Characterisation of a papain-like proteinase domain encoded by ORF1a of the coronavirus IBV and determination of the C-terminal cleavage site of an 87 kDa protein

Our previous studies have shown that two overlapping papain-like proteinase domains (PLPDs) encoded by the IBV sequence from nucleotides 4155 to 5550 is responsible for cleavage of the ORF la polyprotein to an 87 kDa protein. In this study, we demonstrate that only the more 5' one of the two domains, PLPD-1 encoded between nucleotides 4155 and 5031, is required for processing to the 87 kDa protein. Site-directed mutagenesis studies have shown that the Cys(1274) and His(1435) residues are essential for the PLPD-1 activity, suggesting that they may be the components of the catalytic centre of this proteinase. Coexpression and immunoprecipitation studies have further revealed that PLPD can interact with the 87 kDa protein. Meanwhile, data obtained from the construction and expression of a series of deletion mutants have indicated that the 87 kDa protein is encoded by the 5'- most 2600 bp part of ORF la. Further deletion and mutagenesis studies are underway to determine precisely the C-terminal cleavage site of the 87 kDa protein.