Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a New Cleavage Site

Abstract

Coronavirus infectious bronchitis virus (IBV) encodes a trypsin-like proteinase (3C-like proteinase) by ORF 1a, which has been demonstrated to play a pivotal role in proteolytic processing of gene 1-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 H²⁸²⁰ and C²⁹²² residues. It is released from the 1a and 1a/1b polyproteins by autoprocessing at two Q–S dipeptide bonds (Q²⁷⁷⁹–S²⁷⁸⁰ and Q³⁰⁸⁶–S³⁰⁸⁷). In this report, further characterization of the two cleavage sites demonstrates that the N-terminal Q²⁷⁷⁹–S²⁷⁸⁰ 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 Q²⁷⁷⁹–S²⁷⁸⁰ site is the first cleavage event mediated by this proteinase. This is followed by cleavage at the Q³⁰⁸⁶–S³⁰⁸⁷ 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 (Q³³⁷⁹–A³³⁸⁰) dipeptide bond encoded by nucleotides 10,663 to 10,668 was demonstrated to be a cleavage site of the proteinase.