IDENTIFICATION OF THE ACTIVE-SITE RESIDUES OF THE 3C PROTEINASE OF FOOT-AND-MOUTH-DISEASE VIRUS

To identify the active-site residues of the 3C proteinase of foot-and- mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substra tes. Based on alignment of FMDV 3C with other picornavirus 3C proteina ses and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, inv olved in substrate binding, or present in nonconserved regions. Wild-t ype and mutant 3C proteins were expressed in Escherichia coil and test ed for their ability to cleave synthetic substrates corresponding to d ifferent portions of the viral genome. Substitutions at His-46 (cataly tic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) p roduced enzymes unable to process P1, P2, or P3 substrates in trans, w hereas a change in the conserved Asp-98 had no effect on enzyme activi ty. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzym e that retained activity on some substrates, while a substitution of G ly at this position resulted in a completely inactive enzyme. The kine tics of trans processing of translation products from a transcript enc oding the P1 and P2 coding regions and the 2C/3A cleavage site with wi ld-type 3C or a transcript encoding P1 with 3C mutants revealed that t he order of cleavage was VP3-VP1, VPO-VP3, VP1-2A, 2C-3A, and 2B-2C. M utations in 3C that resulted in a partially active enzyme were individ ually introduced into full-length FMDV cDNA and RNA transcripts were t ranslated in a cell-free system and used to transfect cells. In all ca ses the virus that was rescued had reverted to the wild-type 3C codon. (C) 1995 Academic Press, Inc.