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. 2002 Aug 6;99(16):10736-41.
doi: 10.1073/pnas.112338099. Epub 2002 Jul 29.

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza

Gary K Geiss  1 , Mirella Salvatore, Terrence M Tumpey, Victoria S Carter, Xiuyan Wang, Christopher F Basler, Jeffery K Taubenberger, Roger E Bumgarner, Peter Palese, Michael G Katze, Adolfo García-Sastre
Affiliations

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza

Gary K Geiss et al. Proc Natl Acad Sci U S A. .

Abstract

The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-kappaB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

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Figures

Fig 1.
Fig 1.
Increased NF-κB activation and IFN-β production in delNS1 virus-infected A549 cells results in activation of target genes. (A) Electrophoretic mobility-shift assay analysis of NF-κB binding to its cognate DNA element. An oligonucleotide corresponding to the NF-κB binding site was labeled with 32P and incubated with nuclear extracts from mock-infected or virus-infected A549 cells at 16 h postinfection, as indicated. Cells were also infected with Sendai virus as a positive control of this experiment. The position of the NF-κB/DNA complex is shown. (B) Northern blot analysis of IFN-β and cellular genes with known NF-κB or IFN responsive elements. A β-actin control Northern is also shown. The moi and the postinfection times that were used in these experiments are indicated on the top. The average fold change and P value observed by microarray analysis at 8 h postinfection of repeated experiments is shown on the right. The IFN-β gene was not present on microarrays used (N/A). The asterisk indicates the microarray results for α-actin because the β-actin cDNA was not represented on the array. It should be noted that, in contrast to some other cell lines, such as MDCK cells, no severe cytopathic effect was induced in A549 cells after influenza virus infection.
Fig 2.
Fig 2.
Ven diagram showing the distribution of differentially regulated genes during infection with wt or mutant NS1 influenza PR8 viruses relative to mock-infected cells. Differentially expressed genes were selected from combined data of multiple experiments (see Materials and Methods). For a gene to fall into multiple categories, it had to meet the fold change and P value selection criteria outlined in Materials and Methods (≥2.0) and P value (<0.01). Some differentially regulated genes that are shared between viruses may exhibit different degrees of regulation (see Table 2). Area shaded in black represents the number of genes that met the statistical and fold change criteria for all virus infections. Hatched areas represent the number of genes that met the criteria in two of three viruses, and areas shaded in gray represent the number of genes differentially regulated by a single virus type.
Fig 3.
Fig 3.
Summary of cellular genes that are regulated by wt and NS1 mutant PR8 viruses. The cellular genes represented in each panel are as follows: (A) genes regulated by all three viruses; (B) genes regulated by both mutant viruses but not by wt virus; (C) genes regulated in NS1 (1–126) mutant virus-infected cells; and (D) genes regulated in delNS1 virus-infected cells. The subset of genes that each image corresponds to is shaded in the Venn diagrams below each panel. Genes and ESTs that lacked sufficient functional annotation and those enhanced in wt virus are not shown (see Fig. 8, which is published as supporting information on the PNAS web site). Columns represent data obtained for individual replica experiments. Red, Expression was induced in infected cells relative to mock; green, expression was repressed in infected cells relative to mock; black, expression was not changed; and gray, gene was not present on the array. The shade of red or green represents the degree of change. The scale represents log10 ratios and is the same for all four images. *, IFN-stimulated gene; •, potential antiviral gene; #, tripartite motif-containing protein.
Fig 4.
Fig 4.
Pathways and gene families regulated by PR8 virus infection. (A) Relative mRNA levels of suppressor of cytokine gene family and associated genes during PR8, WSN, and NS 1918 virus infections. Colors represent relative change in mRNA levels relative to mock-infected cells. See legend for Fig. 3 for details on color scheme. Genes whose expression was up-regulated by PR8 virus but down-regulated by 1918 NS virus (and also by wt WSN virus, although to a lesser extent) are with solid arrow. (B) Relative mRNA levels of TRIM proteins and their corresponding chromosomal locations. Hierarchical clustering of 19 TRIM genes was performed (Fig. 12), and the cluster representing induced genes is shown. EST_810133 was identified as a TRIM gene by a blast search of the human genome. Chromosomal locations were obtained from the LocusLink database (38).
Fig 5.
Fig 5.
Comparison of expression ratios for cellular genes during infection with wt influenza A/WSN/33 and NS/1918 viruses vs. mock-infected cells. Scatter plot of log2 ratios for genes that exhibited P values ≤0.01 in at least one of the two infections. Blue, Genes that were significantly up- or down-regulated during infection with 1918 NS recombinant virus; magenta, genes that were significantly up- or down-regulated during infection with wt A/WSN/33 virus; and black, genes that had P values ≤0.01 during both infections. Examples of genes with known cellular function and whose expression differs considerably between the two viruses are indicated by arrows and Human Genome Organization (HUGO) names. ESTs that exhibited differences in gene expression between WSN and NS 1918 virus-infected cells are indicated with solid arrow heads: 1, EST_134682; 2, EST_32376 (LOC51103); 3, EST_427797; 4, EST_207649 (KIAA1272); 5, EST_283495; 6, EST_795255.
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