细胞质内模式识别受体及其抗病毒免疫研究

先天性免疫监视机制的核心是通过模式识别受体(pattern recognition receptors,PRRs)识别病毒分子诱导抗病毒防御,使宿主免受感染。PRRs表达在不同类型细胞的不同细胞区室,包括细胞膜、内体膜、溶酶体膜和胞质。病毒进入细胞区室后将被一个或多个模式识别受体所识别并激活机体的免疫反应。主要对细胞质内模式识别受体视黄酸诱导基因I样受体(retinoic acid-inducible gene I(RIG-I)-like receptors,RLRs)、核苷酸结合寡聚化结构域样受体(nucleotide-binding oligomerization domain(NOD)-like receptors,NLRs)、DEXDc螺旋酶受体(DLRs)及最近发现的DNA模式识别分子——DAI(DNA-dependent activator of interferonregulatory factors)识别病毒核酸并诱导I型干扰素产生的分子机制作一综述。     

A novel selective autophagy receptor,CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection

Autophagy is a conserved process that delivers cytosolic substances to the lysosome for degradation, but its direct role in the regulation of antiviral innate immunity remains poorly understood. Here, through high-throughput screening, we discovered that CCDC50 functions as a previously unknown autophagy receptor that negatively regulates the type I interferon (IFN) signaling pathway initiated by RIG-I-like receptors (RLRs), the sensors for RNA viruses. The expression of CCDC50 is enhanced by viral infection, and CCDC50 specifically recognizes K63-polyubiquitinated RLRs, thus delivering the activated RIG-I/MDA5 for autophagic degradation. The association of CCDC50 with phagophore membrane protein LC3 is confirmed by crystal structure analysis. In contrast to other known autophagic cargo receptors that associate with either the LIR-docking site (LDS) or the UIM-docking site (UDS) of LC3, CCDC50 can bind to both LDS and UDS, representing a new type of cargo receptor. In mouse models with RNA virus infection, CCDC50 deficiency reduces the autophagic degradation of RIG-I/MDA5 and promotes type I IFN responses, resulting in enhanced viral resistance and improved survival rates. These results reveal a new link between autophagy and antiviral innate immune responses and provide additional insights into the regulatory mechanisms of RLR-mediated antiviral signaling.Subject terms: Macroautophagy, Ubiquitylation, RIG-I-like receptors     

RLRs介导的抗鲤春病毒血症病毒作用研究进展

鲤春病毒血症病毒(SVCV)是水生动物病毒中重要的病原体,常引起鲤科鱼类疾病暴发。近些年研究发现,维甲酸诱导基因I样受体家族(RLRs)信号通路在SVCV免疫过程中起到重要的作用。主要功能是在识别病原体相关模式,激活下游信号分子,诱导天然免疫的产生,以及控制病毒的早期复制。当病毒进入机体时会形成病毒-RLRs-IFN互联反馈回路,RLRs相关基因识别SVCV的RNA,最终引起Ⅰ型干扰素(IFN-I)表达量升高,并且RLRs族内成员相互作用增强抗病毒作用。RLRs不仅可以活化天然免疫信号通路,还可增强适应性免疫效应,在控制病毒感染过程中发挥重要作用。介绍RLRs家族,RLRs抗病毒信号调控因子,干扰素诱导的鱼类Mx (myxovirus resistant)蛋白对鲤春病毒血症病毒的抑制作用。     

Mechanisms of RIG-I-Like Receptor Activation and Manipulation by Viral Pathogens

RIG-I-like receptors (RLRs) play important roles in the host defense to numerous viral pathogens. Since they were discovered, much light has been shed on the molecular details of how these cytoplasmic viral RNA receptors sense viral infection and orchestrate antiviral innate immunity. Intriguingly, in addition to viral RNA binding, a series of posttranslational modifications (PTMs) is required for the rapid activation of RLRs and, inversely, for the prevention of aberrant innate immune signaling. Recent discoveries have shown that viruses manipulate the PTMs of RLRs to escape innate immune detection. This article highlights some of these recent findings in this fast-evolving field.     

Interplay between Innate Immunity and Negative-Strand RNA Viruses: towards a Rational Model

Summary: The discovery of a new class of cytosolic receptors recognizing viral RNA, called the RIG-like receptors (RLRs), has revolutionized our understanding of the interplay between viruses and host cells. A tremendous amount of work has been accumulating to decipher the RNA moieties required for an RLR agonist, the signal transduction pathway leading to activation of the innate immunity orchestrated by type I interferon (IFN), the cellular and viral regulators of this pathway, and the viral inhibitors of the innate immune response. Previous reviews have focused on the RLR signaling pathway and on the negative regulation of the interferon response by viral proteins. The focus of this review is to put this knowledge in the context of the virus replication cycle within a cell. Likewise, there has been an expansion of knowledge about the role of innate immunity in the pathophysiology of viral infection. As a consequence, some discrepancies have arisen between the current models of cell-intrinsic innate immunity and current knowledge of virus biology. This holds particularly true for the nonsegmented negative-strand viruses (Mononegavirales), which paradoxically have been largely used to build presently available models. The aim of this review is to bridge the gap between the virology and innate immunity to favor the rational building of a relevant model(s) describing the interplay between Mononegavirales and the innate immune system.     

