title: Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30.
abstract: BACKGROUND
  The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.
  
  
  METHODS
  NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.
  
  
  RESULTS
  Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.
  
  
  CONCLUSIONS
  The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada.
authors.forename: Samar K
authors.initials: SK
authors.lastname: Dankar
authors.forename: Elena
authors.initials: E
authors.lastname: Miranda
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Forbes
authors.forename: Martin
authors.initials: M
authors.lastname: Pelchat
authors.forename: Ali
authors.initials: A
authors.lastname: Tavassoli
authors.forename: Mohammed
authors.initials: M
authors.lastname: Selman
authors.forename: Jihui
authors.initials: J
authors.lastname: Ping
authors.forename: Jianjun
authors.initials: J
authors.lastname: Jia
authors.forename: Earl G
authors.initials: EG
authors.lastname: Brown
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D039223:Cleavage And Polyadenylation Specificity Factor / Q000378:metabolism*; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D005260:Female; D054884:Host-Pathogen Interactions; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology* / Q000302:isolation & purification / Q000472:pathogenicity*; D007372:Interferons / Q000037:antagonists & inhibitors*; D008168:Lung / Q000473:pathology / Q000821:virology; D017563:Lung Diseases, Interstitial / Q000473:pathology / Q000821:virology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D059386:Reverse Genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D039223:Cleavage And Polyadenylation Specificity Factor; C085820:INS1 protein, influenza virus; D050505:Mutant Proteins; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors; D007372:Interferons; C503356:Ddx58 protein, mouse; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases