae / Q000378:metabolism*; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011817:Rabbits; D013460:Sulfur Isotopes; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism
D010455:Peptides; D012367:RNA, Viral; D013460:Sulfur Isotopes; D014764:Viral Proteins; D012343:RNA, Transfer; D008715:Methionine; D014357:Trypsin