title: Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus.
abstract: To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mänz
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Götz
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wunderlich
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Eisel
authors.affiliation: the Department of Pharmaceutical Chemistry, University of Innsbruck, A-6020 Innsbruck, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kirchmair
authors.affiliation: the Friedrich-Loeffler-Institut, D-17493 Greifswald-Insel Riems, Germany, and.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Stech
authors.affiliation: the Friedrich-Loeffler-Institut, D-17493 Greifswald-Insel Riems, Germany, and.
authors.forename: Olga
authors.initials: O
authors.lastname: Stech
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Chase
authors.affiliation: the Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.affiliation: From the Department of Virology, University of Freiburg, 79104 Freiburg, Germany,. Electronic address: martin.schwemmle@uniklinik-freiburg.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Schwemmle
mesh_terms: D000818:Animals; D004285:Dogs; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D053126:Influenza A Virus, H7N7 Subtype / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D007252:Influenza Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007251:Influenza, Human / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D016898:Interferon-alpha / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016899:Interferon-beta / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D009154:Mutation; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014613:Vaccines, Attenuated / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics / Q000276:immunology
chemical_list: D007252:Influenza Vaccines; D016898:Interferon-alpha; C061290:PA protein, influenza viruses; D014613:Vaccines, Attenuated; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D016899:Interferon-beta; D012324:RNA-Dependent RNA Polymerase