Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site

Coronavirus infectious bronchitis Virus (IBV) encodes a trypsin-like proteinase (BC-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene I-encoded polyproteins. In our previous studies, the proteinase was identified as a 33-kDa protein in IBV-infected cells, and its catalytic center was shown to consist of H-2820 and C-2922 residues. It is released from the la and 1a/1b polyproteins by autoprocessing at two Q-S dipeptide bonds (Q(2779)-S-2780 and Q(3086)-S-3087). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q(2779)-S-2780 site is tolerant to mutations at the P1 position. Deletion of the C-terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids, suggesting that the extreme C-terminal region may be dispensable for the proteolytic activity of the proteinase. Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q(2779)-S-2780 Site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q(3086)-S-3087 site. The occurrence of both cleavage events in intact cells is potentially rapid and efficient, as no intermediate cleavage products covering the proteinase were detected in either IBV-infected or transfected cells. Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV-infected cells and in cells expressing the 3C-like proteinase alone, indicating that additional roles in viral replication might be played by this protein. Finally, a Q-A(Q(3379)-A(3380)) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase. (C) 2000 Academic Press.