Picornavirus 3C proteinases (3Cpro) are cysteine proteinases but recent sequence analyses have shown that they are related to trypsin-like serine proteinases. Two models of 3Cpro structure have been presented. Both models indicate that residues His40 and Cys147 are members of the catalytic triad but the models differ in the designation of the third member of the catalytic triad, which is assigned as either Glu71 or Asp85. To test the importance of these four residues in the catalytic activity of 3Cpro of coxsackievirus B3, a member of the enterovirus subgroup of the picornavirus family, single amino acid substitutions were introduced at each of the four sites. All of these mutations resulted in the reduction or inactivation of autocatalytic cleavage of the 3C precursor protein expressed in Escherichia coli, suggesting that all of these residues are essential for the proteolytic reaction. The substitution of Cys147 with Ala abolished 3Cpro activity while the mutant in which Cys147 was replaced with Ser retained reduced proteolytic activity both in cis and in trans. Our results strongly support the proposal that Cys147 of 3Cpro functions as a nucleophile analogous to Ser195 of trypsin-like serine proteinases.
The substrate requirements of coxsackievirus 3C proteinase (3Cpro) were investigated on the C-terminal side of the scissile bond using C-terminal truncated peptides of the substrate peptide Ac-EALFQGPPV. Not only the Gln-Gly bond of Ac-EALFQG-NH2 but also the C-terminal amide group of Ac-EALFQ-NH2 was hydrolyzed by 3Cpro, suggesting that the essential residues for cleavage by coxsackievirus 3Cpro would exist within the N-terminal 5 residues.
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