Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA.
An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.
D005537:Aphthovirus / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D012156:Reticulocytes; D014764:Viral Proteins / Q000096:biosynthesis*
D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D014764:Viral Proteins; D008715:Methionine