Fig 1.
Generation of influenza PA-X and NS1 mutant viruses.
(A) Cal mutant viruses contain mutations in either the frame-shift motif of PA gene or indicated NS1 amino acids or both. (B) Multi-step viral growth kinetics of the viruses in MDCK cells. Cells were infected with the viruses at an MOI of 0.05 and cultured in the presence of trypsin for up to 48 h. Virus samples in the cultures were titrated in MDCK cells. The dashed line indicates the detection limit of the assay. (C) A single-step virus growth in A549 and DF-1 cells. Cells were infected with the viruses at MOI 2 and cultured at 37°C. Released viruses in the culture supernatant were collected at 2 and 18 h pi and titrated. Fold increase of the virus titer at 18 h pi compared to 2 h pi was calculated. The data represent averages with standard deviations (n = 3). *, P< 0.05.
Fig 2.
Shutoff activity on host protein synthesis of human and avian cultured cells by mutant viruses.
(A) A549 or DF-1 cells were left uninfected or infected with the viruses at MOI of 2. At 16 h pi, cells were labeled with 35S-Met/Cys for 30 min and the radiolabeled lysates were resolved by SDS-PAGE. As loading controls, cellular actin was detected by Western blot analysis (lower panel). (B) Densitometric traces of labeled proteins shown in (A). (C) Quantities of cellular β-actin mRNAs in infected A549 or DF-1 cells at 12 h pi as determined by qRT-PCR. The data represent averages with standard deviations (n = 3). *, P< 0.05, **, P<0.01, ***, P<0.001, NS, not significant. (D) Shutoff activity of the viruses in A549 cells at an early and late time points. Cells were labeled with 35S-Met/Cys at 6 or 24 h pi and total lysates were analyzed by SDS-PAGE as (A).
Table 1.
Number of differentially expressed genes between virus-infected cells.
Fig 3.
Analysis of genes suppressed by active NS1 or PA-X.
Genes suppressed by shutoff-active NS1 (A) or PA-X (B) are shown. A Venn diagram demonstrates number of genes suppressed by NS1 or PA-X at least two-fold. Heatmaps show the 493 (A) or 156 (B) DEGs. Each row represents each gene (blue: low expression; red: high expression; Z-score scaling from -2 to 2). Each column represents one replicate from each virus group. Shutoff activity elicited from PA-X and active NS1 was noted under each virus-infected conditions. Hierarchical clustering was performed and cluster numbers based on their expression profiles were demonstrated on the left of the heatmap (A).
Fig 4.
Clustering and pathway analysis of genes suppressed by active NS1.
Bar graphs demonstrate the enriched biological processes associated with cluster 1, 3, and 4 in Fig 3, which were plotted against the combined score (Enrichr) on the y-axis. Cluster 2 showed no significant enriched biological process.
Fig 5.
Induction of representative genes in infected cells.
Fold change of genes involved in (A) innate signaling and IFNs, (B) ISGs, and (C) cytokines, chemokines and MHC class I molecules are shown. Bar graphs demonstrate fold change of the host genes in response to the virus infection compared to mock as revealed by the RNA-Seq analysis.
Fig 6.
Validation of RNA-Seq data by qRT-PCR and ELISA.
IFN-λ1, IFN-β1, and IL-6 mRNAs were quantitated by qRT-PCR and compared with RNA-Seq data. Secreted IFN-λ1 and IL-6 in the culture medium were quantified by ELISA at 16 and 24 h pi.
Fig 7.
Inhibitory activity of NS1 on innate signaling.
(A) Interaction between NS1 and TRIM25 was determined by co-immunoprecipitation assay. Infected cell lysates were reacted with anti-NS1 antibody, and the immunoprecipitated materials were analyzed for the presence of TRIM25 and NS1. Left figure is a representative of four independent experiments and bar graph on right shows relative ratio of co-immunoprecipitated proteins (n = 4). (B) Activation of IRF3 in infected cells. (Left) A549 cells infected with the viruses were determined for IRF phosphorylation by Western blot analysis. (Right) Translocation of IRF3 to the nucleus in infected cells was determined by immunofluorescence assay using anti-IRF3 antibody (red) counterstained with DAPI (blue).
Fig 8.
RNA-Seq analysis of influenza viral gene expression.
(A) Viral gene expression data obtained from RNA-Seq analysis demonstrates levels of normalized influenza viral mRNA counts of PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, and NEP mRNAs. DESeq2 performs an internal normalization where geometric mean is calculated for each gene across all infected samples. (B) Impact of shutoff-active NS1 on viral gene expression was analyzed by a direct comparison between viruses expressing shutoff-active and inactive NS1, and was demonstrated in fold difference of viral mRNAs in log2 scale. (C) Impact of PA-X on viral gene expression was analyzed by a direct comparison between viruses expressing normal and reduced amount of PA-X, and was demonstrated in fold difference of viral mRNAs in log2 scale.
Fig 9.
Expression of mRNA, vRNA and proteins of NS1 and HA, and growth of the viruses in Calu-3 cells.
A549 cells were infected with the viruses at MOI of 2 and viral mRNA (A) and vRNA (B) were quantitated by qRT-PCR at various times after infection. The data represent averages with standard deviations (n = 3). *, P<0.05. (C) HA and NS1 protein expression in infected A549 cells were determined by Western blot analysis. Cellular actin protein was detected as a loading control. Left figure is a representative of three independent experiments and bar graph on right shows relative expression level (n = 3). *, P<0.05, **, P<0.01. (D) Multi-step growth of the viruses were determined in human airway Calu-3 cells. Cells were infected at MOI of 0.1 and released progeny virions in the culture supernatants at various time points were titrated. The data represent averages with standard deviations (n = 3).