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.     

Virus-induced expression of type I interferon genes

Intracellular double-stranded (ds) RNA is a major sign of replication for many viruses. Host mechanisms detect the dsRNA and provoke antiviral responses. Recently, we identified retinoic acid inducible gene-I (RIG-I), which encodes a DExD/H box RNA helicase containing the caspase recruitment domain (CARD) as a critical regulator for dsRNA-induced signaling. The helicase domain with intact ATPase activity is responsible for recognition of dsRNA, and the CARD transmits downstream signals, resulting in the activation of genes including type I interferons. In this review, we discuss the function of RIG-I in antiviral innate immunity.     

Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA

RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.     

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.     

Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.     

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.     

The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I. We identify a series of mutations that additively decouple the Pincer motif from the ATPase core and show that this decoupling results in impaired signaling. Through enzymological and biophysical analysis, we further show that the Pincer domain controls coupled enzymatic activity of the protein through allosteric control of the ATPase core. Further, we show that select regions of the HEL1 domain have evolved to potentiate interactions with the Pincer domain, resulting in an adapted ATPase cleft that is now responsive to adjacent domains that selectively bind viral RNA.     

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.     

DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).     

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.     

Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-�� Production

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-β mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-β production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-β or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.     

Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway

The cytoplasmic helicase protein RIG-I (retinoic acid-inducible gene I) and downstream signaling molecules, MAVS (mitochondrial antiviral signaling protein), TRAF3 (TNF-receptor-associated factor 3) and TBK1 (TANK-binding kinase 1), have significant roles in the recognition of cytoplasmic 5'-triphosphate ssRNA and short dsRNA, and phosphorylation of IRF-3 (interferon regulatory factor 3) and IRF-7 which is responsible for the induction of type I interferons (IFN). In the present study, the full-length cDNAs of RIG-I, MAVS, TRAF3 and TBK1 were cloned and identified in common carp (Cyprinus carpio L.). The deduced protein of carp RIG-I is of 946 aa (amino acids), consisting of two CARDs (caspase-recruitment domain), a DEXDc (DExD/H box-containing domain), a HELICc (helicase superfamily c-terminal domain) and a RD (regulatory domain). Carp MAVS is of 585 aa, containing a CARD, a proline-rich region and a TM (transmembrane domain). Carp TRAF3 encodes a protein of 573 aa, including a RING (really interesting new gene), two TRAF-type zinc fingers, a coiled coil and a MATH-TRAF3 (meprin and TRAF homology) domain. Carp TBK1 is of 727 aa and contains a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Carp RIG-I, MAVS, TRAF3 and TBK1 mRNAs are ubiquitously expressed in all tissues examined. In response to SVCV infection, carp RIG-I and MAVS mRNAs were up-regulated at different levels in spleen, head kidney and intestine tissues at different time points. Similarly, both carp IRF-3 and IRF-7 mRNAs were significantly up-regulated in the detected tissues. Especially in intestine, the IRF-3 and IRF-7 mRNAs of carp increased and reached 25.3-fold (at 3 dpi) and 224.7-fold (at 5 dpi). Noteworthily, a significant growth of carp TRAF3 and TBK1 mRNA was also mainly found in intestine (7.0-fold and 11.3-fold at 5 dpi, respectively). These data implied that the expression profiles of IRF-3/-7 mRNAs in carp correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK, and carp RIG-I and MAVS may be involved in antiviral responses through the RIG-I viral recognition signaling pathway in a TRAF3/TBK1-dependent manner.     

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.     

Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling

Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.     

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.     

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.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.