title: Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells.
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.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8007, USA. ss216m@nih.gov
authors.forename: Stanislav V
authors.initials: SV
authors.lastname: Sosnovtsev
authors.forename: Gaël
authors.initials: G
authors.lastname: Belliot
authors.forename: Kyeong-Ok
authors.initials: KO
authors.lastname: Chang
authors.forename: Victor G
authors.initials: VG
authors.lastname: Prikhodko
authors.forename: Larissa B
authors.initials: LB
authors.lastname: Thackray
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Karst
authors.forename: Herbert W
authors.initials: HW
authors.lastname: Virgin
authors.forename: Kim Y
authors.initials: KY
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017250:Caliciviridae Infections / Q000821:virology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D029322:Norovirus / Q000378:metabolism*; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D020815:Polyproteins; D017361:Viral Nonstructural Proteins; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases