Danying Chen

ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization

We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non–self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

SIKE is an IKKε/TBK1‐associated suppressor of TLR3‐ and virus‐triggered IRF‐3 activation pathways

Viral infection or TLR3 engagement causes activation of the transcription factors IRF‐3 and NF‐κB, which collaborate to induce transcription of type I IFN genes. IKKε and TBK1 are two IKK‐related kinases critically involved in virus‐ and TLR3‐triggered activation of IRF‐3. We identified a protein termed SIKE (for Suppressor of IKKε) that interacts with IKKε and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKε or TBK1 with TRIF, RIG‐I and IRF‐3, components in virus‐ and TLR3‐triggered IRF‐3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3‐triggered NF‐κB activation pathway. Consistently, overexpression of SIKE inhibited virus‐ and TLR3‐triggered interferon‐stimulated response elements (ISRE) but not NF‐κB activation. Knockdown of SIKE potentiated virus‐ and TLR3‐triggered ISRE but not NF‐κB activation. Moreover, overexpression of SIKE inhibited IKKε‐ and TBK1‐mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKε and TBK1 and plays an inhibitory role in virus‐ and TLR3‐triggered IRF‐3 but not NF‐κB activation pathways.

Negative regulation of MDA5- but not RIG-I-mediated innate antiviral signaling by the dihydroxyacetone kinase

Viral infection leads to activation of the transcription factors interferon regulatory factor-3 and NF-κB, which collaborate to induce type I IFNs. The RNA helicase proteins RIG-I and MDA5 were recently identified as two cytoplasmic viral RNA sensors that recognize different species of viral RNAs produced during viral replication. In this study, we identified DAK, a functionally unknown dihydroacetone kinase, as a specific MDA5-interacting protein. DAK was associated with MDA5, but not RIG-I, under physiological conditions. Overexpression of DAK inhibited MDA5- but not RIG-I- or TLR3-mediated IFN-β induction. Overexpression of DAK also inhibited cytoplasmic dsRNA and SeV-induced activation of the IFN-β promoter, whereas knockdown of endogenous DAK by RNAi activated the IFN-β promoter, and increased cytoplasmic dsRNA- or SeV-triggered activation of the IFN-β promoter. In addition, overexpression of DAK inhibited MDA5- but not RIG-I-mediated antiviral activity, whereas DAK RNAi increased cytoplasmic dsRNA-triggered antiviral activity. These findings suggest that DAK is a physiological suppressor of MDA5 and specifically inhibits MDA5- but not RIG-I-mediated innate antiviral signaling.

Negative feedback regulation of cellular antiviral signaling by RBCK1-mediated degradation of IRF3

Viral infection causes host cells to produce type I interferons (IFNs), which are critically involved in viral clearance. Previous studies have demonstrated that activation of the transcription factor interferon regulatory factor (IRF)3 is essential for virus-triggered induction of type I IFNs. Here we show that the E3 ubiquitin ligase RBCC protein interacting with PKC1 (RBCK1) catalyzes the ubiquitination and degradation of IRF3. Overexpression of RBCK1 negatively regulates Sendai virus-triggered induction of type I IFNs, while knockdown of RBCK1 has the opposite effect. Plaque assays consistently demonstrate that RBCK1 negatively regulates the cellular antiviral response. Furthermore, viral infection leads to induction of RBCK1 and subsequent degradation of IRF3. These findings suggest that the cellular antiviral response is controlled by a negative feedback regulatory mechanism involving RBCK1-mediated ubiquitination and degradation of IRF3.

REUL Is a Novel E3 Ubiquitin Ligase and Stimulator of Retinoic-Acid-Inducible Gene-I

RIG-I and MDA5 are cytoplasmic sensors that recognize different species of viral RNAs, leads to activation of the transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons. In this study, we identified REUL, a RING-finger protein, as a specific RIG-I-interacting protein. REUL was associated with RIG-I, but not MDA5, through its PRY and SPRY domains. Overexpression of REUL potently potentiated RIG-I-, but not MDA5-mediated downstream signalling and antiviral activity. In contrast, the RING domain deletion mutant of REUL suppressed Sendai virus (SV)-induced, but not cytoplasmic polyI:C-induced activation of IFN-β promoter. Knockdown of endogenous REUL by RNAi inhibited SV-triggered IFN-β expression, and also increased VSV replication. Full-length RIG-I, but not the CARD domain deletion mutant of RIG-I, underwent ubiquitination induced by REUL. The Lys 154, 164, and 172 residues of the RIG-I CARD domain were critical for efficient REUL-mediated ubiquitination, as well as the ability of RIG-I to induce activation of IFN-β promoter. These findings suggest that REUL is an E3 ubiquitin ligase of RIG-I and specifically stimulates RIG-I-mediated innate antiviral activity.

The Ret Finger Protein Inhibits Signaling Mediated by the Noncanonical and Canonical IκB Kinase Family Members

IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical IκB kinase (IKK) family members, IKKε and TRAF family-associated NF-κB activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKKε-interacting proteins, we identified Ret finger protein (RFP) as an IKKε-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKKε and TRAF family-associated NF-κB activator-binding kinase-1 as well as the two canonical IKK family members, IKKβ and IKKα. RFP inhibited activation of the IFN-stimulated response element and/or NF-κB mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-κB triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.

TNF receptor‐associated factor‐1 (TRAF1) negatively regulates Toll/IL‐1 receptor domain‐containing adaptor inducing IFN‐β (TRIF)‐mediated signaling

Toll‐like receptor 3 (TLR3) plays an important role in antiviral responses through recognizing viral double‐stranded RNA produced during viral infection and mediating induction of type I IFN. TRIF is a Toll/IL‐1 receptor (TIR) domain‐containing adaptor protein that is associated with TLR3 and critically involved in TLR3‐mediated signaling. In yeast two‐hybrid screens, we identified TNF receptor‐associated factor (TRAF)1 as a TRIF‐interacting protein. The TRAF‐C domain of TRAF1 and the TIR domain of TRIF were responsible for their interaction. Overexpression of TRAF1 inhibited TRIF‐ and TLR3‐mediated activation of NF‐κB, IFN‐stimulated response element and the IFN‐β promoter. Overexpression of TRIF caused caspase‐dependent cleavage of TRAF1. The cleaved N‐terminal but not C‐terminal fragment of TRAF1 was responsible for inhibiting TRIF signaling. Mutation of the caspase cleavage site of TRAF1 or addition of the caspase inhibitor crmA inhibited TRAF1 cleavage and abolished the ability of TRAF1 to inhibit TRIF signaling, suggesting that TRIF‐induced cleavage of TRAF1 is required for its inhibition of TRIF signaling. Our findings provide a novel mechanism for negative regulation of TRIF‐mediated signaling.

TRIP6 is a RIP2-associated common signaling component of multiple NF-κB activation pathways

Receptor-interacting protein 2 (RIP2) is a member of the RIP kinase family that has been shown to be crucially involved in inflammation, innate and adaptive immune responses. The physiological and pathological roles of RIP2 are mediated through its involvement in multiple NF-κB activation pathways, including those triggered by tumor necrosis factor (TNF), interleukin 1 (IL-1), Toll-like receptor 2 (TLR2), TLR3, TLR4 and Nod1. In this report, we identified the LIM-domain-containing protein TRIP6 as a RIP2-interacting protein in yeast two-hybrid screens. In mammalian cells, TRIP6 interacts with RIP2 in a TNF- or IL-1-dependent manner. Overexpression of TRIP6 potentiates RIP2-mediated NF-κB activation in a dose-dependent manner. The LIM domains of TRIP6 are responsible for its interaction with RIP2. TRIP6 also interacts with TRAF2, a protein that is crucially involved in TNF signaling, as well as the IL-1 receptor, TLR2 and Nod1. Overexpression of TRIP6 potentiates NF-κB activation by TNF, IL-1, TLR2 or Nod1, whereas a dominant negative mutant or RNA-interference construct of TRIP6 inhibits NF-κB activation by TNF, IL-1, TLR2 or Nod1. Moreover, TRIP6 also potentiates RIP2- and Nod1-mediated ERK activation. These data have established a physical and functional association between TRIP6 and RIP2, and suggest that RIP2s involvement in multiple NF-κB and ERK activation pathways is mediated through TRIP6.

Crystal structure of ISG54 reveals a novel RNA binding structure and potential functional mechanisms

Interferon-stimulated gene 56 (ISG56) family members play important roles in blocking viral replication and regulating cellular functions, however, their underlying molecular mechanisms are largely unclear. Here, we present the crystal structure of ISG54, an ISG56 family protein with a novel RNA-binding structure. The structure shows that ISG54 monomers have 9 tetratricopeptide repeat-like motifs and associate to form domain-swapped dimers. The C-terminal part folds into a super-helical structure and has an extensively positively-charged nucleotide-binding channel on its inner surface. EMSA results show that ISG54 binds specifically to some RNAs, such as adenylate uridylate (AU)-rich RNAs, with or without 5′ triphosphorylation. Mutagenesis and functional studies show that this RNA-binding ability is important to its antiviral activity. Our results suggest a new mechanism underlying the antiviral activity of this interferon-inducible gene 56 family member.

NIK is a component of the EGF/heregulin receptor signaling complexes

Nuclear factor κB-inducing kinase (NIK) is a member of the MAP kinase kinase kinase family that was first identified as a component of the TNF-R1-induced NF-κB activation pathway (TNF, tumor necrosis factor; nuclear factor kappaB, NF-κB). Gene knockout study, however, suggests that NIK is dispensable for TNF-R1- but required for lymphotoxin-β receptor-induced NF-κB activation. A NIK kinase inactive mutant is a potent inhibitor of NF-κB activation triggered by various stimuli, suggesting that NIK is involved in a broad range of NF-κB activation pathways. To unambiguously identify signaling pathways that NIK participates in, we screened antibody arrays for proteins that are associated with NIK. This effort identified ErbB4, one of the EGF/heregulin receptors, and Grb7, an adapter protein associated with ErbB4 (ErbB, epidermal growth factor receptor family protein; EGF, epidermal growth factor; Grb, growth factor receptor bound). Coimmunoprecipitation experiments demonstrated that NIK interacted with Grb7, as well as Grb10 and Grb14, but not Grb2. Domain mapping experiments indicated that the central GM domain of Grb7 was sufficient for its interaction with NIK. Coimmunoprecipitation experiments also indicated that Grb7 and NIK could be simultaneously recruited into signaling complexes of all known EGF/heregulin receptors, including EGFR, ErbB2, ErbB3, and ErbB4. In reporter gene assays, NIK could potentiate Grb7, ErbB2/ErbB4, and EGF-induced NF-κB activation. A NIK kinase inactive mutant could block ErbB2/ErbB4 and EGF-induced NF-κB activation. Moreover, EGF/heregulin receptors activated NF-κB in wild-type, but not NIK−/− embryonic fibroblasts. Our findings suggest that NIK is a component of the EGF/heregulin receptor signaling complexes and involved in NF-κB activation triggered by these receptors.