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1、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 K. P. Lim and D. X. Liu1 Institute of Molecular Agrobiology, The National University of Singa
2、pore, 1 Research Link, Singapore 117604 Received December 10, 1997; returned to author for revision January 16, 1998; accepted March 26, 1998 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
3、/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 th
4、e 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
5、 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 59-most 2.6 kb of ORF 1a. Deletion and amino acid substitution mutation studies demonstrated that the Gly673Gly674dipeptide bond was most l
6、ikely the cleavage site responsible for releasing the C-terminus of the 87-kDa protein from the 1a and 1a/1b polyproteins. 1998 Academic Press INTRODUCTION Avian infectious bronchitis virus (IBV) is the proto- type of Coronaviridae, a family of enveloped viruses which possess a large continuous posi
7、tive-stranded RNA genome. The genomic RNA is 27.6 kb in length and contains at least 10 distinct open reading frames (ORFs) (Boursnell et al., 1987). Most of these ORFs do not appear to be translated from the genomic RNA but are expressed from a set of subgenomic mRNAs. Six mRNA species, including a
8、 genome-length mRNA (mRNA 1) and five subgenomic mRNA species (mRNA 2-6), are produced in virus-infected cells. These mR- NAs range in length from about 2 to 27.6 kb and have been shown to form a 39-coterminal nested structure (Stern and Sefton, 1984). Approximately 70% of the coding potential of th
9、e IBV genome is contained within the 59-terminal unique re- gion of mRNA 1. Nucleotide sequencing has shown that mRNA 1 contains two large overlapping ORFs (1a and 1b), with ORF 1a having the potential to encode a polyprotein of 441 kDa and 1b a polyprotein of 300 kDa (Boursnell et al., 1987). The d
10、ownstream ORF 1b is pro- duced as a fusion protein of 741 kDa with 1a by a ribosomal frameshifting mechanism (Brierley et al., 1987, 1989). These polyproteins are expected to be cleaved by viral or cellular proteinases to produce functional prod- ucts associated with viral replication. Recently, pro
11、ducts encoded by these two ORFs have been identified. These include 87-, 10-, and 100-kDa polypeptides (Liu et al., 1994, 1995, 1997; Liu and Brown, 1995). Three ORF 1a-encoding proteinase domains, i.e., two overlapping papain-like proteinase domains (PLPDs) encoded by the IBV sequence between nucle
12、otides 4243 and 5553 and a picornavirus 3C-like proteinase domain encoded between nucleotides 8937 and 9357, were dem- onstrated to be involved in the production of these ma- ture viral proteins (Liu et al., 1994, 1995, 1997; Liu and Brown, 1995). Proteolytic processing of the 1a and 1a/1b polyprote
13、ins by the equivalent proteinase activities of human coronavirus and mouse hepatitis virus has also been documented. So far, four mature products have been identified in cells infected by human coronavirus strain 229E. These include a 34-kDa protein representing the 3C-like proteinase, a 105-kDa pro
14、tein corresponding to the 100-kDa protein of IBV, a 71-kDa protein with an ATPase activity, and a 41-kDa protein processed from the 1b polyprotein (Ziebuhr et al., 1995; Grotzinger et al., 1996; Heusipp et al., 1997a, b). Similarly, three mature products, including two N-terminal cleavage products o
15、f 28 and 65 kDa and a 27-kDa protein identified as the 3C-like proteinase, have been reported for mouse hep- atitis virus (Denison and Perlman, 1987; Denison et al., 1995; Lu et al., 1995). In this communication, we report that of the two pre- dicted PLPDs of IBV, only the first one, PLPD-1, encoded
16、 1To whom reprint requests should be addressed. E-mail: liudx ima.org.sg. VIROLOGY245, 303312 (1998) ARTICLE NO. VY989164 0042-6822/98 $25.00 Copyright 1998 by Academic Press All rights of reproduction in any form reserved. 303 between nucleotides 4243 and 5019, is required for cleavage of the 87-kDa protein at its C-terminus. Site- directed mutagenesis studies confirmed that the previ- ously predicted Cys1274and His1437residues of PLPD-1 are essential for the proteinase activity, suggesting t
