A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo.

The PB1-F2 protein of influenza A virus can contribute to viral pathogenesis of influenza virus strains. Of note, an N66S amino acid mutation in PB1-F2 has been shown to increase the pathogenesis associated with H5N1 Hong Kong/1997 and H1N1 Brevig Mission/1918 influenza viruses. To identify the mechanism of enhanced immunopathology, we evaluated the host response to two isogenic viruses that differ by a single amino acid at position 66 of the PB1-F2 protein. Various components of the adaptive immune response were ruled out as factors contributing to pathogenesis through knockout mouse studies. Transcriptional profiling of lungs from PB1-F2 66S-infected mice revealed an early delay in innate immune responses. In particular, enhanced activation of type I interferon (IFN) pathway genes, including IFN-β, RIG-I, and numerous interferon-inducible genes, was not observed until day 3 postinfection. The N66S mutant virus caused increased cellularity in the lungs, as a result of monocyte and neutrophil infiltration. Furthermore, numerous cytokines and chemokines related to monocyte and neutrophil migration and maturation were upregulated. The cellular infiltration and increased cytokine expression corresponded to increased PB1-F2 66S titer. These data suggest that PB1-F2 N66S may contribute to the delay of innate immune responses, allowing for unchecked viral growth and ultimately severe immunopathology observed in the lungs.