title: Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases.
abstract: The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Lin
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Hills Road CB2 2QQ, UK.
authors.forename: Lucy
authors.initials: L
authors.lastname: Thorne
authors.affiliation: Alios BioPharma, Inc., 260 East Grand Avenue South, San Francisco, CA 94080, USA.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Laboratory for Biological Mass Spectrometry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Loubna A
authors.initials: LA
authors.lastname: Hammad
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Serena
authors.initials: S
authors.lastname: Li
authors.affiliation: Alios BioPharma, Inc., 260 East Grand Avenue South, San Francisco, CA 94080, USA.
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Hills Road CB2 2QQ, UK.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Goodfellow
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA ckao@indiana.edu.
authors.forename: C Cheng
authors.initials: CC
authors.lastname: Kao
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016679:Genome, Viral; D051379:Mice; D009154:Mutation; D029322:Norovirus / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D050436:Regulatory Elements, Transcriptional; D061785:Transcription Initiation, Genetic
chemical_list: D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase