CHARACTERIZATION IN-VITRO OF AN AUTOCATALYTIC PROCESSING ACTIVITY ASSOCIATED WITH THE PREDICTED 3C-LIKE PROTEINASE DOMAIN OF THE CORONAVIRUS AVIAN INFECTIOUS-BRONCHITIS VIRUS

A region of the infectious bronchitis virus (IBV) genome between nucle otide positions 8693 and 10927 which encodes the predicted 3C-like pro teinase (3CLP) domain and several potential cleavage sites has been cl oned into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detect able processing activity of the nascent polypeptide unless the transla tion was carried out in the presence of microsomal membrane preparatio ns. 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 t o the predicted size of 3CLP. Efficient processing was dependent on th e presence of membranes during translation; processing was found to oc cur when microsomes were added posttranslationally, but only after ext ended periods of incubation. C-terminal deletion analysis of the encod ed polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hyd rophobic 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 t he predicted Q/S release sites implicated them in the release of the p 35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3C LP is sensitive to inhibition by both serine and cysteine protease cla ss-specific inhibitors. These data reveal the identity of the IBV 3C-l ike proteinase, which exhibits characteristics in common with the 3C p roteinases of picornaviruses.