Analysis of genes induced by Sendai virus infection of mutant cell lines reveals essential roles of interferon regulatory factor 3, NF-kappaB, and interferon but not toll-like receptor 3

Abstract

Sendai virus (SeV) infection causes the transcriptional induction of many cellular genes that are also induced by interferon (IFN) or double-stranded RNA (dsRNA). We took advantage of various mutant cell lines to investigate the putative roles of the components of the IFN and dsRNA signaling pathways in the induction of those genes by SeV. Profiling the patterns of gene expression in SeV-infected cells demonstrated that Toll-like receptor 3, although essential for gene induction by dsRNA, was dispensable for gene induction by SeV. In contrast, Jak1, which mediates IFN signaling, was required for the induction of a small subset of genes by SeV. NF-kappaB and interferon regulatory factor 3 (IRF-3), the two major transcription factors activated by virus infection, were essential for the induction of two sets of genes by SeV. As expected, some of the IRF-3-dependent genes, such as ISG56, were more strongly induced by SeV in IRF-3-overexpressing cells. Surprisingly, in those cells, a number of NF-kappaB-dependent genes, such as the A20 gene, were induced poorly. Using a series of cell lines expressing increasing levels of IRF-3, we demonstrated that the degree of induction of A20 mRNA, upon SeV infection, was inversely proportional to the cellular level of IRF-3, whereas that of ISG56 mRNA was directly proportional. Thus, IRF-3 can suppress the expression of NF-kappaB-dependent genes in SeV-infected cells.

FIG. 1.

Effect of TLR3 on the regulation of cellular genes by SeV and dsRNA. Shown are global changes in mRNA levels in 293 cells (A and C) or 293 cells expressing TLR3 (B and D) 6 h after SeV infection at 1 hemagglutinating unit/4.0 × 10³ cells (A and B) or dsRNA treatment at 100 μg/ml (C and D) as determined by cDNA microarray experiment. Each point on the scatter plots represents the expression of an individual mRNA message, as determined by units of fluorescent intensity, in untreated cells (x axis) plotted against its expression 6 h after SeV infection or dsRNA treatment (y axis). The central diagonal line (black) represents equal expression in treated and untreated samples, while twofold differences in expression are indicated by the two flanking blue lines. (E) Regulation of specific genes by dsRNA and SeV in 293 (−) and 293/TLR3 (+) cells. The tiles show the increase (n-fold) in mRNA expression for specific genes in SeV- or dsRNA-treated cells relative to untreated cells as a function of color. Green, expression was unchanged; yellow to red, expression was induced to increasing degrees. (F) RPA of ISG56 induction in untreated (lanes 1 and 4), dsRNA-treated (lanes 2 and 5), or SeV-infected (lanes 3 and 6) 293 cells (lanes 1 to 3) or 293/TLR3 cells (lanes 4 to 6).

FIG. 2.

Differential induction of genes by SeV and dsRNA. Select genes were differentially regulated by SeV and dsRNA in 293 cells expressing (+) or not expressing (−) TLR3. Colors represent induction (n-fold) by SeV (left column) or dsRNA (right column) as described in the legend to Fig. 1.

FIG. 3.

IFN-dependent regulation of SeV-induced genes. Select SeV-regulated genes display differential induction patterns in wt (2fTGH) and Jak1^−/− (U4C) cells. (A) Microarray data showing average increase (n-fold) in mRNA expression 6 h after SeV infection in 2fTGH and U4C cells. Genes are grouped based on their independence of, dependence on, or impairment by Jak1 for induction. (B) RPA (A20) and Northern (OAS, p69) analysis of mRNA expression in 2fTGH and U4C cells, before or 6 h after SeV infection.

FIG. 4.

Requirement for NF-κB for gene induction by SeV. Genes were regulated by SeV in wt (2fTGH) and NF-κB null (2F-SR) cells. (A) Average induction (n-fold) of SeV-regulated genes, grouped by dependence on NF-κB, as determined by microarray. (B) Quantitative analysis of NOXA, ISG56, and A20 mRNA induction by RPA.

FIG. 5.

IRF-3-dependent gene induction. Shown are examples of genes regulated by SeV in U4C cells and P2.1 cells in which IRF-3 expression is impaired. Genes are grouped by their dependence on IRF-3 for induction.

FIG. 6.

Regulation of SeV-induced genes by IRF-3 and altered expression of SeV-regulated genes in U4C cells and U4C.2 cells, which express high levels of IRF-3. (A and B) Microarray data for the induction (n-fold) of select SeV-regulated genes in U4C and U4C.2 cells. Genes are grouped as follows: IRF-3 enhanced, genes with augmented expression in IRF-3-overexpressing cells (i.e., induced more strongly in U4C.2 cells); IRF-3 neutral, genes unaffected by cellular IRF-3 levels (induced at equivalent levels in both cell lines); IRF-3 repressed, SeV-induced genes negatively regulated by IRF-3 (induced in U4C but not U4C.2 cells). Also shown are quantitative RPA analyses of follistatin (C), NOXA (D), and A20 (E) mRNA induction in U4C and U4C.2 cells before or after SeV infection.

FIG. 7.

Modulation of A20 mRNA and ISG56 mRNA expression by cellular levels of IRF-3. Cell lines derived from U4C and P2.1 cells and expressing different levels of IRF-3 protein were infected with SeV and analyzed for the expression of A20 mRNA and ISG56 mRNA. (A) Western blot showing the relative levels of IRF-3 expression in the different cell lines relative to actin. (B) Percent maximum induction (n-fold) of A20 mRNA (white bars) and ISG56 mRNA (black bars) in cells 6 h after infection, normalized to actin mRNA expression as determined by RPA.

FIG. 8.

Negative regulation of SeV-induced A20 mRNA expression by IRF-3 in wt cells. (A) Western analysis of IRF-3 expression in HT1080 and 1080.10 cells. (B) RPA comparing A20 mRNA induction in untreated (−) and SeV-infected (+) HT1080 and 1080.10 cells.