title: Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.
abstract: Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Thierry
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Guilligay
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Kosinski
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bock
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Gaudon
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Alexander
authors.initials: A
authors.lastname: Pflug
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Narin
authors.initials: N
authors.lastname: Hengrung
authors.affiliation: Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Kamel
authors.initials: K
authors.lastname: El Omari
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Florence
authors.initials: F
authors.lastname: Baudin
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beck
authors.affiliation: European Molecular Biology Laboratory Grenoble Outstation and Unit of Virus Host-Cell Interactions, University Grenoble Alpes-CNRS-EMBL, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France. Electronic address: cusack@embl.fr.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology* / Q000235:genetics; D009981:Influenza B virus / Q000201:enzymology* / Q000235:genetics; D009982:Gammainfluenzavirus / Q000201:enzymology* / Q000235:genetics; D007834:Lasers; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D012367:RNA, Viral / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D053838:Scattering, Small Angle; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; C057027:PB2 protein, Influenzavirus A; C502893:PB2 protein, influenza virus; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase