The Ebola virus (EBOV) VP35 protein antagonizes the early antiviral alpha/beta interferon (IFN-alpha/beta) response. We previously demonstrated that VP35 inhibits the virus-induced activation of the IFN-beta promoter by blocking the phosphorylation of IFN-regulatory factor 3 (IRF-3), a transcription factor that is crucial for the induction of IFN-alpha/beta expression. Furthermore, VP35 blocks IFN-beta promoter activation induced by any of several components of the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA-5)-activated signaling pathways including RIG-I, IFN-beta promoter stimulator 1 (IPS-1), TANK-binding kinase 1 (TBK-1), and IkappaB kinase epsilon (IKKepsilon). These results suggested that VP35 may target the IRF kinases TBK-1 and IKKepsilon. Coimmunoprecipitation experiments now demonstrate physical interactions of VP35 with IKKepsilon and TBK-1, and the use of an IKKepsilon deletion construct further demonstrates that the amino-terminal kinase domain of IKKepsilon is sufficient for interactions with either IRF-3 or VP35. In vitro, either IKKepsilon or TBK-1 phosphorylates not only IRF-3 but also VP35. Moreover, VP35 overexpression impairs IKKepsilon-IRF-3, IKKepsilon-IRF-7, and IKKepsilon-IPS-1 interactions. Finally, lysates from cells overexpressing IKKepsilon contain kinase activity that can phosphorylate IRF-3 in vitro. When VP35 is expressed in the IKKepsilon-expressing cells, this kinase activity is suppressed. These data suggest that VP35 exerts its IFN-antagonist function, at least in part, by blocking necessary interactions between the kinases IKKepsilon and TBK-1 and their normal interaction partners, including their substrates, IRF-3 and IRF-7.