Structural insights into RNA recognition and activation of RIG-I-like receptors

RIG-I like receptors (RLR) that recognize non-self RNA play critical roles in activating host innate immune pathways in response to viral infections. Not surprisingly, RLRs and their associated signaling networks are also targeted by numerous antagonists that facilitate viral pathogenesis. Although the role of RLRs in orchestrating antiviral signaling has been recognized for some time, our knowledge of the complex regulatory mechanisms that control signaling through these key molecules is incomplete. A series of recent structural studies shed new light into the structural basis for dsRNA recognition and activation of RLRs. Collectively, these studies suggest that the repression of RLRs is facilitated by a cis element that makes multiple contacts with domains within the helicase and that RNA binding initiated by the C-terminal RNA binding domain is important for ATP hydrolysis and release of the CARD domain containing signaling module from the repressed conformation. These studies also highlight potential differences between RIG-I and MDA5, two RLR members. Together with previous studies, these new results bring us a step closer to uncovering the complex regulatory process of a key protein that protects host cells from invading pathogens.     

Emerging complexity and new roles for the RIG-I-like receptors in innate antiviral immunity

Innate immunity is critical for the control of virus infection and operates to restrict viral susceptibility and direct antiviral immunity for protection from acute or chronic viral-associated diseases including cancer. RIG-I like receptors (RLRs) are cytosolic RNA helicases that function as pathogen recognition receptors to detect RNA pathogen associated molecular patterns (PAMPs) of virus infection. The RLRs include RIG-I, MDA5, and LGP2. They function to recognize and bind to PAMP motifs within viral RNA in a process that directs the RLR to trigger downstream signaling cascades that induce innate immunity that controls viral replication and spread. Products of RLR signaling also serve to modulate the adaptive immune response to infection. Recent studies have additionally connected RLRs to signaling cascades that impart inflammatory and apoptotic responses to virus infection. Viral evasion of RLR signaling supports viral outgrowth and pathogenesis, including the onset of viral-associated cancer.     

USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

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.     

Critical Role of an Antiviral Stress Granule Containing RIG-I and PKR in Viral Detection and Innate Immunity

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.     

Regulation of RIG-I-like receptor signaling by host and viral proteins

Vertebrate innate immunity is characterized by an effective immune surveillance apparatus, evolved to sense foreign structures, such as proteins or nucleic acids of invading microbes. RIG-I-like receptors (RLRs) are key sensors of viral RNA species in the host cell cytoplasm. Activation of RLRs in response to viral RNA triggers an antiviral defense program through the production of hundreds of antiviral effector proteins including cytokines, chemokines, and host restriction factors that directly interfere with distinct steps in the virus life cycle. To avoid premature or abnormal antiviral and proinflammatory responses, which could have harmful consequences for the host, the signaling activities of RLRs and their common adaptor molecule, MAVS, are delicately controlled by cell-intrinsic regulatory mechanisms. Furthermore, viruses have evolved multiple strategies to modulate RLR-MAVS signal transduction to escape from immune surveillance. Here, we summarize recent progress in our understanding of the regulation of RLR signaling through host factors and viral antagonistic proteins.     

TBK1-associated Protein in Endolysosomes (TAPE)/CC2D1A Is a Key Regulator Linking RIG-I-like Receptors to Antiviral Immunity

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key RNA viral sensors for triggering antiviral immunity. The underlying mechanisms for RLRs to trigger antiviral immunity have yet to be explored. Here we report the identification of TAPE (TBK1-associated protein in endolysosomes) as a novel regulator of the RLR pathways. TAPE functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-β promoter. TAPE knockdown impairs IFN-β activation induced by RLRs but not IPS-1. TAPE-deficient cells are defective in cytokine production upon RLR ligand stimulation. During RNA virus infection, TAPE knockdown or deficiency diminishes cytokine production and antiviral responses. Our data demonstrate a critical role for TAPE in linking RLRs to antiviral immunity.     

Genomic analysis and functional characterization of immune genes from the RIG-I- and MAVS-mediated antiviral signaling pathway in lamprey

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are well-known viral RNA sensors in the cytoplasm. RIG-I-mediated antiviral signals are activated by interacting with the adapter protein mitochondrial antiviral signaling (MAVS), which triggers interferon (IFN) responses via a signaling cascade. Although the complete RIG-I receptor signaling pathway has been traced back to teleosts, definitive evidence of its presence in lampreys is lacking. Here, we identified 13 pivotal molecules in the RIG-I signaling pathway in lamprey, and demonstrated that the original RIG-I/MAVS signaling pathway was activated and mediated the expression of unique immunity factors such as RRP4, to inhibit viral proliferation after viral infection in vivo and in vitro. This study confirmed the conservation of the RIG-I pathway, and the uniqueness of the RRP4 effector molecule in lamprey, and further clarified the evolutionary process of the RIG-I antiviral signaling pathway, providing evidence on the origins of innate antiviral immunity in vertebrates.     

