Abstract: Retinoic acid inducible-gene I (RIG-I) is a cytosolic multidomain protein that detects viral RNA and elicits an antiviral immune response Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal, and the regulatory domain prevents signaling in the absence of viral RNA 5'-triphosphate and double-stranded RNA (dsRNA) are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA However, the function of the DExH box helicase domain that is also required for activity is less clear Using single-molecule protein-induced fluorescence enhancement, we discovered a robust adenosine 5'-triphosphate-powered dsRNA translocation activity of RIG-I The CARDs dramatically suppress translocation in the absence of 5'-triphosphate, and the activation by 5'-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses

Abstract: Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-β (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, 10-kDa IFN-γ-inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-β, IFN-λ1, IFN-λ2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.

Abstract: RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-I-like receptor, LGP2, differentially regulates RIG-I- and MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5'-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6 A crystal structure of LGP2 RD along with in vitro and in vivo functional analyses and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5'-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

Abstract: gC1qR is one of the C1q receptors implicated in the regulation of innate and adaptive immunity. We found that gC1qR inhibits RIG-I and MDA5-dependent antiviral signaling. Double stranded RNA and virus trigger the translocation of gC1qR to the mitochondrial outer membrane leading to the interaction of gC1qR with the RIG-I and MDA5 adaptor, VISA/MAVS/IPS-1/Cardif. The interaction of gC1qR with VISA/MAVS/IPS-1/Cardif at mitochondria results in the disruption of RIG-I and MDA5 signaling and the promotion of virus replication. Knockdown of endogenous gC1qR enhances RIG-I-dependent antiviral signaling, and augments the inhibition of virus proliferation. Therefore, gC1qR is a physiological inhibitor of the RIG-I and MDA5-mediated antiviral signaling pathway. These data uncover a new viral mechanism used to negatively control antiviral signaling in host cells.

