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Updated: Aug 6, 2026
Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography
Published on: April 19, 2016
Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells
Stanislav V Sosnovtsev1, Gaël Belliot, Kyeong-Ok Chang
1Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8007, USA. ss216m@nih.gov
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.
Insights
Murine norovirus (MNV) polyprotein processing was mapped, revealing 3C-like proteinase (Pro) cleavage sites. Caspase 3 also processes the N-terminal protein in infected cells, aiding norovirus replication studies.
Area of Science:
- Virology
- Molecular Biology
Background:
- Murine norovirus (MNV) is the only cultivable norovirus, making it a key model for studying Caliciviridae replication.
- Understanding viral polyprotein processing is crucial for elucidating replication mechanisms.
Purpose of the Study:
- To determine the proteolytic processing strategy of the MNV ORF1 polyprotein during cell culture replication.
- To identify the viral and host factors involved in MNV polyprotein cleavage.
Main Methods:
- Generation of a proteolytic cleavage map for the MNV ORF1 polyprotein.
- Bacterial expression and in vitro cleavage assays of MNV 3C-like proteinase (Pro).
- Immunoprecipitation and Western blot analysis of proteins from MNV-infected cells.
- Site-directed mutagenesis to map caspase 3 cleavage sites.
Main Results:
- The MNV 3C-like proteinase (Pro) mediates cleavage at five specific sites, defining six viral proteins.
- Bacterially expressed MNV 3CL Pro accurately processed the polyprotein in vitro.
- Efficient cleavage of the proteinase-polymerase precursor was observed in infected cells.
- Caspase 3 was identified as a host factor involved in processing the N-terminal protein, with specific cleavage sites mapped.
Conclusions:
- A comprehensive cleavage map of the MNV nonstructural polyprotein was established.
- Both viral (3CL Pro) and host (caspase 3) proteases play roles in MNV polyprotein processing.
- The availability of this map and a permissive cell culture system will advance norovirus replication research.