RIG-I family RNA helicases: cytoplasmic sensor for antiviral innate immunity

Viral infection is detected by cellular sensors as foreign nucleic acid and initiates innate antiviral responses, including the activation of type I interferon (IFN) and proinflammatory cytokines. Recent advances in cytoplasmic virus sensors highlight their essential role in the induction of innate immunity. Moreover, it is intriguing to understand how they can discriminate innate RNA from viral foreign RNA. In this mini-review, we focus on these cytoplasmic virus sensors, termed retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), and discuss their function in the innate immune system.     

Mitochondrial dynamics regulate the RIG‐I‐like receptor antiviral pathway

The intracellular retinoic acid‐inducible gene I‐like receptors (RLRs) sense viral ribonucleic acid and signal through the mitochondrial protein mitochondrial antiviral signalling (MAVS) to trigger the production of type I interferons and proinflammatory cytokines. In this study, we report that RLR activation promotes elongation of the mitochondrial network. Mimicking this elongation enhances signalling downstream from MAVS and favours the binding of MAVS to stimulator of interferon genes, an endoplasmic reticulum (ER) protein involved in the RLR pathway. By contrast, enforced mitochondrial fragmentation dampens signalling and reduces the association between both proteins. Our finding that MAVS is associated with a pool of mitofusin 1, a protein of the mitochondrial fusion machinery, suggests that MAVS is capable of regulating mitochondrial dynamics to facilitate the mitochondria–ER association required for signal transduction. Importantly, we observed that viral mitochondria‐localized inhibitor of apoptosis, a cytomegalovirus (CMV) antiapoptotic protein that promotes mitochondrial fragmentation, inhibits signalling downstream from MAVS, suggesting a possible new immune modulation strategy of the CMV.     

Autoimmunity caused by constitutive activation of cytoplasmic viral RNA sensors

RIG-I-like receptors (RLRs) are well-known viral sensors that trigger the antiviral interferon (IFN) response by recognizing the non-self signatures of viral RNAs. The proper induction of the IFN response is known to play a crucial role in protecting against viral infections, whereas aberrant activation can lead to autoimmune disorders. We herein provided an overview of the antiviral IFN response and autoimmunity, with a focus on recent studies describing autoimmunity caused by mutations in the cytoplasmic viral RNA sensor, melanoma differentiation-associated gene 5 (MDA5).     

Autophagic control of RLR signaling

Innate immunity to viral infection is initiated within the infected cells through the recognition of unique viral signatures by pattern recognition receptors (PRRs) that mediate the induction of potent antiviral factor, type I interferons (IFNs). Infection with RNA viruses is recognized by the members of the retinoic acid inducible gene I (RIG-I)-like receptor (RLR) family in the cytosol. Our recent study demonstrates that IFN production in response to RNA viral ligands is increased in the absence of autophagy. The process of autophagy functions as an internal clean-up crew within the cell, shuttling damaged cellular organelles and long-lived proteins to the lysosomes for degradation. Our data show that the absence of autophagy leads to the amplification of RLR signaling in two ways. First, in the absence of autophagy, mitochondria accumulate within the cell leading to the build up of mitochondrial associated protein, IPS-1, a key signaling protein for RLRs. Second, damaged mitochondria that are not degraded in the absence of autophagy provide a source of reactive oxygen species (ROS), which amplify RLR signaling in Atg5 knockout cells. Our study provides the first link between ROS and cytosolic signaling mediated by the RLRs, and suggests the importance of autophagy in the regulation of signaling emanating from mitochondria.     

Regulation of virus-triggered type I interferon signaling by cellular and viral proteins

Host pattern recognition receptors (PRRs) recognize invading viral pathogens and initiate a series of signaling cascades that lead to the expression of type I interferons (IFNs) and inflammatory cytokines. During the past decade, significant progresses have been made to characterize PRRs such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) and elucidate the molecular mechanisms of TLR- and RLR-mediated signaling. To avoid excessive and harmful immune effects caused by over-activation of these signaling pathways, host cells adopt a number of strategies to regulate them. In addition, invading viruses also employ a variety of mechanisms to inhibit the production of type I IFNs, thereby evading the supervision and clearance by the host. In this review, we briefly summarize the TLR- and RLR-mediated type I IFN signaling and then focus on the mechanisms by which host cellular and viral components regulate the expression of type I IFNs.     

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease.