The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

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.     

RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.     

Negative Regulation of Melanoma Differentiation-associated Gene 5 (MDA5)-dependent Antiviral Innate Immune Responses by Arf-like Protein 5B

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon β promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5.     

Structure and function of LGP2, a DEX(D/H) helicase that regulates the innate immunity response

RNA recognition receptors are important for detection of and response to viral infections. RIG-I and MDA5 are cytoplasmic DEX(D/H) helicase proteins that can induce signaling in response to RNA ligands, including those from viral infections. LGP2, a homolog of RIG-I and MDA5 without the caspase recruitment domain required for signaling, plays an important role in modulating signaling by MDA5 and RIG-I, presumably through heterocomplex formation and/or by serving as a sink for RNAs. Here we demonstrate that LGP2 can be coexpressed with RIG-I to inhibit activation of the NF-kappaB reporter expression and that LGP2 protein produced in insect cells can bind both single- and double-stranded RNA (dsRNA), with higher affinity and cooperativity for dsRNA. Electron microscopy and image reconstruction were used to determine the shape of the LGP2 monomer in the absence of dsRNA and of the dimer complexed to a 27-bp dsRNA. LGP2 has striking structural similarity to the helicase domain of the superfamily 2 DNA helicase, Hef.     

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.     

Identification of Loss of Function Mutations in Human Genes Encoding RIG-I and MDA5: IMPLICATIONS FOR RESISTANCE TO TYPE I DIABETES*

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential for detecting viral RNA and triggering antiviral responses, including production of type I interferon. We analyzed the phenotype of non-synonymous mutants of human RIG-I and MDA5 reported in databases by functional complementation in cell cultures. Of seven missense mutations of RIG-I, S183I, which occurs within the second caspase recruitment domain repeat, inactivated this domain and conferred a dominant inhibitory function. Of 10 mutants of MDA5, two exhibited loss of function. A nonsense mutation, E627*, resulted in deletion of the C-terminal region and double-stranded RNA (dsRNA) binding activity. Another loss of function mutation, I923V, which occurs within the C-terminal domain, did not affect dsRNA binding activity, suggesting a novel and essential role for this residue in the signaling. Remarkably, these mutations are implicated in resistance to type I diabetes. However, the A946T mutation of MDA5, which has been implicated in type I diabetes by previous genetic analyses, affected neither dsRNA binding nor IFN gene activation. These results provide new insights into the structure-function relationship of RIG-I-like receptors as well as into human RIG-I-like receptor polymorphisms, antiviral innate immunity, and autoimmune diseases.Innate and adaptive immune systems constitute the defense against infections by pathogens. Immediately after an infection occurs, various cells in the body sense the virus and initiate antiviral responses in which type I IFN² plays a critical role, both in viral inhibition and in the subsequent adaptive immune response (1). The production of IFN is initiated when sensor molecules such as Toll-like receptors (TLRs) and RLRs detect virus-associated molecules. TLRs detect pathogen-associated molecular patterns (PAMPs) at the cell surface or in the endosome in immune cells such as dendritic cells and macrophages (2). RLRs sense viral RNA in the cytoplasm of most cell types and induce antiviral responses, including the activation of IFN genes (3). RLRs include RIG-I, MDA5, and laboratory of genetics and physiology 2 (LGP2).It is proposed that RLRs sense and activate antiviral signals through the coordination of their functional domains (4). The N-terminal region of RIG-I and MDA5 is characterized by two repeats of CARD and functions as an activation domain (3). This domain is responsible for the transduction of signals downstream to IFN-β promoter stimulator 1 (IPS-1) (also known as MAVS, VISA, and Cardif). The primary sequence of the CTD, consisting of ∼140 amino acids, is conserved among RLRs. The CTD of RIG-I functions as a viral RNA-sensing domain as revealed by biochemical and structural analyses (5, 6). Both dsRNA and 5′-ppp-ssRNA, which are generated in the cytoplasm of virus-infected cells, are recognized by a basic cleft structure of RIG-I CTD. In addition to its RNA recognition function, the CTD of RIG-I and LGP2 functions as a repression domain through interaction with the activation domain. The repression domain is responsible for keeping RIG-I inactive in non-stimulated cells (3, 7). The helicase domain, with DEXD/H box-containing RNA helicase motifs, is the largest domain found in RLRs. Once dsRNA or 5′-ppp-ssRNA is recognized by the CTD, the helicase domain causes structural changes to release the activation domain. ATP binding and/or its hydrolysis is essential for the conformational change because Walker''s ATP-binding site within the helicase domain is essential for signaling by RIG-I and MDA5.Analyses of knock-out mice have revealed that RIG-I and MDA5 recognize distinct RNA viruses (8, 9). Picornaviruses are detected by MDA5, but many other viruses such as influenza A, Sendai, vesicular stomatitis, and Japanese encephalitis are detected by RIG-I. The difference is based on the distinct non-self RNA patterns generated by viruses, as demonstrated by the finding that RIG-I is selectively activated by dsRNA or 5′-ppp ssRNA, whereas MDA5 is activated by long dsRNA (10–12).Single nucleotide polymorphisms (SNPs) of the human RIG-I and MDA5 genes including several non-synonymous SNPs (nsSNPs), which potentially alter the function of the proteins encoded, are reported in databases. In this report, we investigated the functions of nsSNPs of RIG-I and MDA5 by functional complementation using respective knock-out cells. We identified loss of function mutations of RIG-I and MDA5. Notably, two MDA5 mutations, E627* and I923V, recently reported to have a strong association with resistance to T1D (13), were severely inactive. The results suggest a novel molecular mechanism for the activation of RLRs and will contribute to our understanding of the functional effects of RLR polymorphisms and the critical relationship between RLR nsSNPs and diseases.     

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.     

Uridine Composition of the Poly-U/UC Tract of HCV RNA Defines Non-Self Recognition by RIG-I

Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5′-triphosphate (5′-ppp) on the viral RNA in association with a 3′ poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine “core” of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5′-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5′-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5′-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.     

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.     

Antiviral RNA recognition and assembly by RLR family innate immune sensors

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1], [2], [3], [4]. One important group of cytosolic RNA sensors known as the RIG-I-like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5], [6].     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

Regulation of RLR-mediated innate immune signaling--it is all about keeping the balance

The current view of cytoplasmic RNA-mediated innate immune signaling involves the differential activation of the RNA helicases retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology-2 (LGP2) by distinct RNA viruses. RIG-I, MDA5 and LGP2 form the RIG-I like receptor family (RLR). Since the initial characterization of the RLRs rapid progress has been made in the understanding of the molecular mechanisms that upon virus infection lead to the activation of downstream signaling cascades and the subsequent induction of type I interferon (IFN) and proinflammatory cytokines by these receptors. However, antiviral responses must be tightly regulated in order to prevent uncontrolled production of type I IFN that might have deleterious effects on the host. Exploring the structural and molecular mechanisms that underlie RLR signaling thus was accompanied by the discovery of how RLR-dependent antiviral responses are modulated. This article summarizes the current understanding of endogenous regulation in RLR signaling by various intrinsic molecules that exert their regulatory function in both the steady state or upon viral infection by targeting multiple steps of the signaling cascade.     

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