The human DEAD box protein 3 (DDX3) has been implicated in different processes contributing to gene expression. Interestingly, DDX3 is required as an essential host factor for the replication of HIV and hepatitis C virus (HCV) and is therefore considered a potential drug target. On the other hand, DDX3 interacts with IB kinase (IKK) and TANK-binding kinase 1 (TBK1) and contributes to the induction of antiviral type I interferons (IFNs). However, the molecular mechanism by which DDX3 contributes to IFN induction remains unclear. Here we show that DDX3 mediates phosphorylation of interferon regulatory factor 3 (IRF3) by the kinase IKK. DDX3 directly interacts with IKK and enhances its autophosphorylation and activation. IKK then phosphorylates several serine residues in the N terminus of DDX3. Phosphorylation of DDX3 at serine 102 (S102) is required for recruitment of IRF3 to DDX3, facilitating its phosphorylation by IKK. Mutation of S102 to alanine disrupted the interaction between DDX3 and IRF3 but not that between DDX3 and IKK. The S102A mutation failed to enhance ifnb promoter activation, suggesting that the DDX3-IRF3 interaction is crucial for this effect. Our data implicates DDX3 as a scaffolding adaptor that directly facilitates phosphorylation of IRF3 by IKK. DDX3 might thus be involved in pathway-specific activation of IRF3. The human DEAD box RNA helicase DDX3 is a multifunctional cellular protein which has been implicated in various processes linked to gene expression (1). Its RNA helicase activity is coopted by viruses that require DDX3 as an essential host factor for their replication, such as HIV and HCV (2-4). In contrast, we and others have previously demonstrated that DDX3 contributes to antiviral innate immune signaling pathways leading to ifnb induction (5-9). This function of DDX3 is independent of its ATPase and helicase activity (6, 7). ifnb promoter induction requires activation of the transcription factors IRF3 and IRF7, which occurs only downstream of certain, mostly antiviral, pattern recognition receptors (PRRs). Antiviral PRRs comprise the endosomal Toll-like receptors TLR3, TLR7, TLR8, and TLR9, cytosolic RIG-like helicases (RLHs), and several newly discovered cytosolic DNA receptors (10). These receptors recognize different species of viral nucleic acids and induce type I IFNs in response (11). TLR3 and TLR4 engage the TRIF (TIR domain-containing adaptor inducing IFN-␤)-dependent pathway for activation of IRF3 (12). In this pathway, phosphorylation-dependent activation of IRF3 is mediated by the IKK-related kinases TBK1 and IKKε (13,14). The RLHs also utilize TBK1 and IKKε for IRF3 activation after engaging the adaptor molecule MAVS (mitochondrial antiviral signaling) (15, 16). DNA receptors activate IRF3 through TBK1 and the adaptor molecule STING (stimulator of IFN genes).While these signaling pathways converge on IKKε and TBK1 for IRF3 activation, not all receptors that activate IKKε and/or TBK1 lead to IRF3 activation (17). This suggests that additional factors are required for li...

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.

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