title: The Influenza A Virus Genotype Determines the Antiviral Function of NF-κB.
abstract: The role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-β) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB.
  
  
  
  This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier.
authors.affiliation: Institute of Biochemistry, Medical Faculty, Justus Liebig University, Giessen, Germany German Center for Lung Research, Justus Liebig University, Giessen, Germany.
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Dam
authors.affiliation: German Center for Lung Research, Justus Liebig University, Giessen, Germany Rudolf-Buchheim Institute of Pharmacology, Justus Liebig University, Giessen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kracht
authors.affiliation: German Center for Lung Research, Justus Liebig University, Giessen, Germany Institute of Medical Virology, Justus Liebig University, Giessen, Germany German Center for Infection Research, Justus Liebig University, Giessen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Pleschka
authors.affiliation: Institute of Biochemistry, Medical Faculty, Justus Liebig University, Giessen, Germany German Center for Lung Research, Justus Liebig University, Giessen, Germany lienhard.schmitz@biochemie.med.uni-giessen.de.
authors.forename: M Lienhard
authors.initials: ML
authors.lastname: Schmitz
mesh_terms: D000818:Animals; D002460:Cell Line; D005838:Genotype; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D051379:Mice; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D059386:Reverse Genetics; D014779:Virus Replication
chemical_list: D052002:NF-kappa B p50 Subunit; C073701:Nfkb1 protein, mouse