title: Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus.
abstract: A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.
authors.affiliation: Department of Pathology, University of Cambridge, United Kingdom.
authors.forename: K W
authors.initials: KW
authors.lastname: Tibbles
authors.forename: I
authors.initials: I
authors.lastname: Brierley
authors.forename: D
authors.initials: D
authors.lastname: Cavanagh
authors.forename: T D
authors.initials: TD
authors.lastname: Brown
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D000086782:Coronavirus 3C Proteases; D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D001351:Infectious bronchitis virus / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational
chemical_list: D004151:Dipeptides; D003546:Cysteine Endopeptidases; C000706208:3C-like proteinase, Avian infectious bronchitis virus; D000086782:Coronavirus 3C Proteases; D003545:Cysteine