Hepatitis C Virus Core Protein Abrogates the DDX3 Function That Enhances IPS-1-Mediated IFN–Beta Induction

Oshiumi, Hiroyuki; Ikeda, Masanori; Matsumoto, Misako; Watanabe, Ayako; Takeuchi, Osamu; Akira, Shizuo; Kato, Naoya; Shimotohno, Kunitada; Seya, Tsukasa

doi:10.1371/journal.pone.0014258

Cited by 70 publications

(55 citation statements)

References 47 publications

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“…DDX3 is ubiquitously expressed in a variety of cells and exerts its positive effect as a part of TBK-1-and/or IKK-ε-containing complexes that activate IRF-3 (41). DDX3 also binds to RIG-I and IPS-1 and promotes the activation of those proteins (28,32). Our study showed that another non-RLR helicase, DDX60, is also involved in RLR-dependent pathways.…”

Section: Discussionmentioning

confidence: 57%

DDX60, a DEXD/H Box Helicase, Is a Novel Antiviral Factor Promoting RIG-I-Like Receptor-Mediated Signaling

Miyashita

Oshiumi

Matsumoto

et al. 2011

Molecular and Cellular Biology

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244

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The cytoplasmic viral RNA sensors RIG-I and MDA5 are important for the production of type I interferon and other inflammatory cytokines. DDX60 is an uncharacterized DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, a cofactor of RNA exosome, which is a protein complex required for the integrity of cytoplasmic RNA. Expression of DDX60 increases after viral infection, and the protein localizes at the cytoplasmic region. After viral infection, the DDX60 protein binds to endogenous RIG-I protein. The protein also binds to MDA5 and LGP2 but not to the downstream factors IPS-1 and IB kinase (IKK-). Knockdown analysis shows that DDX60 is required for RIG-I-or MDA5-dependent type I interferon and interferoninducible gene expression in response to viral infection. However, DDX60 is dispensable for TLR3-mediated signaling. Purified DDX60 helicase domains possess the activity to bind to viral RNA and DNA. Expression of DDX60 promotes the binding of RIG-I to double-stranded RNA. Taken together, our analyses indicate that DDX60 is a novel antiviral helicase promoting RIG-I-like receptor-mediated signaling.RIG-I and MDA5 are cytoplasmic viral RNA sensors belonging to the group of RIG-I-like receptors (RLRs), which includes LGP2 (57-59). RIG-I recognizes RNAs from vesicular stomatitis virus (VSV), hepatitis C virus (HCV), Sendai virus (SeV), and influenza A virus (21, 36, 37), while MDA5 recognizes RNA from picornaviruses such as encephalomyocarditis virus and poliovirus (PV) (3,19,21). RLRs are also involved in the recognition of cytoplasmic B-DNA. RNA polymerase III transcribes cytoplasmic AT-rich double-stranded DNA (dsDNA), and the transcribed RNA is recognized by RIG-I (1, 6). In contrast, Choi et al. have reported that RIG-I associates with dsDNA (7).When RIG-I or MDA5 is activated by viral infection, the N-terminal caspase recruitment domains (CARDs) associate with the adaptor protein IPS-1 (also called MAVS/Cardif/ VISA) on the outer mitochondrial membrane (22,26,42,55). After this association occurs, IPS-1 activates TBK1 and IB kinase ε (IKK-ε) and signals interferon (IFN) regulatory factor 3 (IRF-3)-and NF-B-responsive genes, such as those for type I IFNs or other inflammatory cytokines (22,23,26,42,44,55).Both the helicase and C-terminal domain (CTD) of RIG-I bind to RNA, but it is the CTD that is responsible for the recognition of the 5Ј triphosphate double-stranded structure typical of viral RNA (16,39,40). Recently, Rehwinkel et al. showed that the physiological ligand of RIG-I during influenza A virus or SeV infection is the full-length viral genomic single-stranded RNA (ssRNA), which possesses base-paired regions or defective interfering (DI) genomes (35). In contrast to RIG-I, MDA5 recognizes long viral double-stranded RNA (dsRNA) (21). The RNA binding activity of the MDA5 CTD is relatively weak compared with that of the RIG-I CTD, because the basic surface of the MDA5 CTD has a more extensive flat region than the RIG-I CTD (8,45,46). Although the RNA binding activity of the MDA5 CTD is weak, t...

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Section: Discussionmentioning

confidence: 57%

DDX60, a DEXD/H Box Helicase, Is a Novel Antiviral Factor Promoting RIG-I-Like Receptor-Mediated Signaling

Miyashita

Oshiumi

Matsumoto

et al. 2011

Molecular and Cellular Biology

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244

287

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“…HCV genotype 1b 39 UTR RNA, including the polyU/UC region, was synthesized using T7 and SP6 RNA polymerase and purified with TRIzol, as described previously (20). RNA was i.v.…”

Section: Hydrodynamic Injectionmentioning

confidence: 99%

IPS-1 Is Essential for Type III IFN Production by Hepatocytes and Dendritic Cells in Response to Hepatitis C Virus Infection

Okamoto

Oshiumi

Azuma

et al. 2014

The Journal of Immunology

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Hepatitis C virus (HCV) is a major cause of liver disease. The innate immune system is essential for controlling HCV replication, and HCV is recognized by RIG-I and TLR3, which evoke innate immune responses through IPS-1 and TICAM-1 adaptor molecules, respectively. IL-28B is a type III IFN, and genetic polymorphisms upstream of its gene are strongly associated with the efficacy of polyethylene glycol–IFN and ribavirin therapy. As seen with type I IFNs, type III IFNs induce antiviral responses to HCV. Recent studies established the essential role of TLR3–TICAM-1 pathway in type III IFN production in response to HCV infection. Contrary to previous studies, we revealed an essential role of IPS-1 in type III IFN production in response to HCV. First, using IPS-1 knockout mice, we revealed that IPS-1 was essential for type III IFN production by mouse hepatocytes and CD8+ dendritic cells (DCs) in response to cytoplasmic HCV RNA. Second, we demonstrated that type III IFN induced RIG-I but not TLR3 expression in CD8+ DCs and augmented type III IFN production in response to cytoplasmic HCV RNA. Moreover, we showed that type III IFN induced cytoplasmic antiviral protein expression in DCs and hepatocytes but failed to promote DC-mediated NK cell activation or cross-priming. Our study indicated that IPS-1–dependent pathway plays a crucial role in type III IFN production by CD8+ DCs and hepatocytes in response to HCV, leading to cytoplasmic antiviral protein expressions.

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“…Oshiumi et al suggested that DDX3 directly senses HCV RNA and then triggers MAVS-dependent signaling (5). In a follow-up study, those authors also suggested that the HCV core protein, which had previously been shown to interact with DDX3 (21)(22)(23), blocks the interaction between DDX3 and MAVS and thereby prevents initiation of this pathway (24). In summary, there is now strong evidence that human DDX3 contributes to ifnb promoter induction, in particular with respect to the RIG-I pathway.…”

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confidence: 99%

“…Several studies, including ours, placed DDX3 downstream of TBK1/IKKε (6-9). However, Oshiumi et al placed it upstream of MAVS, as a viral RNA sensor that functions in conjunction with RIG-I/MAVS (5,24). It is also unclear whether the effect of DDX3 is located upstream of IRF3 activation and/or whether it binds directly to the ifnb promoter to enhance transcription (6,7).…”

mentioning

confidence: 99%

Human DEAD Box Helicase 3 Couples IκB Kinase ε to Interferon Regulatory Factor 3 Activation

Fullam

Brennan

et al. 2013

Molecular and Cellular Biology

110

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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...

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Hepatitis C Virus Core Protein Abrogates the DDX3 Function That Enhances IPS-1-Mediated IFN–Beta Induction

Cited by 70 publications

References 47 publications

DDX60, a DEXD/H Box Helicase, Is a Novel Antiviral Factor Promoting RIG-I-Like Receptor-Mediated Signaling

DDX60, a DEXD/H Box Helicase, Is a Novel Antiviral Factor Promoting RIG-I-Like Receptor-Mediated Signaling

IPS-1 Is Essential for Type III IFN Production by Hepatocytes and Dendritic Cells in Response to Hepatitis C Virus Infection

Human DEAD Box Helicase 3 Couples IκB Kinase ε to Interferon Regulatory Factor 3 Activation

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