Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.     

Sensing of viral nucleic acids by RIG-I: from translocation to translation

The innate immune system is a first layer of defense against infection by pathogens. It responds to pathogens by activating host defense mechanisms via interferon and inflammatory cytokine expression. Pathogen associated molecular patterns (PAMPs) are sensed by specific pattern recognition receptors. Among those, the ATP dependent helicase related RIG-I like receptors RIG-I, MDA5 and LGP2 sense the presence of viral RNA in the cytoplasm of host cells. While the precise PAMPs and functions of MDA5 or LGP2 are still unclear, RIG-I senses predominantly viral RNA containing a 5′-triphosphate along with dsRNA regions. Here we review our current knowledge of how these PAMPs are sensed and integrated by RIG-I, and how RIG-I's innate immune function can be used in translational medical approaches.     

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain

Background  

IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region.     

Retinoic acid inducible gene-I,more than a virus sensor

Retinoic acid inducible gene-I (RIG-I) is a caspase recruitment domain (CARD) containing protein that acts as an intracellular RNA receptor and senses virus infection. After binding to double stranded RNA (dsRNA) or 5′-triphosphate single stranded RNA (ssRNA), RIG-I transforms into an open conformation, translocates onto mitochondria, and interacts with the downstream adaptor mitochondrial antiviral signaling (MAVS) to induce the production of type I interferon and inflammatory factors via IRF3/7 and NF-κB pathways, respectively. Recently, accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events, such as inflammation and inflammation related diseases, cell proliferation, apoptosis and even senescence. Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.     

The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response

RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response.     

Activation of RIG-I-like receptor signal transduction

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed.     

Activation of RIG-I-like receptor signal transduction

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed.     

Positive Evolutionary Selection On the RIG-I-Like Receptor Genes in Mammals

The mammalian RIG-I-like receptors, RIG-I, MDA5 and LGP2, are a family of DExD/H box RNA helicases responsible for the cytoplasmic detection of viral RNA. These receptors detect a variety of RNA viruses, or DNA viruses that express unusual RNA species, many of which are responsible for a great number of severe and lethal diseases. Host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. Using six codon-based Maximum Likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. The highest number of positively selected codons was detected in MDA5, but a great percentage of these codons were located outside of the currently defined protein domains for MDA5, which likely reflects the imposition of both functional and structural constraints. Additionally, our results support LGP2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. On the other hand, the preponderance of positively selected codons for RIG-I were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this RNA helicase. Furthermore, the RIG-I repressor domain, the region responsible for recognizing and binding to its RNA substrates, exhibited the strongest evidence of selective pressures. Branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. In conclusion, by looking for evidence of positive evolutionary selection on mammalian RIG-I-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the RIG-I gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens.     

Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I

Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.     

DDX60, a DEXD/H box helicase, is a novel antiviral factor promoting RIG-I-like receptor-mediated signaling

The cytoplasmic viral RNA sensors RIG-I and MDA5 are important for the production of type I interferon and other inflammatory cytokines. DDX60 is an uncharacterized DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, a cofactor of RNA exosome, which is a protein complex required for the integrity of cytoplasmic RNA. Expression of DDX60 increases after viral infection, and the protein localizes at the cytoplasmic region. After viral infection, the DDX60 protein binds to endogenous RIG-I protein. The protein also binds to MDA5 and LGP2 but not to the downstream factors IPS-1 and IκB kinase ε (IKK-ε). Knockdown analysis shows that DDX60 is required for RIG-I- or MDA5-dependent type I interferon and interferon-inducible gene expression in response to viral infection. However, DDX60 is dispensable for TLR3-mediated signaling. Purified DDX60 helicase domains possess the activity to bind to viral RNA and DNA. Expression of DDX60 promotes the binding of RIG-I to double-stranded RNA. Taken together, our analyses indicate that DDX60 is a novel antiviral helicase promoting RIG-I-like receptor-mediated signaling.     

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.     

Induction of apoptosis in colon cancer cells by a novel topoisomerase I inhibitor TopIn

In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735–925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747–801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a “closed” structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.     

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.     

Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses

A DExD/H protein, RIG-I, is critical in innate antiviral responses by sensing viral RNA. Here we show that RIG-I recognizes two distinct viral RNA patterns: double-stranded (ds) and 5'ppp single-stranded (ss) RNA. The binding of RIG-I with dsRNA or 5'ppp ssRNA in the presence of ATP produces a common structure, as suggested by protease digestion. Further analyses demonstrated that the C-terminal domain of RIG-I (CTD) recognizes these RNA patterns and CTD coincides with the autorepression domain. Structural analysis of CTD by NMR spectroscopy in conjunction with mutagenesis revealed that the basic surface of CTD with a characteristic cleft interacts with RIG-I ligands. Our results suggest that the bipartite structure of CTD regulates RIG-I on encountering viral RNA patterns.     

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.     

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.