Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase.
The genome of tobacco etch virus contains a single open reading frame with the potential to encode a 346-kilodalton (kDa) polyprotein. The large polyprotein is cleaved at several positions by a tobacco etch virus genome-encoded, 49-kDa proteinase. The locations of the 49-kDa proteinase-mediated cleavage sites flanking the 71-kDa cytoplasmic pinwheel inclusion protein, 6-kDa protein, 49-kDa proteinase, and 58-kDa putative polymerase have been determined by using cell-free expression, proteolytic processing, and site-directed mutagenesis systems. Each of these sites is characterized by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly (in which cleavage occurs after the Gln residue). The amino acid residue (Gln) predicted to occupy the -1 position relative to the scissile bond has been substituted, by mutagenesis of cloned cDNA, at each of four cleavage sites. The altered sites were not cleaved by the 49-kDa proteinase. A series of synthetic polyproteins that contained the 49-kDa proteinase linked to adjoining proteins via defective cleavage sites were expressed, and their proteolytic activities were analyzed. As part of a polyprotein, the proteinase was found to exhibit cis (intramolecular) and trans (intermolecular) activity.
Carrington
J C
JC
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Cary
S M
SM
Dougherty
W G
WG
D000595:Amino Acid Sequence; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000378:metabolism*
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D004247:DNA; D010447:Peptide Hydrolases