(A) Three P58^IPK−/− and wild-type mice were infected with 10 or 100 PFU of the PR8 strain of influenza virus. The number of surviving mice, as determined by having at least 20% of starting weight, at each day is shown. (B) Three P58^IPK−/− and wild-type mice infected with 10³ PFU of PR8 were sacrificed at 1, 3, or 5 days post infection. Levels of infectious virions in diaphragmatic lung homogenates were determined by triplicate plaque assay on MDCK cells. The results represent the mean activity of 3 independent samples±standard deviation.

P58^IPK−/− and wild-type (WT) mice were infected with 10³ (A,B, E–H), 10⁴ (I,J), or 10⁵ (C,D) PFU of the PR8 strain of influenza virus and stained for hematoxalin and eosin (H&E) (A,B), periodic acid-Schiff (PAS) (C,D), F4/80 (E,F), influenza virus NP (G,H), or cleaved caspase 3 (I,J) at 5 days post infection. This shows that, while both P58^IPK−/− and wild-type mouse lungs contain similar amounts of infected cells, P58^IPK−/− mice exhibit increased alveolitis and peribrochiolitis, marked by macrophage and neutrophil infiltration, hyaline membrane formation, and caspase activation. Arrows denote hyaline membranes. Bar = 50 µm.

Three P58^IPK−/− and wild-type mice were infected with 10⁴ (A,B) or 10³ (C,D) PFU of the PR8 strain of influenza virus. At 1 and 3 days post infection, azygous and apical lung lobes were excised and homogenized. (A) Equivalent concentrations of protein in lung homogenates were subjected to SDS-10% PAGE. The levels of phosphorylated or total eIF2α and actin were determined by immunoblot analysis. (B) Densitometry analysis of (A). The density of the eIF2α-P band was normalized to the density of the total eIF2α band, which is represented graphically. (C) 400 µg of total protein from lung homogenates was used for immunoprecipitation with an antibody for total PKR. Following immunoprecipitation, protein samples were clarified by SDS-10% PAGE and the levels of phosphorylated or total PKR were determined by immunoblot analysis. (D) Densitometry analysis of (C). The density of the P-PKR band was normalized to the density of the total PKR band, which is represented graphically. The results represent the mean activity of 3 biologically independent samples±standard deviation. P-values from a two-tailed t-test assuming non-equal variance are indicated (*: P<0.05, **: P<0.05).

Gene Set Enrichment Analysis (GSEA) analysis was performed on a sample set derived from P58^IPK−/− and wild-type mice infected with the PR8 strain of influenza virus in triplicate at 1 day post infection. All infected samples were compared to genotype-matched mock-infected samples via microarrays analysis. Replicate samples were then in silico error-weighted pooled and re-ratioed to compare P58^IPK−/− gene expression to wild-type gene expression. Following GSEA analysis using only those genes which were significantly regulated, the top gene ontology categories related to the immune response were selected (see for entire table). An edge is placed between gene ontology categories if they share common genes, and the edge's thickness increases as the number of common genes increases. The log₁₀ ratio of P58^IPK to wild-type gene regulation is noted for selected gene ontology categories.

Three P58^IPK−/− and wild-type mice were mock infected or infected with 10³ (A,B) or 10⁴ (C,D) PFU of the PR8 strain of influenza virus. At 1, 3, or 5 days post infection, blood serum was isolated, azygous and apical lung lobes, or left lung lobes were excised and homogenized. Equivalent concentrations of protein in serum (A) or azygous and apical lung homogenates (B) were subjected to ELISA using an IL6 antibody. Total RNA was isolated from left lung lobes for reverse transcription to generate cDNA. Quantitative RT-PCR was used to determine the amount of mouse IL6 (C) and IFNβ (D) mRNA in each sample. The results represent the mean activity of 3 independent samples (with technical triplicates)±standard deviation. P values from a two-tailed t-test assuming non-equal variance are indicated (*: P<0.05, **: P<0.01, ***: P<1×10⁻⁴).

(A) Three P58^IPK−/− and wild-type mice were infected with 10⁵ or 5×10⁶ PFU of the r1918 strain of influenza virus. The number of surviving mice, as determined by having at least 20% of starting weight, at each day is shown. (B) Three P58^IPK−/− and wild-type mice infected with 10⁴ PFU of r1918 were sacrificed at 3 days post infection. Levels of infectious virions in diaphragmatic lung homogenates were determined by triplicate plaque assay on MDCK cells. The results represent the mean activity of 3 independent samples±standard deviation.

(A) Venn diagram analysis was performed on a sample set derived from P58^IPK−/− and wild-type (WT) mice infected with the PR8 or r1918 strains of influenza virus in triplicate at 1 day post infection. All infected samples were compared to genotype-matched mock-infected samples via microarrays analysis. Replicate samples were then pooled and error-weighted in silico. For each virus, a gene set was isolated which included genes which were up-regulated in P58^IPK−/− mice as compared to mock and as compared to wild-type mice. The intersection of the sets for both PR8 and r1918 infection was isolated for IPA. Of the 113 genes in this set, 65 had annotation, and 47 of those had functions related to the inflammatory and cell death responses; log₁₀ ratio regulation of these genes in P58^IPK−/− and wild-type mice as compared to mock is represented for both viral infections. The bar graph represents the log₁₀ ratio of P58^IPK−/− to wild-type gene regulation, showing that each gene is more up-regulated in P58^IPK−/− mice. (B) IPA network analysis of the middle set of genes in (A) highlights a subset of genes that are up-regulated in wild-type mice during r1918 infection but not PR8 infection. This diagram shows the direct or indirect interactions reported for these cell-death- (blue shading) and inflammatory-response-related (yellow shading) genes. Genes shaded orange fit into both categories.

During influenza virus infection, P58^IPK and PKR are activated. While P58^IPK-mediated inhibition of PKR results in decreased apoptotic and inflammatory responses, the absence of P58^IPK results in the amplification of these responses via increased Caspase, eIF2α, and NF-κB activation. Molecules shaded light grey were up-regulated in infected P58^IPK−/− mice at the transcript level while those shaded dark grey were up-regulated at the protein level. In tandem, the enhancement of these responses results in increased mortality in P58^IPK−/− mice.