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1、JOURNAL OFVIROLOGY, 0022-538X/00/$04.0010 Feb. 2000, p. 16741685Vol. 74, No. 4 Copyright 2000, American Society for Microbiology. All Rights Reserved. Identifi cation of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Inf
2、ectious Bronchitis Virus and Characterization of the Cleavage Products K. P. LIM, LISA F. P. NG,ANDD. X. LIU* Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604, Singapore Received 26 July 1999/Accepted 6 November 1999 The coronavirus Avian infectious bronchitis v
3、irus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identifi ed the fi rst cleavage event as proteolysis at the Gly673-Gly674 dipeptide bond mediated by the fi rst papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this repo
4、rt, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mu- tagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly2265-Gly2266dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleava
5、ge product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detec
6、ted in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modifi ed by N-linked glycosylation at the Asn2313residue encoded by nucleotides 7465 to 7467. By using a region-specifi c
7、 antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifi cally immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a p
8、reviously predicted cleavage site (Q2583-G2584) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product. Avian infectious bronchitis virus (IBV), the prototype of the family of enveloped viruses Coronavir
9、idae, is now categorized under the newly established order Nidovirales, which comprises the families Coronaviridae and Arteriviridae. The name Nidovi- rales came from the Latin nidus, for nest, referring to the 39-coterminal “nested” set of subgenomic mRNAs produced during viral infection (33). Two
10、overlapping open reading frames (ORFs), 1a and 1b, are located at the 59-end unique region of the genome and are expected to encode polyproteins of 441 and 300 kDa, respectively (2). By a unique frameshifting mechanism, the downstream ORF 1b is expressed as a fusion protein of 741 kDa with ORF 1a (3
11、, 4). The 441-kDa 1a and 741-kDa 1a/1b polyproteins are proteolytically processed into smaller mature products required for RNA synthesis and other aspects of viral replication. Nucleotide sequence analyses of coronaviruses from three different antigenic groups have predicted the presence of three p
12、roteinases (2, 7, 9, 10, 16), namely, two overlapping papain- like proteinase domains (PLPDs) and a picornavirus 3C-like proteinase domain (3CLP). To date, both PLPD-1 and 3CLP have been demonstrated to be active, and their catalytic prop- erties have been characterized. PLPD-1 is involved in proteo
13、- lytic cleavage of the N-terminal region of the 1a and 1a/1b polyproteins. For mouse hepatitis virus (MHV), it was shown that PLPD-1 mediates cleavage at Gly247-Val248and Ala832- Gly833dipeptide bonds to release p28 and p65, respectively (1, 6, 13). The Cys1137and His1288 residues were defi ned as
14、the catalytic dyad of this proteinase (1, 6, 13). For human corona- virus (HCV) 229E, it was reported that PLPD-1 is required for releasing p9 upon cleavage at the Gly111-Asn112dipeptide bond, and the Cys1137and His1205 residues were identifi ed as its catalytic dyad (11). No experimental evidence h
15、as so far shown that PLPD-2 is functionally active for both viruses. Multiple proteinase domains have also been described for ar- teriviruses. For example, nsp1, nsp2, and nsp4 of equine ar- teritis virus were shown to have proteolytic activities (32). In our previous reports, IBV PLPD-1 was shown t
16、o be re- sponsible for cleavage of the 1a and 1a/1b polyproteins at the Gly673-Gly674dipeptide bond to release an 87-kDa N-terminal cleavage product (17, 21). The Cys1274and His1437residues were revealed to be the catalytic dyad of this proteinase (17). In this paper, we report yet another cleavage event performed by PLPD-1. Our results show that PLPD-1 is responsible for the cleavage of the product encoded by nucleotides 2548 to 8865 (which is located between the 87-kDa protein and 3CLP) (Fig.
