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

Abstract

Viral infection leads to activation of the transcription factors interferon regulatory factor-3 and NF-B, 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-␤ induction. Overexpression of DAK also inhibited cytoplasmic dsRNA and SeV-induced activation of the IFN-␤ promoter, whereas knockdown of endogenous DAK by RNAi activated the IFN-␤ promoter, and increased cytoplasmic dsRNA-or SeV-triggered activation of the IFN-␤ promoter. In addition, overexpression of DAK inhibited MDA5-but not RIG-Imediated 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. The innate immune system has developed at least two distinct mechanisms for the recognition of viral RNAs (2, 3, 9). One is mediated by Toll-like receptor 3 (TLR3), which recognizes viral dsRNA released by infected cells (10). Engagement of TLR3 by dsRNA triggers TRIF-mediated signaling pathways, leading to IRF-3 and NF-B activation (11-16). The second mechanism involves two RNA helicase proteins, RIG-I and MDA5, which function as cytoplasmic viral RNA sensors (17, 18). Both RIG-I and MDA5 contain two CARD modules at their N terminus and a DexD/H-box helicase domain at their C terminus. The helicase domains of RIG-I and MDA5 serve as intracellular viral RNA receptors, whereas the CARD modules are responsible for transmitting signals to downstream CARD-containing adaptor VISA/ MAVS/IPS-1/Cardif, which in turn activates TAK1-IKK␤ and TBK1/IKK kinases, leading to activation of NF-B and IRF-3 and induction of type I IFNs (19)(20)(21)(22)(23)(24).Although MDA5 and RIG-I share a similar structural architecture, and their signaling pathways converge at the adaptor level, gene-knockout studies indicate that the two proteins are required for responding to distinct species of RNA viruses. RIG-I responds to in vitro-transcribed dsRNA, vesicular stomatitis virus (VSV), Newcastle disease virus, and influenza virus in mice. In contrast, MDA5 recognizes poly(I:C) and is essential for the antiviral response to the picornavirus encephalomyocarditis virus (25,26). Recently, it was demonstrated that RIG-I, but not MDA5, recognizes single-strand RNA bearing 5Ј phosphate (27,28). In addition, it has been shown that signaling mediated by RIG-I-and MDA5-mediated signaling is differentially regulated. For example, the V protein of paramyxovirus appears to selectively target MDA5-but not RIG-I-mediated IFN response (29)(30)(31).In the present study, we identified D...

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

“…However, uncontrolled excessive production of type I IFNs significantly contributes to chronic inflammation and autoimmune diseases, underscoring the importance of negative regulation of type I IFN [35][36][37][38]. The host innate immune system has developed distinct strategies to ensure proper production of type I IFNs following viral infection [ [18][19][20][21][22][23][24][25][26]. In the present work, we identified the E3 ubiquitin ligase RBCK1 as a negative feedback regulator of the cellular antiviral response.…”

“…The cellular protein DAK is associated with MDA5, and specifically inhibits MDA5-mediated, but not RIG-I-mediated, signaling [22]. The coiled-coil protein SIKE is associated with TBK1/inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε) and keeps these kinases inactive under physiological conditions [23].…”

Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-β induction by disrupting the VISA-associated complex

“…Tumor-necrosis factor-a and IL-1b (R&D Systems, Minneapolis, MN, USA), mouse monoclonal antibodies against Flag and HA (Sigma, St Louis, MO, USA) and rabbit polyclonal antibodies against IRF3 and MDA5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were purchased from the indicated manufacturers; SeV and VSV were previously described. 7,36,37 Plasmids A plasmid carrying a greater-than-unit-length (129%) HBV genome (payw1.2; subtype ayw) and the same plasmid with a stop codon replacing the coding sequence for amino acid 7 of HBx (payw*7) were previously described. 34,35 The vector pEGFP-C1 was a gift provided by Yan Wu.…”

“…Luciferase reporter plasmids for NF-kB, ISRE and the IFN-b promoters, as well as mammalian expression plasmids for HA-or Flag-tagged RIG-I, RIG-I-N, MDA5, MDA5-N, MDA5-C, VISA, TBK1, MITA and IRF3, were previously described. 7,11,36,37 Expression plasmids for human HA-, Flag-or GFP-tagged HBx and RFP-tagged RIG-I, MDA5 and VISA were constructed by standard molecular biology techniques.…”

“…Coimmunoprecipitation, immunoblot analysis, native polyacrylamide gel electrophoresis (PAGE) and VSV plaque assays 7,36,37 Coimmunoprecipitation and western blot analysis. For transient transfection and coimmunoprecipitation experiments, 293 cells (,1310 6 ) plated on 10-cm dishes were transfected with indicated plasmids for 24 h. The transfected cells were lysed in 800 ml of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 ml/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride).…”