DEAD/H BOX 3 (DDX3) helicase binds the RIG‐I adaptor IPS‐1 to up‐regulate IFN‐β‐inducing potential

Retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLR) are members of the DEAD box helicases, and recognize viral RNA in the cytoplasm, leading to IFN‐β induction through the adaptor IFN‐β promoter stimulator‐1 (IPS‐1) (also known as Cardif, mitochondrial antiviral signaling protein or virus‐induced signaling adaptor). Since uninfected cells usually harbor a trace of RIG‐I, other RNA‐binding proteins may participate in assembling viral RNA into the IPS‐1 pathway during the initial response to infection. We searched for proteins coupling with human IPS‐1 by yeast two‐hybrid and identified another DEAD (Asp‐Glu‐Ala‐Asp) box helicase, DDX3 (DEAD/H BOX 3). DDX3 can bind viral RNA to join it in the IPS‐1 complex. Unlike RIG‐I, DDX3 was constitutively expressed in cells, and some fraction of DDX3 is colocalized with IPS‐1 around mitochondria. The 622‐662 a.a DDX3 C‐terminal region (DDX3‐C) directly bound to the IPS‐1 CARD‐like domain, and the whole DDX3 protein also associated with RLR. By reporter assay, DDX3 helped IPS‐1 up‐regulate IFN‐β promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN‐β induction. This activity was conserved on the DDX3‐C fragment. DDX3 only marginally enhanced IFN‐β promoter activation induced by transfected TANK‐binding kinase 1 (TBK1) or I‐kappa‐B kinase‐ε (IKKε). Forced expression of DDX3 augmented virus‐mediated IFN‐β induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS‐1 enhancer.