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Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli

Liu, B.L., Viljoen, GJ, Clarke, I.N. and Lambden, P.R. (1999) Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli Journal of General Virology, 80, (2), pp. 291-296. (PMID:10073687).

Record type: Article

Abstract

Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.

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Published date: February 1999

Identifiers

Local EPrints ID: 194183
URI: http://eprints.soton.ac.uk/id/eprint/194183
ISSN: 0022-1317
PURE UUID: f5387d34-5a2d-40ac-892b-4b43238262b5
ORCID for I.N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 25 Jul 2011 15:20
Last modified: 18 Jul 2017 11:27

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Contributors

Author: B.L. Liu
Author: GJ Viljoen
Author: I.N. Clarke ORCID iD
Author: P.R. Lambden

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