Characterization of the Two Overlapping Papain-like Proteinase Domains Encoded in Gene 1 of the Coronavirus Infectious Bronchitis Virus and Determination of the C-Terminal Cleavage Site of an 87-kDa Protein

In a previous report, we showed that proteolytic processing of an 87-kDa mature viral protein from the coronavirus infectious bronchitis virus (IBV) 1a and 1a/1b polyproteins was mediated by two putative overlapping papain-like proteinase domains (PLPDs) encoded within the region from nucleotides 4243 to 5553 of ORF 1a (Liuet al.,1995). In this study, we demonstrate that only the first domain, PLPD-1, is responsible for this cleavage, as deletion of the second domain did not affect the formation of the 87-kDa protein. Site-directed mutagenesis studies further showed that a previously predicted nucleophilic cysteine residue (Cys(1274)) and a histidine residue (His(1437)) were essential for the proteinase activity, indicating that they may be important components of the catalytic center of the proteinase. Meanwhile, expression of a series of deletion mutants revealed that the 87-kDa protein was encoded by the 5′-most 2.6 kb of ORF 1a. Deletion and amino acid substitution mutation studies demonstrated that the Gly(673)–Gly(674)dipeptide bond was most likely the cleavage site responsible for releasing the C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins.