P132 The human dead-box helicase 3 couples IKK-epsilon to IRF3 activation

Abstract

Introduction The human DEAD-box RNA helicase DDX3 has been implicated in different cellular processes including transcription, splicing, mRNA nuclear export and translation. We and others previously showed that DDX3 is involved in innate immune signalling pathways which trigger the induction of type I interferon through the kinases IKKe or TBK1 [1] , [2] , [3] . We demonstrated that DDX3 interacts with IKKe, and that it contributes to IRF3 activation. Other studies have confirmed a role for DDX3 in IFN-s induction, but placed DDX3 upstream in the MAVS-mediated signalling pathway, or suggested that DDX3 binds to the IFN-s promoter directly. Therefore, our study here aimed at placing DDX3 more firmly in the signalling pathway(s) leading to IFN-s induction, by dissecting its molecular interaction with IKKe and the functional consequences of this interaction. Methods The DDX3, IKKe and IRF3 interaction studies were performed using co-immunoprecipitation (Co-IP) and His pulldown assays. In vitro kinase assays were employed for investigating DDX3 and IRF3 phosphorylation by IKKe. IKKe- phosphorylation sites in DDX3 were identified using Mass Spectrometry. Luciferase reporter assays were used to study effects of phospho-deficient and –mimetic DDX3 mutants on IFN-s promoter induction. Results We show here that DDX3 directly interacts with, and is phosphorylated by IKKe. Phosphorylation of DDX3 occurs mainly in its N-terminal region, which is required for IFN-s induction, and is targeted by the vaccinia virus protein K7 in a viral immune evasion strategy. In agreement with this, we found that K7 specifically inhibits DDX3-phosphorylation by IKKe. Furthermore, we have identified one serine residue in the N-terminus of DDX3 that is crucial for the effect of DDX3 on the IFN-s promoter. We also show that DDX3 directly enhances IKKe activity and phosphorylation of IRF3 in an in vitro reconstitution assay. In addition, we demonstrate that DDX3 directly binds to IRF3, and that this interaction is crucial for the DDX3 effect on IFN-s induction. Phosphorylation of DDX3 by IKKe facilitates the subsequent recruitment of IRF3. IRF3 and IKKe have adjacent and potentially overlapping, but not identical, binding sites in the N-terminus of DDX3. Conclusion Our study has shown that the N-terminus of DDX3 directly interacts with IKKe and IRF3 and that this mediates their activation and functional interaction. The interaction between DDX3 and IRF3 contributes to IRF3 activation and is required for the effect of DDX3 on IFN-s promoter induction. The effects of DDX3 on IKKe activation and IRF3 activation can be uncoupled by mutation of a single serine residue in the N-terminus of DDX3.Our work implicates DDX3 as a bridging adaptor for IKKe and IRF3 in RLH and TLR signalling pathways.