Abstract: Detection of pathogens by the mammalian innate immune system is mediated by pattern recognition receptors. For viruses, nucleic acids are often the trigger for innate responses that culminate in antiviral gene expression, including the production of type I interferon (IFN-α/β). Foreign nucleic acids outside the cell can be recognized by transmembrane Toll-like receptors at the cell surface or in the lumen of endocytic vesicles (9). Intracellular nucleic acids are recognized by cytoplasmic receptor proteins (24). In both cases, receptor binding to the nucleic acid ligand triggers a signal transduction cascade that activates immediate transcriptional responses, including the induction of the antiviral cytokines in the IFN family. An important class of receptors for the detection of cytosolic nonself RNAs is represented by the proteins encoded by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (24). These proteins share functional domains, including an amino-terminal protein interaction motif that functions in signal transduction and is homologous to the caspase activation and recruitment domain (CARD) (8) and a C-terminal DECH-box family RNA helicase domain. The helicase domains share approximately 30 to 40% overall sequence identity. Experimental and structural studies have demonstrated a third functional region at the extreme C terminus of RIG-I that forms a zinc-mediated fold and functions as a regulatory domain for the recognition and discrimination of RNA 5′ ends (6, 20). Current evidence supports a model in which binding to RNA ligand induces a change in accessibility to the CARD, allowing it to interact with downstream signaling proteins, including the mitochondrial resident IPS-1 (also identified as MAVS, VISA, and Cardiff [12, 15, 21, 28]). This association coordinates a serine kinase-mediated cascade that activates latent transcription factors, including NF-κB and IFN regulatory factor 3, culminating in the expression of IFN-β and a number of other crucial antiviral effector genes (8). A third helicase protein, LGP2, resembles RIG-I and MDA5 in the helicase domain, also exhibiting 30 to 40% sequence identity. This overall level of similarity may underestimate the relatedness of these proteins, as they are more identical in their key helicase sequence motifs and encompassing domains (2). Significantly, LGP2 lacks the N-terminal CARD homology region. As a result, LGP2 expression is not typically associated with the ability to directly activate downstream signaling. Ectopic expression of LGP2 interferes with double-stranded RNA (dsRNA) and virus-induced antiviral signaling, while reduction of LGP2 expression by RNA interference results in enhanced IFN-β synthesis and antiviral responses. Expression of LGP2, like that of RIG-I and MDA5, is induced by virus infection, nucleic acid transfection, and IFN stimulation, suggesting it has the properties of a negative feedback inhibitor (14, 19, 20, 29). The importance of these RNA helicase proteins in antiviral responses is validated by the phenotypes of mice harboring targeted disruptions in these genes (9). Deficiency in RIG-I leads to a widespread enhancement in replication of many RNA virus types, due to suppressed IFN biosynthesis and antiviral responses (11). Cells deficient in RIG-I exhibit a general defect in the ability to respond to foreign dsRNAs or single-stranded RNAs (ssRNAs) bearing phosphorylated 5′ ends. MDA5 deficiency showed a more specific phenotype, resulting in increased susceptibility to picornavirus infection and insensitivity to the synthetic dsRNA analog poly(I:C) (7). LGP2 deficiency leads to a more complex phenotype (27). LGP2-deficient mice are more sensitive to IFN induction by cytosolic poly(I:C), an MDA5 ligand, and more resistant to vesicular stomatitis virus, a virus that triggers RIG-I signaling. Negative regulation of the IFN response remains intact overall, indicating that LGP2 may not be the primary negative regulator of type I IFN production. However, LGP2 deficiency results in a suppressed IFN response to infection with encephalomyocarditis virus, a picornavirus that has been demonstrated to trigger MDA5-mediated antiviral signaling. These data suggest that LGP2 executes both negative and positive regulatory functions related to RIG-I and MDA5 signaling. The disparate effects of LGP2 deficiency are difficult to interpret without a better mechanistic understanding of LGP2 functions in cellular innate antiviral immunity. Further affirmation of the antiviral role of the helicase proteins derives from the fact that individual viruses have evolved means to evade or disrupt their activity, either by direct interference or by antagonizing the signaling intermediates. The large Paramyxovirus family of negative-strand RNA viruses is well known to evade IFN antiviral responses. Most viruses within this family encode a protein, called V, that is essential for IFN signaling evasion. In addition to suppressing the activity of signal transducer and activator of transcription (STAT) proteins to compromise IFN signal transduction, paramyxovirus V proteins can also limit the induction of IFN gene expression by interfering with the MDA5 protein (1, 3). Associations between the V protein C-terminal domain (CTD), a 60- to 70-residue zinc binding fold that is the conserved hallmark of V proteins, and the MDA5 helicase domain prevents signal transduction downstream of poly(I:C), a synthetic RNA ligand that is a known MDA5 activator (1, 3). This inhibition was observed for all tested V proteins of the large Paramyxovirus family (3). Despite the potential importance of the V protein-MDA5 interface as an antiviral target, the mechanistic basis and consequences for MDA5 interference by V proteins are poorly understood and difficult to reconcile with the lack of phenotype in MDA5-deficient mice. Recent evidence indicates that MDA5 is involved in the detection of Sendai virus-defective interfering (DI) genomes, providing plausible biological relevance to the observed V protein interference that may not have been apparent from gene disruption studies (31). Identification of the MDA5 target recognized by paramyxovirus V proteins led to the discovery that LGP2 is also a target for V protein antagonism. Results demonstrate that the helicase C domain of both proteins functions as a V protein binding region common to both MDA5 and LGP2 but absent in RIG-I. We demonstrate that interaction with the V protein interferes with the catalytic activity of both target helicases, providing a biochemical basis for V protein helicase antagonism.

Abstract: The striking difference in evolution of type I IFN genes of fish and mammals poses the question of whether these genes are induced through similar or different signalling pathways in the two vertebrate groups. Previous work has shown that expression of both Atlantic salmon (Salmo salar) IFNa1 and mammalian IFN-beta genes is dependent on IRF and NF-kappaB elements in their promoters. In mammals, IFN-beta transcription is induced through the RIG-I/MDA5 pathway where the adaptor protein IPS-1 plays a key role in the signal transduction. In this work we show that an Atlantic salmon homologue of IPS-1 (AsIPS-1) mediates activation of the salmon IFNa1 promoter and an NF-kappaB driven promoter. AsIPS-1 shares only 18% identity in amino acid sequence with human IPS-1, but possesses the CARD, proline-rich and transmembrane domains found in mammalian IPS-1. Overexpression of AsIPS-1 resulted in induction of an antiviral state in the cells apparently due to induction of IFN. Deletion of the CARD and transmembrane domains of AsIPS-1 abolished its ability to activate the IFNa1 promoter and the NF-kappaB driven promoter, and thus its ability to induce an antiviral state. AsIPS-1 is located to mitochondria similar to human IPS-1. Taken together, IPS-1 plays a key role in the induction of Atlantic salmon IFNa1, which appears to be the first and major IFN induced in host cells upon recognition of viral dsRNA.

