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 (Liu et 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 (Cys1274) and a histidine residue (His1437) 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 Gly673-Gly674 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.
Lim
K P
KP
Institute of Molecular Agrobiology, National University of Singapore, Singapore.
Liu
D X
DX
D000086882:Coronavirus Papain-Like Proteases; D015967:Gene Expression Regulation, Viral; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000235:genetics*; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; D000086882:Coronavirus Papain-Like Proteases; D010206:Papain