A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity.

The virulence of influenza virus is a multigenic trait. One determinant of virulence is the multifunctional NS1 protein that functions in several ways to defeat the cellular innate immune response. Recent large-scale genome sequence analysis of avian influenza virus isolates indicated that four C-terminal residues of the NS1 protein is a PDZ ligand domain of the X-S/T-X-V type and it was speculated that it may represent a virulence determinant. To test this hypothesis, by using mice as a model system, the four C-terminal amino acid residues of a number of influenza virus strains were engineered into the A/WSN/33 virus NS1 protein by reverse genetics and the pathogenicity of the viruses determined. Viruses containing NS1 sequences from the 1918 H1N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses demonstrated increased virulence in infected mice compared with wt A/WSN/33 virus, as characterized by rapid loss of body weight, decreased survival time, and decreased mean lethal dose. Histopathological analysis of infected mouse lung tissues demonstrated severe alveolitis, hemorrhaging, and spread of the virus throughout the entire lung. The increase in pathogenicity was not caused by the overproduction of IFN, suggesting the NS1 protein C terminus may interact with PDZ-binding protein(s) and modulate pathogenicity through alternative mechanisms.