Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase

Mutational Analysis of Plum Pox Potyvirus Polyprotein Processing By the NIa Protease in Escherichia Coli

“…The 151 amino acid deletion of ASX protease rendered it inactive at all cleavage sites analysed, as did the Gin or Leu substitutions for His 239. As a Tyr for His 234 substitution had been previously shown to render the TEV 49K protease inactive for its autocatalytic processing (Dougherty et al, 1989b), our results support the previous identification of this His residue as part of the catalytic triad of the potyviral protease (Dougherty et al, 1989b). Dougherty et al (1989b) also reported that substituting Glu for Asp at position 269 of the TEV 49K protease resulted in an anomalous proteolytic activity capable of cutting at the 49K N-terminal cleavage site but not at the C-terminal one.…”

“…As a Tyr for His 234 substitution had been previously shown to render the TEV 49K protease inactive for its autocatalytic processing (Dougherty et al, 1989b), our results support the previous identification of this His residue as part of the catalytic triad of the potyviral protease (Dougherty et al, 1989b). Dougherty et al (1989b) also reported that substituting Glu for Asp at position 269 of the TEV 49K protease resulted in an anomalous proteolytic activity capable of cutting at the 49K N-terminal cleavage site but not at the C-terminal one. Interestingly, the effect of the replacement of the Asp 274 of PPV NIa, which corresponds to Asp 269 of TEV (Garcia et al, 1989a), by Glu also depended on the cleavage site analysed: D274E NIa protease cleaved at the NIb-CP junction almost as efficiently as the wildtype protease and at the CI-6K site with low efficiency, whereas cleavage at the C terminus of the NIa protease was undetectable in our system (Fig.…”

“…NIa-mediated cleavages occurring only in cis have also been reported for tobacco vein mottling virus (Hellmann et al, 1988); trans cleavage at the NIb-CP junction of PPV has been demonstrated in an E. coli expression system (Garcia et al, 1989a). On the other hand, computer-generated modelling and site-directed mutagenesis have shown that the active site of the TEV 49K protease is located in the carboxyl half of the molecule and includes His 234, Asp 269 and Cys 339 (Dougherty et al, 1989b). Expression of PPV cDNA in E. coli has allowed us to investigate further, by a procedure different from the one used for TEV studies, the proteolytic processing pathway of the potyviral polyprotein.…”

“…The frameshift-inducing deletions of plasmids pPPV23AB5, AB7 and AB10 introduced termination codons near the end of the NIa cistron, which produced proteins in which some amino acids had been removed and others had been added (Garcia et al, 1989a). pPPV23 H239Q, pPPV23 H239L and pPPV23 m74E contained point mutations at His 239 and Asp 274 which are predicted to be part of the active centre of the NIa protease on the basis of homology to the TEV 49K protease (Dougherty et al, 1989b).…”

“…In this paper we have studied the different substrate susceptibilities of several cleavage sites of the PPV polyprotein using these mutants as well as NIa proteases with point mutations as His 239 and Asp 274, which correspond to the His and Asp proposed to form part of the active centre of TEV 49K protease (Dougherty et al, 1989b). Trans activity of the wild-type protease in the presence of an excess of the mutant proteins has also been analysed.…”