title: A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing.
abstract: Mature viral-encoded proteins of tobacco etch virus (TEV) arise by proteolytic processing of a large precursor. The proteinase responsible for most of these cleavages is a viral-encoded 49-kDa protein. All known or predicted cleavage sites in the TEV polyprotein are flanked by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly, with the scissile bond located between the Gln-Ser or Gly dipeptide. By using cell-free systems to manipulate and express cloned cDNA sequences, a 25-amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the 49-kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site. The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. A protein that harbored an insertion of the conserved 7-amino acid segment was cleaved by the 49-kDa TEV proteinase. Cleavage of the synthetic precursor was shown to occur accurately between the expected Gln-Ser dipeptide by microsequence analysis. Proteins containing insertions that generated only the Gln-Ser, or only the serine moiety of the conserved sequence, were insensitive to the 49-kDa proteinase.
authors.affiliation: Department of Microbiology, Oregon State University, Corvallis 97331.
authors.forename: J C
authors.initials: JC
authors.lastname: Carrington
authors.forename: W G
authors.initials: WG
authors.lastname: Dougherty
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010942:Plant Viruses / Q000235:genetics*; D010947:Plants, Toxic; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D014026:Nicotiana; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D014764:Viral Proteins; D010447:Peptide Hydrolases