Abstract: Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-β promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-β promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60–90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.

Abstract: The type I interferon (IFN) is a host defense factor against microbial pathogens in vertebrates. In mammals, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm are regarded as sensors for double-stranded RNA (dsRNA) and trigger IFN regulatory factor-3 (IRF-3) activation followed by type I IFN induction through the mitochondrial antiviral signaling (MAVS) adapter. This intrinsic pathway appears to link the main protective responses against RNA virus infection in mammals. On the other hand, human Toll-like receptor 3 (TLR3) is localized in the endosomal membrane or cell surface and signals the presence of extrinsic dsRNA. In response to RNA stimulation, TLR3 recruits the Toll-interleukin 1 receptor domain (TIR)-containing adapter molecule 1 (TICAM-1) adapter and induces IRF-3 activation followed by IFN-beta promoter activation. Human TLR3 is localized limitedly extent in myeloid dendritic cells, fibroblasts, and epithelial cells. The TICAM-1 and cytoplasmic MAVS pathways converge at the IRF-3-activating kinase in human cells. The reason for the involvement of this extrinsic mode of IFN-inducing pathways in the dsRNA response remains unknown. In fish, two TLRs, i.e. endoplasmic TLR3 and cell surface TLR22, participate in teleost IFN production without the activation of IRF-3. TLR22 is distinct from mammalian TLR3 in terms of cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute for human cell surface TLR3 and may serve as a surveillance molecule for detecting dsRNA virus infection and alerting the immune system for antiviral protection in fish. In this review, we discuss the fundamentals of the extrinsic dsRNA recognition system, which has evolved to induce cellular effectors to cope with dsRNA virus infection across different vertebrate species.

Abstract: Toll-like receptor 3 and RIG-I like receptors (RLRs; MDA5, RIG-I) are involved in cell growth inhibition and apoptosis. However, the toll-like receptor 3-related apoptotic pathway is insensitive to direct polyinosinic-polycytidylic acid (dsRNA analog) stimulation in hepatoma cells. To determine whether the strategy of transferring polyinosinic-polycytidylic acid into cells (polyinosinic-polycytidylic acid-liposome) could induce apoptosis in hepatoma cells through cytoplasm receptors, we examined the responses of innate immune receptors RLRs and toll-like receptor 3 in response to different stimulation. We found that the apoptosis could exclusively be detected under polyinosinic-polycytidylic acid-liposome stimulation, which involved the activation of the caspase pathway. Besides, the expression of RIG-I, MDA5, IFNβ and interferon-stimulated gene 15 was increased significantly at an early stage. Moreover, the growth inhibition of polyinosinic-polycytidylic acid-liposome was confirmed in a mouse model. Taken together, these results suggest polyinosinic-polycytidylic acid-liposome could be used as a potential apoptotic agent in hepatocellular carcinoma cells and imply a potential therapeutic strategy. (Cancer Sci 2009; 100: 529–536)

Abstract: Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM3CSK4. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM3CSK4-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM3CSK4), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.

Abstract: Viral inflammation and infection of mesothelial cells (MC) are a major problem in several organ systems including pleura, pericardium and peritoneum. Toll-like receptors (TLRs) are an essential part of the innate immune system for early recognition of pathogen-associated molecular patterns. TLRs recognise molecular patterns associated with microbial pathogens and induce an immune response. TLR3 recognises dsRNA of viral origin as exemplified by poly (I:C) RNA, a synthetic analogue of viral dsRNA. The helicases RIG-I and MDA5 may also act as sensors of viral infections. MC exhibit an expression of TLR3, RIG-I and MDA5. Poly (I:C) RNA stimulation resulted in an up-regulation of proinflammatory cytokines and chemokines as well as type I interferons. This novel finding of functional expression of viral sensors on human MC may indicate a novel link between viral infections and mesothelial inflammation and indicates a pathophysiologic role of viral receptors in these processes.

Abstract: Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.

