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

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.

Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification

“…It is likely that some NIa functions require a polyprotein context. The proteinase, for example, exhibits a high preference for cis cleavage at certain sites in vitro (6,8). The VPg activity, as stated above, is proposed to function within a polyprotein containing the 6-kDa protein, although neither cells expressing the 6/NIa polyprotein nor cells expressing the CI/6/NIa polyprotein were able to rescue the NIa-defective mutants.…”

“…The P1 and HC-Pro proteinases catalyze autoproteolytic cleavages within the Nterminal region of the polyprotein (7,42). A third TEV proteinase, NIa, resembles the picornavirus 3C proteinase and catalyzes cis-and trans-proteolytic reactions within the C-terminal two-thirds of the polyprotein (6,8,9,16,18,21). The 3C-like proteinases contain a polypeptide fold resembling chymotrypsin, but with a cysteine rather than serine occupying the nucleophilic position within the active site (1,5,20,26).…”

Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro

“…This mode of genome expression has been demonstrated also for other potyviruses, including tobacco vein mottling virus (11,19), papaya ringspot virus (31), and soybean mosaic virus (27). Most of the TEV polyprotein cleavage events occur at Gln-Gly or Gln-Ser dipeptides and are catalyzed by a 49-kilodalton (kDa) proteinase (6,9,10). This enzyme autoexcises from the polyprotein precursor by a monomolecular mechanism (9) and cleaves at additional sites in bimolecular or trans-catalytic reactions (6, 10).…”

Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants

“…, 1990). NIa is responsible for proteolytic cleavage of viral proteins at conserved heptapeptide sequences located in the viral polyprotein, at sites other than the COOH‐termini of P1 and HC‐Pro (Carrington and Dougherty, 1987, 1988; Carrington et al. , 1988; Dougherty et al.…”