The Influenza A Virus Genotype Determines the Antiviral Function of NF-kappaB

UNLABELLED: The role of NF-kappaB 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-kappaB pathway. Cells devoid of either the central NF-kappaB essential modulator (NEMO) scaffold protein and thus defective in IkappaB kinase (IKK) activation or cells not expressing the NF-kappaB 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-kappaB was not relevant for replication of SC35M, the absence of NF-kappaB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-kappaB 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-kappaB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-beta) gene. To identify the viral proteins responsible for NF-kappaB 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-kappaB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-kappaB. IMPORTANCE: This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-kappaB 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-kappaB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-kappaB function greatly improved the growth of a nonadapted IAV, suggesting that NF-kappaB may contribute to the maintenance of the host species barrier.

• Dam, S.; Kracht, M.; Pleschka, S.; Schmitz, M. L.