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Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by double-stranded (ds) RNA, which culminates in the activation of the transcription factor NF-kappaB and induction of NF-kappaB-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-kappaB pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-kappaB was partially activated. It was released from IkappaB and translocated to the nucleus but failed to bind to the kappaB site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-kappaB, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 --> Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-kappaB release and the other leading to its phosphorylation.

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.