Abstract: Thromb Haemost 2009; 101: 993–994 Toll-like receptors (TLR) constitute a protein family of 13 different mammalian subtypes of heterodimeric immune receptors which are activated by a diverse range of molecules specific to certain microbial hazards. Among them, TLR subtype receptor 3 (TLR3) is unique because it is activated by double-stranded RNA (dsRNA) of viral pathogens. Polyinosinic:polycytidylic acid (poly I:C) serves as a well-known activator of TLR3 in cell culture studies to mimic the agonistic effect of dsRNAs (1). In general, TLRs are expressed on immune cells of monoblastic or myeloblastic origin, such as dendritic cells, lymphocytes or monocytes and play a pivotal role in innate immunity. Moreover, certain TLRs have been described on certain epithelial cell types and in particular TLR3 was found to be present on epithelial and neuronal cells, in the kidney and also on mesothelial cells (2–5). In contrast to most TLRs, signalling of TLR3 is independent of the common TLR adaptor protein MyD88 but involves the adaptor protein Trif (TIR-domain-containing adapter-inducing interferon-β, TICAM-1), which ultimately induces transcriptional activation of interferon-α and -β, both of which play a substantial role in the inflammatory immune response following viral infections. In this issue of Thrombosis and Haemostasis, Wornle et al. describe a novel link between viral infection of serosal cavities and regulation of fibrin generation and fibrinolysis (6). In a cell culture model of mesothelial cells isolated from explanted human omental tissue, they show that stimulation with poly I:C leads to both a concentration-dependent increase of plasminogen activator inhibitor 1 (PAI-1) synthesis and a concentration-dependent decrease of tissue plasminogen activator (t-PA) synthesis. Aside from TLR3, mesothelial cells also express the helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5),which may also act as sensors of viral infections through recognition of viral dsRNA and may up-regulate type I interferons. The specifity of the involvement of TLR3, RIG-I and MDA5 in these observations is confirmed by the use of RNA interference technology. These results indicate that the effect of poly I:C on increased PAI-1 but decreased t-PA synthesis is mainly mediated by TLR3. The authors interpret their findings to be of potential importance for the pathogenesis of viral diseases in serous tissue such as the mesothelium, pleura or pericardium because fibrin deposition at these sites could be promoted through the described mechanism. This could ultimately determine the course of the infectious disease e.g. by leading to pleural or pericardial fibrosis. In the mesothelium, similar mechanisms may be responsible for the failure of the peritoneal space to contribute to permeability changes. Hence, in this specific context, activation of mesothelial viral receptors followed by disturbance of the fibrinolytic system could explain why patients undergoing chronic abdominal peritoneal dialysis only profit from this type of treatment for a limited time span. From the data obtained by Wornle et al., such conclusions seem straightforward and comprehensible, yet another implication of the findings presented in this work could be related to the change in gene expression of adjacent vascular tissue. Since under guiescent conditions in vivo the ratio in gene expression between t-PA and PAI-1 in vascular endothelium is very high, poly I:C could dramatically shift this value resulting in the upregulation of PAI-1. This would lead to a more prothrombotic cellular phenotype as recently documented by the poly I:C induced up-regulation of tissue factor and down-regulation of thrombomodulin in the vessel wall (7). Indeed, increased levels of t-PA and PAI-1 have long been known to be present in the early stages of atherosclerosis and to be independent risk predictors for myocardial infarction (8, 9). However, according to present data, they seem to be markers of increased endothelial activation rather than playing an active role in promoting atherosclerosis (10). Intriguingly, besides being expressed in the above mentioned tissues, TLR3 also appears to be expressed on cells of the vascular endothelium, where it most likely acts as a receptor for viral dsRNAs. Thus, activation of endothelial innate immune receptors such as TLR3 could actually represent an active promoting mechanism in atherosclerosis and its thrombotic complications because it might not only result in dysregulation of the t-PA-PAI-1 system, but could also activate other signalling pathways within endothelial cells that directly promote endothelial dysfunction. Indeed, in a very recent report by Fischer et al., the authors describe endothelial activation through extracellular RNA (but not DNA) leading to binding of vascular endothelial growth factor (VEGF) to neuropilin-1, © 2009 Schattauer GmbH, Stuttgart

