title: DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS.
abstract: The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3-MAVS and TRAF3-IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling.
authors.affiliation: Institute of Immunology, Maynooth University, Maynooth, County Kildare, Ireland.
authors.forename: Lili
authors.initials: L
authors.lastname: Gu
authors.affiliation: Institute of Immunology, Maynooth University, Maynooth, County Kildare, Ireland.
authors.forename: Anthony
authors.initials: A
authors.lastname: Fullam
authors.affiliation: Institute of Immunology, Maynooth University, Maynooth, County Kildare, Ireland.
authors.forename: Niamh
authors.initials: N
authors.lastname: McCormack
authors.affiliation: Institute of Immunology, Maynooth University, Maynooth, County Kildare, Ireland.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Höhn
authors.affiliation: Institute of Immunology, Maynooth University, Maynooth, County Kildare, Ireland martina.schroeder@nuim.ie.
authors.forename: Martina
authors.initials: M
authors.lastname: Schröder
mesh_terms: D000072283:A549 Cells; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000071457:DEAD Box Protein 58 / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D007372:Interferons / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic; D029082:Sendai virus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015398:Signal Transduction; D048008:TNF Receptor-Associated Factor 3 / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C505371:MAVS protein, human; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D048008:TNF Receptor-Associated Factor 3; C090656:TRAF3 protein, human; D007372:Interferons; D051550:I-kappa B Kinase; C119332:DDX3X protein, human; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases
keywords: DEAD-box proteins; RIG-like helicases; TRAFs; interferons; pattern recognition receptors; ubiquitin signalling