title: In vitro cleavage at or near the N-terminus of the helper component protein in the tobacco vein mottling virus polyprotein.
abstract: Translation of tobacco vein mottling virus (TVMV) RNA in a wheat germ system resulted in two products that were not observed in a rabbit reticulocyte system. One of these was the N-terminal protein, based on its being the most abundant product and its migration on SDS-PAGE at about 34 kDa. The second product was similar or identical to helper component (HC) isolated from TVMV-infected plants, based on co-migration with HC on SDS-PAGE and immunoprecipitation with anti-HC antibodies. The N-terminus of this product was determined by radiochemical Edman degradation to be Ser-257 of the polyprotein. This assignment was supported by peptide mapping with a tryptophan-specific reagent. A similar cleavage was observed when tobacco etch virus was translated in a wheat germ system. Comparison with homologous regions in five other potyviruses indicated conservation of amino acid residues on both sides of the proposed cleavage site. Conversion of Phe-256 to Met, Pro, Arg, His, or Trp by site-directed mutagenesis of a TVMV RNA transcription template inhibited cleavage in the wheat germ system. These results suggest that in vitro cleavage occurs between Phe-256 and Ser-257 and that this cleavage is the same as the in vivo cleavage which liberates the N-terminus of HC.
authors.affiliation: Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
authors.forename: G
authors.initials: G
authors.lastname: Mavankal
authors.forename: R E
authors.initials: RE
authors.lastname: Rhoads
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010942:Plant Viruses / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D012328:RNA Viruses / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus