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1、Virus Research 60 (1999) 137145 Activity of a purifi ed His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus K.W. Tibbles 1,*, D. Cavanagh2, T.D.K. Brown1 1Di6ision of Virology, Department of Pathology, Uni6ersity of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK 2Inst
2、itute for Animal Health, Compton Laboratory, Newbury, Berks RG20 7NN, UK Received 23 October 1998; accepted 12 January 1999 Abstract Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), con
3、fi rmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein me
4、diated by 3CLpro, the proteinase was expressed in bacteria, purifi ed and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing matu
5、re 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purifi cation. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we
6、were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identifi ed sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro. 1999 Elsevier Science B.V. All rights reserved.
7、 Keywords:Coronavirus; 3CLproteinase; His-tagged; Bacterial expression; Trans processing 1. Introduction Thecoronavirusinfectiousbronchitisvirus (IBV) encodes the major portion of its non-struc- tural proteins within two large open reading frames, ORFs 1a and 1b, situated at the 5 end and occupying
8、the major portion of the 27.6 kb single-strandedpositive-senseRNAgenome (Brown and Brierley, 1995). The ORFs overlap slightly with ORF1b accessed via programmed ribosomal frameshifting (Brierley et al., 1989) re- sulting in ORF1a and ORF1a/1b (a fusion) trans- * Corresponding author. Tel.: +44-1223-
9、336917; fax: +44- 1223-336926. E-mail address:kwtmole.bio.cam.ac.uk (K.W. Tibbles) 0168-1702/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1702(99)00011-8 K.W. Tibbles et al./Virus Research60 (1999) 137145138 lation products. The large size of the ORFs sug- gests
10、thatthetranslationproductsare polyproteins that are processed to generate a set of mature non-structural proteins. In common with all other coronaviruses so far examined, IBV encodes a 3C-like proteinase domain which is located towards the C-terminus of the ORF1a polyprotein and approximately centra
11、lly in the ORF1a/1b fusion product. The 3CLP domain was fi rst identifi ed as a possible component of IBV polyproteins (Gorbalenya et al., 1989) due to sequence similarity to picornavirus 3C proteinases (reviewed in Ryan and Flint, 1997). In the same study, predictions were made as to likely target
12、dipeptide cleavage sites for a 3C-like proteinase within the ORF1 polyproteins, the large number suggested implicating this proteinase in a pivotal role in the maturation of the polyproteins. Similar domains were subsequently identifi ed in mouse hepatitis virus (MHV) (Lee et al., 1991) and hu- man
13、coronavirus (HCV 229E) (Herold et al., 1993). There are no cellular counterparts for the activities of the 3C-like proteinases, therefore they are essential to the successful maturation of their respective polyproteins and subsequent replication events. This, and the comparatively high degree of con
14、servation (at functional level), makes the 3C- like proteinases attractive targets for antiviral intervention. Given the essential role played by the 3C-like proteinase of the coronaviruses in maturation of the polymerase polyproteins, there has been con- siderable interest in characterising the enz
15、yme, with initial emphasis on defi ning the processing events involving the proteinase. Such studies are already at an advanced stage for both IBV (Liu et al., 1994, 1998) and HCV 229E ORF1b regions (Grotzinger et al., 1996; Heusipp et al., 1997a,b). 3C-like proteinase activities have been confi rme
16、d recently for three representatives of the coro- navirus group, MHV (Lu et al., 1995), HCV 229E (Ziebuhr et al., 1995) and IBV (Tibbles et al., 1996) and the protein has been identifi ed in vivo during viral infection in the case of HCV (Ziebuhr et al., 1995) and MHV (Lu et al., 1996). In addition, the proteinases from these two viruses have been purifi ed and partially characterised (Seybert et al., 1997; Ziebuhr et al., 1997). The identifi cation of the IBV 3CLpro was fi rst realised invit
