Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.