Figure 1. The dsRNA binding activity of the influenza B virus NS1 protein is dispensable for efficient viral replication and pathogenicity in PKR-deficient hosts.

(A) The schematic diagram shows basic amino acid residues in the dsRNA-binding domain of the NS1 protein. NS1 proteins with alanine exchange mutations at the indicated positions have been shown to have strong (+), weak (+/−) or no (−) dsRNA binding activity [21]. Recombinant influenza B viruses expressing NS1 proteins with abolished dsRNA-binding and mutant virus #1 did not inhibit PKR activation and eIF2α phosphorylation (−), whereas viruses expressing dsRNA binding NS1 proteins inhibited PKR as WT virus (+) [16]. (B) PKR^+/+ or PKR^−/− MEFs were infected with WT virus, delNS1 virus or NS1 mutant viruses #1, #2, #3, #4, #6 or #7 at an MOI of 0.1. Virus titers were determined at the indicated time points and represent the average of two independent experiments performed as duplicates. Error bars indicate the standard deviation. (C) For infection studies in mice the indicated representative recombinant influenza B/Lee viruses were chosen according to their ability to block PKR activation. Groups of eight-week-old female PKR^−/− and wild type C57BL6 mice were anesthetized and infected intranasally with 1×10⁵ ffu of the indicated recombinant influenza B/Lee virus. For viral lung titrations, three mice were sacrificed at day 3 and at day 6 post-infection and virus titers were determined in lung homogenates. Error bars indicate the standard deviation. Statistical analysis indicated significant differences between WT and mutant virus titers. *, p<0.05; **, p<0.01; n.d., not detectable. Other recombinant viruses were not tested in this setting.