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. 1988 Jul;62(7):2313–2320. doi: 10.1128/jvi.62.7.2313-2320.1988

Mutational analysis of tobacco etch virus polyprotein processing: cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase.

J C Carrington 1, S M Cary 1, W G Dougherty 1
PMCID: PMC253386  PMID: 3286889

Abstract

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.

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Selected References

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