title: Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR.
abstract: The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Usingin vitroandin vivoapproaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+mice, but replicated to high titers in lungs of PKR-/-mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle.IMPORTANCEInfluenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR-/-mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.
authors.affiliation: Unit 17, Influenza and other Respiratory Viruses, Robert Koch Institut, Berlin, Germany.
authors.forename: Kristina L
authors.initials: KL
authors.lastname: Schierhorn
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt Universität zu Berlin, Berlin, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Jolmes
authors.affiliation: Universities Giessen & Marburg Lung Center, Department of Internal Medicine II, Section of Infectious Diseases, German Center for Lung Research, Giessen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bespalowa
authors.affiliation: Unit 17, Influenza and other Respiratory Viruses, Robert Koch Institut, Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Saenger
authors.affiliation: Universities Giessen & Marburg Lung Center, Department of Internal Medicine II, Section of Infectious Diseases, German Center for Lung Research, Giessen, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Peteranderl
authors.affiliation: Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Dzieciolowski
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt Universität zu Berlin, Berlin, Germany.
authors.forename: Maja
authors.initials: M
authors.lastname: Mielke
authors.affiliation: Unit 17, Influenza and other Respiratory Viruses, Robert Koch Institut, Berlin, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Budt
authors.affiliation: Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Pleschka
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt Universität zu Berlin, Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.affiliation: Universities Giessen & Marburg Lung Center, Department of Internal Medicine II, Section of Infectious Diseases, German Center for Lung Research, Giessen, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Herold
authors.affiliation: Unit 17, Influenza and other Respiratory Viruses, Robert Koch Institut, Berlin, Germany wolfft@rki.de.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wolff
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D054884:Host-Pathogen Interactions; D006801:Humans; D007113:Immunity, Innate; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000472:pathogenicity*; D008168:Lung / Q000821:virology; D051379:Mice; D009154:Mutation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014774:Virulence; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins; D019892:eIF-2 Kinase
keywords: PKR; influenza; innate immunity; virulence regulation