Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus

Tibbles, K. W.; Brierley, Ian; Cavanagh, David; Brown, T. D. K.

doi:10.1128/jvi.70.3.1923-1930.1996

Cited by 47 publications

(61 citation statements)

References 35 publications

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“…The results from the assays in which we used substrates synthesized in rabbit reticulocyte lysates showed that the nsp4 proteinase was able to process its cognate cleavage sites in the nsp6-8 and nsp7-8 substrates, indicating that an active enzyme had been purified. Although this was not a quantitative assay, the EAV nsp4 proteinase displayed kinetics that were comparable to those of other 3C-like proteinases in similar experiments (Grubman et al, 1995;Thole and Hull, 2002;Tibbles et al, 1996Tibbles et al, , 1999Ziebuhr et al, 1995). The unexpected small reaction products visible in Fig.…”

Section: Discussionsupporting

confidence: 60%

Expression, purification, and in vitro activity of an arterivirus main proteinase

et al. 2006

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To allow the biochemical and structural characterization of the chymotrypsin-like "main proteinase" (non-structural protein 4; nsp4) of the arterivirus prototype Equine Arteritis Virus (EAV), we developed protocols for the large-scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase was expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag. Following purification, the nsp4 moiety of MBP-nsp4 was successfully used for structural studies [Barrette-Ng, I.H., Ng, K.K.S., Mark, B.L., van Aken, D., Cherney, M.M., Garen, C, Kolodenko, Y., Gorbalenya, A.E., Snijder, E.J., James, M.N.G, 2002. Structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole. J. Biol. Chem. 277, 39960-39966]. Furthermore, both forms of the EAV proteinase were shown to be proteolytically active in two different trans-cleavage assays. Recombinant nsp4 cleaved the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates. In a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the P9-P7' residues of six nsp4 cleavage sites was investigated. The peptide representing the EAV nsp7/8 junction was used to optimize the reaction conditions (pH 7.5, 25mM NaCl, 30% glycerol at 30 degrees C), which resulted in a maximum turnover of 15% of this substrate in 4h, using a substrate to enzyme molar ratio of 24:1. The assays described in this study can be used for a more extensive biochemical characterization of the EAV main proteinase, including studies aiming to identify inhibitors of proteolytic activity.

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Section: Discussionsupporting

confidence: 60%

Expression, purification, and in vitro activity of an arterivirus main proteinase

et al. 2006

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“…Such studies are already at an advanced stage for both IBV (Liu et al, 1994 and HCV 229E ORF1b regions (Grotzinger et al, 1996;Heusipp et al, 1997a,b). 3C-like proteinase activities have been confirmed recently for three representatives of the coronavirus group, MHV (Lu et al, 1995), HCV 229E (Ziebuhr et al, 1995) and IBV (Tibbles et al, 1996) and the protein has been identified 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 purified and partially characterised (Seybert et al, 1997;Ziebuhr et al, 1997).…”

Section: Introductionmentioning

confidence: 88%

“…Cleavage dipeptides were mutated (to G/S or A/S) by oligonucleotide-directed mutagenesis as described previously (Tibbles et al, 1996). This region was transferred as a SpeI fragment to pGEX4T2 (Pharmacia) and deletions made to express portions containing each target dipeptide individually.…”

Section: Identification Of Possible 3clpro Target Sitesmentioning

confidence: 99%

“…In addition, the proteinases from these two viruses have been purified and partially characterised (Seybert et al, 1997;Ziebuhr et al, 1997). The identification of the IBV 3CLpro was first realised in vitro when a cloned fragment of the polyprotein was expressed in reticulocyte lysate (Tibbles et al, 1996). In the presence of membranes, upon which activity was vitally dependent, processing of the region containing the protease domain was observed with the release of a 35 kDa protein (p35).…”

Section: Introductionmentioning

confidence: 99%

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Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus

1999

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Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed 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 mediated by 3CLpro, the proteinase was expressed in bacteria, purified 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 mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. 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 were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

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“…There has been no report of the cleavage activity of the PCP-2 domain. Re- cently, the activity of the coronavirus 3C-pro domain has been identified for MHV-A59 , human coronavirus 229-E (Ziebuhr et al, 1995) and the avian coronavirus infectious bronchitis virus Tibbles et al, 1996). Using bacterial expression vectors, it has been shown that the coronavirus 3C-like proteinases recognize predicted cleavage sites, Q/S for MHV-A59 and IBV, and Q/A for 229E, to release the 3C domain from the polyprotein precursor.…”

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confidence: 99%

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

1996

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The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

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Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus

Cited by 47 publications

References 35 publications

Expression, purification, and in vitro activity of an arterivirus main proteinase

Expression, purification, and in vitro activity of an arterivirus main proteinase

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

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