CHARACTERIZATION OF THE 2 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-k Da mature viral protein from the coronavirus infectious bronchitis vir us (IBV) 1a and 1a/1b polyproteins was mediated by two putative overla pping papain-like proteinase domains (PLPDs) encoded within the region from nucleotides 4243 to 5553 of ORF 1a (Liu et al., 1995). In this s tudy, we demonstrate that only the first domain, PLPD-1, is responsibl e for this cleavage, as deletion of the second domain did not affect t he formation or the 87-kDa protein. Site-directed mutagenesis studies further showed that a previously predicted nucleophilic cysteine resid ue (Cys(1274)) and a histidine residue (His(1437)) were essential for the proteinase activity, indicating that they may be important compone nts of the catalytic center of the proteinase. Meanwhile, expression o f a series of deletion mutants revealed that the 87-kDa protein was en coded by the 5'-most 2.6 kb of ORF 1a. Deletion and amino acid substit ution mutation studies demonstrated that the Gly(673)-Gly(674) dipepti de bond was most likely the cleavage site responsible for releasing th e C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins. (C) 1998 Academic Press.