These results indicate that the activation and nuclear translocation of IRF3 are independent of TRAF6. was not required for IFN- induction in this process, since normal IFN-/ production was observed in TAK1-deficient mouse embryo 8-Hydroxyguanosine fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN- promoter in response to poly(I:C). Forced expression of MEKK1 8-Hydroxyguanosine in combination with IRF3 was sufficient for the induction of IFN-, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN- by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-B and mitogen-activated protein kinases that are critical for the 8-Hydroxyguanosine optimal induction of type I interferons. The innate immune system serves as a first line defense against viral infection. Host antiviral responses are initiated thorough the recognition of viral components by PRRs,2including TLRs and RIG-I (retinoic acid-inducible gene I)-like helicases (RLHs) (13). Upon recognition, the PRRs trigger intracellular signaling pathways that induce the production of antiviral mediators, such as type I interferons (IFN-/), IFN-stimulated genes, inflammatory cytokines, and chemokines, such as IP-10. The expression of type I IFNs and other antiviral proteins suppresses viral replication and facilitates the adaptive immune responses. dsRNA is one of the viral components recognized by TLR3 and RNA helicases, such as RIG-I and MDA5 (melanoma differentiation-associated protein 5). TLR3 recognizes extracellular viral dsRNA internalized into the endosomes in a certain type of cells, such as DCs, whereas RIG-I and MDA5 detect intracellular viral dsRNA in various types of cells, including fibroblasts (47). The viral recognition by TLR3 and RIG-I/MDA5 results in rapid induction of type I IFNs through the activation of their intracellular signaling molecules (13). For instance, TLR3 interacts with an adaptor molecule, TRIF (8,9), which in turn activates two IKK family proteins, TBK1 (TANK-binding kinase-1) and IKK-i(also known as IKK) (10). Both TBK1 and IKK-isubsequently activate a transcription factor, IRF3, resulting in the initial expression of IFN- (11,12). Another IRF (IFN-regulatory factor) family member, IRF7, which is induced by the initial IFN-, elicits further induction of type I IFN genes, including IFN- and IFN- (13). Stimulation with TLR3 ligand also activates other transcription factors, including NF-B and AP-1, which is thought to synergize with IRF3 to induce type I IFN genes (14,15). On the other 8-Hydroxyguanosine hand, RIG-I/MDA5 bind to intracellular RNA through the C-terminal helicase domain and initiate downstream signaling cascades through the N-terminal CARD domains (46). The CARD domains interact with another CARD containing molecule, IPS-1 (IFN- promoter stimulator-1; also known as MAVS, VISA, and CARDIF) (1619), which activates TBK1/IKK-ivia TRAF3, resulting in the activation of IRF3, IRF7, and NF-B (20,21). Therefore, both TLR3 and RIG-I/MDA5 pathways converge at the TBK1/IKK-ikinase complex. IPS-1 contains multiple TRAF-interacting motifs (TIMs) in the proline-rich region, which can associate with the C-terminal TRAF domain of TRAF3 (22). Furthermore, IPS-1 has been shown to interact with other NEDD4L TRAF family members, such as TRAF6 and its downstream MAP3K, TAK1 (transforming growth factor–activated kinase 1) (18). Both TRAF6 and TAK1 have been demonstrated to play a critical role in the production of proinflammatory cytokines in macrophages and DCs triggered by MyD88 (myeloid differentiation factor 88)-mediated signals from the Toll-like/IL-1 receptor family (23,24). The TRAF6/TAK1 signal activates a canonical IKK complex, IKK//, resulting in the activation of NF-B as well as MAPK cascades leading to the activation of AP-1 (25). Although TRAF6/TAK1 has been implicated in proinflammatory cytokine production induced by TLR ligands, the involvement of these molecules in the regulation of type I IFN production induced by the RLH pathway is largely unknown. In this report, we show that.