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Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is a RNA helicase required for the translation of 5’ structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5’ structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity. [ABSTRACT FROM AUTHOR]

Abstract: The RIG‐I‐like receptors (RLRs) are critical for protection against RNA virus infection, and their activities must be stringently controlled to maintain immune homeostasis. Here, we report that leucine‐rich repeat containing protein 25 (LRRC25) is a key negative regulator of RLR‐mediated type I interferon (IFN) signaling. Upon RNA virus infection, LRRC25 specifically binds to ISG15‐associated RIG‐I to promote interaction between RIG‐I and the autophagic cargo receptor p62 and to mediate RIG‐I degradation via selective autophagy. Depletion of either LRRC25 or ISG15 abrogates RIG‐I‐p62 interaction as well as the autophagic degradation of RIG‐I. Collectively, our findings identify a previously unrecognized role of LRRC25 in type I IFN signaling activation by which LRRC25 acts as a secondary receptor to assist RIG‐I delivery to autophagosomes for degradation in a p62‐dependent manner. [ABSTRACT FROM AUTHOR]

Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity. [ABSTRACT FROM AUTHOR]

Nod-like receptors (NLRs) are intracellular sensors that respond to a variety of pathogen and intracellular danger signals to induce innate immune responses. NLRC5 has recently been identified to be an important regulator of NF-κB, type I interferon (IFN) and inflammasome signaling pathways, but the in vivo function and mechanisms of NLRC5 remain to be defined. Here, we describe the generation and characterization of NLRC5 knockout mice. We show that induction of NLRC5 expression by Toll-like receptor (TLR) ligand or cytokine stimulation requires the signal transducers and activators of transcription (Stat)1-mediated signaling pathway. NLRC5 ablation reduces MHC class I expression, and enhances IKK and IRF3 phosphorylation in response to TLR stimulation or viral infection. Consistent with these observations, we found that NLRC5 deficiency enhanced IL-6 and IFN-β production in mouse embryonic fibroblasts (MEFs), peritoneal macrophages and bone marrow-derived macrophages (BMMs), but not bone marrow-derived dendritic cells (BMDCs) after LPS stimulation or vesicular stomatitis virus (VSV) infection. Furthermore, we found that NLRC5-deficient mice produced higher amounts of IL-6 and IFN-β in the sera when they were challenged with LPS or infected with VSV. Taken together, these results provide in vivo evidence that NLRC5 plays critical roles in MHC class I expression, innate immune signaling and antiviral innate immune responses, thus serving as an important target for modulating innate immune signaling and regulation. [ABSTRACT FROM AUTHOR]

Stringent control of the type I interferon signaling pathway is important for maintaining host immune responses and homeostasis, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. Here we report that the pattern-recognition receptor NLRP4 regulated the activation of type I interferon mediated by double-stranded RNA or DNA by targeting the kinase TBK1 for degradation. NLRP4 recruited the E3 ubiquitin ligase DTX4 to TBK1 for Lys48 (K48)-linked polyubiquitination at Lys670, which led to degradation of TBK1. Knockdown of either DTX4 or NLRP4 abrogated K48-linked ubiquitination and degradation of TBK1 and enhanced the phosphorylation of TBK1 and the transcription factor IRF3. Our results identify a previously unrecognized role for NLRP4 in the regulation of type I interferon signaling and provide molecular insight into the mechanisms by which NLRP4-DTX4 targets TBK1 for degradation. [ABSTRACT FROM AUTHOR]

Summary Type I interferon (IFN) is critical for controlling pathogen infection; however, its regulatory mechanisms in plasmacytoid cells (pDCs) still remain unclear. Here, we have shown that nucleic acid sensors cGAS-, STING-, MDA5-, MAVS-, or transcription factor IRF3-deficient mice produced high amounts of type I IFN-α and IFN-β (IFN-α/β) in the serum and were resistant to lethal plasmodium yoelii YM infection. Robust IFN-α/β production was abolished when gene encoding nucleic acid sensor TLR7, signaling adaptor MyD88, or transcription factor IRF7 was ablated or pDCs were depleted. Further, we identified SOCS1 as a key negative regulator to inhibit MyD88-dependent type I IFN signaling in pDCs. Finally, we have demonstrated that pDCs, cDCs, and macrophages were required for generating IFN-α/β-induced subsequent protective immunity. Thus, our findings have identified a critical regulatory mechanism of type I IFN signaling in pDCs and stage-specific function of immune cells in generating potent immunity against lethal YM infection. [ABSTRACT FROM AUTHOR]

Summary TBK1 is a component of the type I interferon (IFN) signaling pathway, yet the mechanisms controlling its activity and degradation remain poorly understood. Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo. USP38 specifically cleaves K33-linked poly-ubiquitin chains from TBK1 at Lys670, and it allows for subsequent K48-linked ubiquitination at the same position mediated by DTX4 and TRIP. Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signaling. Our findings identify an essential role for USP38 in negatively regulating type I IFN signaling, and they provide insights into the mechanisms by which USP38 regulates TBK1 ubiquitination through the NLRP4 signalosome. [ABSTRACT FROM AUTHOR]

Highlights: [•] TgMAPK1 is a Toxoplasma gondii mitogen-activated protein kinase (MAPK). [•] It affects parasite proliferation in an IFN-γ, iNOS and MKK3-dependent manner. [•] Parasite tissue burden is regulated by TgMAPK1 expression in iNOS-replete tissues. [•] Thus TgMAPK1 ultimately affects virulence by manipulating host IFN-γ-mediated iNOS. [ABSTRACT FROM AUTHOR]

Summary: Stringent control of the NF-κB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-κB-dependent responses by interacting with IKKα and IKKβ and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-κB and its responsive genes, TNF-α and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-κB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity. [ABSTRACT FROM AUTHOR]