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.     

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS*

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.     

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.     

Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5

RIG-I, MDA5 and LGP2 are cytosolic pattern recognition receptors detecting single-stranded or double-stranded RNA in virally infected cells. The activation of RIG-I or MDA5 stimulates the secretion of type I interferons that play key roles in antiviral immune responses. The C-terminal domains (CTD) of RIG-I and LGP2 are responsible for RNA binding; however, it is not clear how MDA5 binds RNA. To understand the structural basis of dsRNA recognition by MDA5, we have determined the 1.45 Å resolution structure of the C-terminal domain of human MDA5. The structure revealed a highly conserved fold similar to the structures of RIG-I and LGP2 CTDs. NMR titration of MDA5 CTD with dsRNA demonstrated that a positively charged surface is involved in dsRNA binding. Mutagenesis and RNA binding studies showed that electrostatic interactions play primary roles in dsRNA recognition by MDA5. Like RIG-I and LGP2, MDA5 CTD preferentially binds dsRNA with blunt ends, but does not associate with dsRNA with either 5′ or 3′ overhangs. Molecular modeling of MDA5 CTD/dsRNA complex suggests that MDA5 CTD may recognize the first turn of blunt-ended dsRNA in a similar manner as LGP2.     

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.     

The C-terminal regulatory domain is the RNA 5'-triphosphate sensor of RIG-I

The ATPase RIG-I senses viral RNAs that contain 5'-triphosphates in the cytoplasm. It initiates a signaling cascade that activates innate immune response by interferon and cytokine production, providing essential antiviral protection for the host. The mode of RNA 5'-triphosphate sensing by RIG-I remains elusive. We show that the C-terminal regulatory domain RD of RIG-I binds viral RNA in a 5'-triphosphate-dependent manner and activates the RIG-I ATPase by RNA-dependent dimerization. The crystal structure of RD reveals a zinc-binding domain that is structurally related to GDP/GTP exchange factors of Rab-like GTPases. The zinc coordination site is essential for RIG-I signaling and is also conserved in MDA5 and LGP2, suggesting related RD domains in all three enzymes. Structure-guided mutagenesis identifies a positively charged groove as likely 5'-triphosphate-binding site of RIG-I. This groove is distinct in MDA5 and LGP2, raising the possibility that RD confers ligand specificity.     

The RIG-I-like Receptor LGP2 Recognizes the Termini of Double-stranded RNA

The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading pathogens; it is the ubiquitous system of defense against microbial infections (1). Toll-like receptors (TLRs)³ and RIG-I (retinoic acid-inducible gene 1)-like receptors (RLRs) play key roles in innate immune response toward viral infection (2-5). Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the endosome following phagocytosis of the pathogens (6). RIG-I-like receptors RIG-I and MDA5 detect viral RNA from replicating viruses in infected cells (3, 7, 8). Stimulation of these receptors leads to the induction of type I interferons (IFNs) and other proinflammatory cytokines, conferring antiviral activity to the host cells and activating the acquired immune responses (4, 9).RIG-I discriminates between viral and host RNA through specific recognition of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp ssRNA) generated by viral RNA polymerases (10, 11). In addition, RIG-I also recognizes double-stranded RNA generated during RNA virus replication (7, 12). Transfection of cells with synthetic double-stranded RNA stimulates the activation of RIG-I (13, 14). Synthetic dsRNA mimics, such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5 when introduced into the cytoplasm of cells. Digestion of poly(I·C) with RNase III transforms poly(I·C) from a ligand for MDA5 into a ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I recognizes short dsRNA (15). Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of these receptors in antiviral immune responses and demonstrated that they sense different sets of RNA viruses (12, 16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N termini, a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory or repressor domain (CTD). The helicase domain and the CTD are responsible for viral RNA binding, whereas the CARDs are required for signaling (3, 8). The current model of RIG-I activation suggests that under resting conditions RIG-I is in a suppressed conformation, and viral RNA binding triggers a conformation change that leads to the exposure of the CARDs for the recruitment of the downstream protein IPS-1 (also known as MAVS, Cardif, or VISA) (14, 17). Limited proteolysis of the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD are involved in dsRNA and 5′ ppp ssRNA binding (14). The CTD of RIG-I overlaps with the C terminus of the previously identified repressor domain (18). The structures of RIG-I and LGP2 (laboratory of genetics and physiology 2) CTD in isolation have been determined by x-ray crystallography and NMR spectroscopy (14, 19, 20). A large, positively charged surface on RIG-I recognizes the 5′ triphosphate group of viral ssRNA (14, 19). RNA binding studies by titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that overlapping sets of residues on this charged surface are involved in RNA binding (14). Mutagenesis of several positively charged residues on this surface either reduces or disrupts RNA binding by RIG-I, and these mutations also affect the induction of IFN-β in vivo (14, 19). However, the exact nature of how the RLRs recognize viral RNA and how RNA binding activates these receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no signaling capability (21, 22). The expression of LGP2 is inducible by dsRNA or IFN treatment as well as virus infection (21). Overexpression of LGP2 inhibits Sendai virus and Newcastle disease virus signaling (21). When coexpressed with RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD with the CARD and the helicase domain of RIG-I (18). LGP2 could suppress RIG-I signaling by three possible ways (23): 1) binding RNA with high affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly with RIG-I to block the assembly of the signaling complex; and 3) competing with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a common binding site on IPS-1. To elucidate the structural basis of dsRNA recognition by the RLRs, we have crystallized human LGP2 CTD (residues 541-678) bound to an 8-bp double-stranded RNA and determined the structure of the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD binds to the termini of dsRNA. Mutagenesis and functional studies showed that dsRNA binding is likely not required for the inhibition of RIG-I signaling by LGP2.     

Origin and evolution of the RIG-I like RNA helicase gene family

Background  

The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria.     

鱼类和哺乳类RLR介导的抗病毒免疫反应的泛素化修饰调控

RLR[retinoic acid-inducible gene Ⅰ(RIG-Ⅰ)-like Receptors]是一类表达在胞浆中的模式识别受体, 在识别细胞质中经病毒复制产生的病毒RNA后, 启动一系列信号级联反应, 以诱导机体Ⅰ型干扰素及干扰素诱导的抗病毒基因的表达, 最后达到清除机体病毒感染的目的。由于在病毒感染时机体干扰素反应必须迅速启动, 当病毒清除后干扰素反应又需要立即恢复到正常本底水平, 因此RLR激活的信号转导途径受到了严格的调控, 其中就包括由E3泛素连接酶参与的泛素化修饰调控和由去泛素化酶参与的去泛素化修饰调控。自2003年成功鉴定出鱼类干扰素基因以来, 鱼类也被发现具有保守的RLR信号转导途径诱导干扰素抗病毒免疫反应, 该信号途径同样受到泛素化修饰的调控。文章总结了近年来泛素化修饰在哺乳类和鱼类RLR介导的抗病毒免疫应答通路中的调节机制。     

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.     

Structural insights into RNA recognition by RIG-I

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.     

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.     

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.