CHARACTERIZATION AND MUTATIONAL ANALYSIS OF AN ORF 1A-ENCODING PROTEINASE DOMAIN RESPONSIBLE FOR PROTEOLYTIC PROCESSING OF THE INFECTIOUS-BRONCHITIS VIRUS 1A 1B POLYPROTEIN/

Coronavirus gene expression involves proteolytic processing of the mRN A 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF Ib-encoded 100-kDa protein is proteol ytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus so proteinase group (3C-like proteinas e). In this report, the 3C-like proteinase has been further analysed b y internal deletion of a 2.3-kb fragment between the 3C-like proteinas e-encoding region and ORF Ib and by substitution mutations of its cata lytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF la seq uences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to t he 100-kDa protein species. Site-directed mutagenesis studies have con firmed that the predicted nucleophilic cysteine residue (Cys(2922)) an d a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 ( His(2820)) are essential for the catalytic activity of the proteinase, and that the Qs(G) dipeptide bonds are its target cleavage sites. Sub stitution mutations of the third component of the putative catalytic t ried, the glutamic acid 2843 (GlU(2843)) residue, however, do not affe ct the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the so-like proteinase is capable of trans-cleav age of the 1a/1b polyprotein. These studies have confirmed the involve ment of the so-like proteinase domain in processing of the 1a/1b polyp rotein, the predicted catalytic centre of the proteinase, and its clea vage sites, (C) 1995 Academic Press, Inc.