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

Abstract

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-b induction through the adaptor IFN-b 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-AlaAsp) 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-b promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN-b induction. This activity was conserved on the DDX3-C fragment. DDX3 only marginally enhanced IFN-b promoter activation induced by transfected TANK-binding kinase 1 (TBK1) or I-kappa-B kinase-e (IKKe). Forced expression of DDX3 augmented virus-mediated IFN-b induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS-1 enhancer. IntroductionRetinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytoplasmic RNA helicases [1][2][3], which signal the presence of viral RNA through the adaptor, IFN-b promoter stimulator-1 (IPS-1) (also known as mitochondrial antiviral signaling protein/caspase recruitment domain (CARD) adaptor inducing IFN-b (Cardif)/virus-induced signaling adaptor) to produce . IPS-1 localizes on the outer membrane of the mitochondria via its C-terminus [6]. Its Nterminus consists of a CARD domain, which interacts with the CARD domains of RIG-I and MDA5. Viral RNA resulting from penetration or replication are believed to assemble in the CARDinteracting helicase complex to activate the cytoplasmic IFNinducing pathway. Although non-infected cells usually express minimal amounts of RIG-I/MDA5, the final output of type I IFN is efficiently induced at an early stage of infection to protect host cells from viral spreading.Once IPS-1 is activated, the kinase complex consisting of TANK-homologous proteins and virus-activated kinases induce nuclear translocation of IFN regulatory factor-3 (IRF-3) to activate the IFN promoter [8]. NAK-associated protein 1, TANKbinding kinase 1 (TBK1) and I-kappa-B kinase-e (IKKe) are components of the kinase complex that phosphorylates IRF-3 to induce type I IFN [9,10]. RIG-I recognizes products of various RNA viruses, while MDA5 recognizes products of picornaviruses 940Frontline [1,11]. RIG-I and MDA5 share the helicase domain, which is classified into the DEAD (Asp-Glu-Ala-Asp) box helicase...

Unexpected roles for DEAD‐box protein 3 in viral RNA sensing pathways

“…The findings in this issue that DDX3 has a positive role in IFN induction [3] are consistent with two previous reports. Schroder et al [7] showed that the vaccinia virus protein K7 inhibited PRRinduced IFN-b promoter induction by binding to DDX3, which led to the discovery that DDX3 had a positive role in the RLR pathway.…”

“…In contrast, the role for DDX3 in RLR signalling identified in this issue by Oshiumi et al [3] seems somewhat distinct from these previous studies. Here, DDX3 is identified as an IPS-1-interacting protein via yeast-2-hybrid.…”

“…Here, DDX3 is identified as an IPS-1-interacting protein via yeast-2-hybrid. The authors confirm DDX3 and IPS-1 interaction via overexpression studies in HEK293FT cells [3]. The results suggest this interaction to be constitutive [3], in contrast to the virus-dependent interaction between DDX3 and IKKe [7].…”

“…The authors confirm DDX3 and IPS-1 interaction via overexpression studies in HEK293FT cells [3]. The results suggest this interaction to be constitutive [3], in contrast to the virus-dependent interaction between DDX3 and IKKe [7]. Furthermore, ectopically expressed DDX3 also interacts with RIG-I and MDA5, and RIG-I-or MDA5-induced IFN-b promoter reporter gene activity was inhibited by DDX3 siRNA [3].…”

“…In this issue of the European Journal of Immunology, Oshiumi et al [3] identify DEAD/H Box 3 (DDX3) as a component of the IPS-1 complex, and a positive regulator of IPS-1-mediated IFN-b induction. Like RIG-I and MDA5, DDX3 (also known as DBX) is a member of the Asp-Glu-Ala-Asp (DEAD)-box family of RNA helicases.…”