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Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells.
- Source :
-
Journal of virology [J Virol] 2006 Aug; Vol. 80 (16), pp. 7816-31. - Publication Year :
- 2006
-
Abstract
- Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.
- Subjects :
- Amino Acid Sequence
Animals
Caspase 3
Caspases metabolism
Cells, Cultured
Immunoprecipitation
Mice
Molecular Sequence Data
Mutagenesis
Polyproteins genetics
Viral Nonstructural Proteins genetics
Virus Replication
Caliciviridae Infections virology
Cysteine Endopeptidases metabolism
Norovirus metabolism
Polyproteins metabolism
Viral Nonstructural Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-538X
- Volume :
- 80
- Issue :
- 16
- Database :
- MEDLINE
- Journal :
- Journal of virology
- Publication Type :
- Academic Journal
- Accession number :
- 16873239
- Full Text :
- https://doi.org/10.1128/JVI.00532-06