Abstract: Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel α-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Abstract: Human parainfluenza virus type 3 (HPIV3) is a respiratory paramyxovirus that infects lung epithelial cells to cause high morbidity among infants and children. To date, no effective vaccine or antiviral therapy exists for HPIV3 and therefore, it is important to study innate immune antiviral response induced by this virus in infected cells. Type-I interferons (IFN, interferon-α/β) and tumor necrosis factor-α (TNFα activated by NFκB) are potent antiviral cytokines that play an important role during innate immune antiviral response. A wide-spectrum of viruses utilizes pattern recognition receptors (PRRs) like toll-like receptors (TLRs) and RLH (RIG like helicases) receptors such as RIGI (retinoic acid inducible gene -I) and Mda5 to induce innate antiviral response. Previously it was shown that both TNFα and IFNβ are produced from HPIV3 infected cells. However, the mechanism by which infected cells activated innate response following HPIV3 infection was not known. In the current study, we demonstrated that RIGI serves as a PRR in HPIV3 infected cells to induce innate antiviral response by expressing IFNβ (via activation of interferon regulatory factor-3 or IRF3) and TNFα (via activation of NF-κB).

Abstract: In plants, SGS3 and RNA-dependent RNA polymerase 6 (RDR6) are required to convert single- to double-stranded RNA (dsRNA) in the innate RNAi-based antiviral response and to produce both exogenous and endogenous short-interfering RNAs. Although a role for RDR6-catalysed RNA-dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA-binding protein with unexpected substrate selectivity favouring 5′-overhang-containing dsRNA. The conserved XS and coiled-coil domains are responsible for RNA-binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi-based host immune response, is a dsRNA-binding protein with similar specificity to SGS3. In competition-binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA-mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.

Abstract: The double-stranded RNA (dsRNA) analogue poly(I:C) is a promising adjuvant for cancer vaccines because it activates both dendritic cells (DCs) and natural killer (NK) cells, concurrently promoting adaptive and innate anticancer responses. Poly(I:C) acts through two dsRNA sensors, Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein-5 (MDA5). Here, we investigated the relative contributions of MDA5 and TLR3 to poly(I:C)-mediated NK cell activation using MDA5−/−, TLR3−/−, and MDA5−/−TLR3−/− mice. MDA5 was crucial for NK cell activation, whereas TLR3 had a minor impact most evident in the absence of MDA5. MDA5 and TLR3 activated NK cells indirectly through accessory cells and induced the distinct stimulatory cytokines interferon-α and interleukin-12, respectively. To identify the relevant accessory cells in vivo, we generated bone marrow chimeras between either wild-type (WT) and MDA5−/− or WT and TLR3−/− mice. Interestingly, multiple accessory cells were implicated, with MDA5 acting primarily in stromal cells and TLR3 predominantly in hematopoietic cells. Furthermore, poly(I:C)-mediated NK cell activation was not notably impaired in mice lacking CD8α DCs, providing further evidence that poly(I:C) acts through diverse accessory cells rather than solely through DCs. These results demonstrate distinct yet complementary roles for MDA5 and TLR3 in poly(I:C)-mediated NK cell activation.

Abstract: Recognition of virus infections by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation associated gene 5 (MDA5), activates signaling pathways, leading to the induction of inflammatory cytokines that limit viral replication To determine the effects of PRR-mediated innate immune response on hepatitis B virus (HBV) replication, a 13mer HBV genome was cotransfected into HepG2 or Huh7 cells with plasmid expressing TLR adaptors, myeloid differentiation primary response gene 88 (MyD88), and TIR-domain-containing adaptor-inducing beta interferon (TRIF), or RIG-I/MDA5 adaptor, interferon promoter stimulator 1 (IPS-1) The results showed that expressing each of the three adaptors dramatically reduced the levels of HBV mRNA and DNA in both HepG2 and Huh7 cells However, HBV replication was not significantly affected by treatment of HBV genome-transfected cells with culture media harvested from cells transfected with each of the three adaptors, indicating that the adaptor-induced antiviral response was predominantly mediated by intracellular factors rather than by secreted cytokines Analyses of involved signaling pathways revealed that activation of NF-κB is required for all three adaptors to elicit antiviral response in both HepG2 and Huh7 cells However, activation of interferon regulatory factor 3 is only essential for induction of antiviral response by IPS-1 in Huh7 cells, but not in HepG2 cells Furthermore, our results suggest that besides NF-κB, additional signaling pathway(s) are required for TRIF to induce a maximum antiviral response against HBV Knowing the molecular mechanisms by which PRR-mediated innate defense responses control HBV infections could potentially lead to the development of novel therapeutics that evoke the host cellular innate antiviral response to control HBV infections