Abstract: The innate immune system recognizes viral dsRNA through two distinct pathways; the Toll-like receptor 3 (TLR3) pathway detects dsRNA phagocytosed in endosomes; the helicases retinoic acid-induced protein I (RIG-I) and melanoma differentiation-associated gene-5 (mda-5) detect cytoplasmic dsRNA generated during viral replication. Both RIG-I and mda-5 can bind polyriboinosinic:polyribocytidylic acid (polyI:C), the synthetic analog of viral dsRNA, and mediate type I IFN responses to polyI:C and multiple RNA viruses in vitro. We generated mda-5-deficient mice and showed that mda-5 is the dominant receptor mediating type I IFN secretion in response to polyI:C in vitro and in vivo. Moreover, mda-5−/− mice exhibited a selectively impaired antiviral response to encephalomyocarditis picornavirus, indicating functional specialization of mda-5 in vivo.

Abstract: Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

Abstract: Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

Abstract: IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

Abstract: Activation of alpha/beta interferon (IFN-α/β) production is a key step in the innate immune response to viral infection. Double-stranded RNA (dsRNA) has long been used as an experimental inducer of IFN-α/β and is potentially synthesized during the replication of many viruses. Thus, viral dsRNA has been hypothesized to be a trigger of cellular antiviral responses (29). A number of cellular dsRNA recognition proteins have been implicated in the IFN-induced antiviral response to infection. These include the dsRNA-dependent protein kinase PKR, the 2′,5′-oligoadenylate synthase, and ADAR1 (20, 44, 59, 63). More recently, two IFN-induced, caspase recruiting domain (CARD)-containing, DExD/H family helicases, the retinoic acid-inducible gene I (RIG-I) protein and the melanoma differentiation-associated gene 5 (MDA-5) protein, have been implicated as key sensors of viral infection (1, 30, 56, 66, 67). These proteins are activated by viral infection, possibly through recognition of dsRNA or of ribonucleoprotein complexes produced during infection, and transduce downstream signaling to activate the IFN-α/β responses (34). The transcription factor interferon regulatory factor 3 (IRF-3) plays a critical role in the activation of the IFN-α/β gene. A cytoplasmic protein in its inactive state, IRF-3 becomes hyperphosphorylated on serine and threonine residues, dimerizes, and accumulates in the nucleus, where it participates in initial IFN-α/β gene expression (37, 68, 69). RIG-I and MDA-5 activate IRF-3 in response to dsRNA or to viral infection upstream of the IRF-3 kinases TBK-1 and IKKɛ (18, 30, 54, 66). This signaling appears to involve the homotypic interaction of the CARDs of the helicases with another CARD-containing protein termed alternatively IPS-1, MAVS, VISA, or Cardif (31, 41, 53, 65). Viruses have evolved a variety of mechanisms to avoid recognition or to block the antiviral responses mediated by IFNs (2, 19). Several such proteins also exhibit dsRNA-binding activity. Examples of dsRNA-binding proteins that counteract cellular antiviral responses include the NS1 proteins of influenza A (NS1A) and B (NS1B) viruses, the E3L protein of vaccinia virus, the σ3 protein of reovirus, and the pTRS1 protein of human cytomegalovirus (5, 11, 12, 14, 16, 17, 22, 27, 57). Although dsRNA binding appears to contribute to IFN antagonist function, NS1A, NS1B, and E3L possess additional dsRNA-binding-independent mechanisms to inhibit the cellular antiviral response. The Ebola virus (EBOV) VP35 protein has been found to inhibit IFN-α/β production and the activation of IRF-3 (3, 4, 9, 23, 48). Recently, it was suggested that VP35 may contain a dsRNA-binding motif similar to that found in the NS1 protein of influenza A virus (23). It was further suggested that this dsRNA-binding activity may be required for the ability of VP35 to inhibit IFN production (23). Here, we provide evidence that VP35 has dsRNA-binding activity that may contribute to IFN antagonism. It is likely that VP35 also possesses a dsRNA-binding-independent mechanism(s) that targets a point at or downstream of the IRF-3 kinases TBK-1 and IKKɛ.

Abstract: Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNA-dependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.

Abstract: TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-β promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-β promoter activation >24 h after poly(I:C) or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-1R domain-containing adapter-inducing IFN-β) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-β promoter activation by RSV infection. Virus-mediated activation of the IFN-β promoter was largely abrogated by the gene silencing of IFN-β promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, IκB kinase-related kinase e and TANK-binding kinase 1 participated in IFN-β induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-β promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-β promoter.

Abstract: Type I interferons (IFNs) were first described several decades ago as soluble factors that were capable of 'interfering' with viral replication when added to infected cells. Type I IFNs have been shown to be induced by recognition of viral DNA and RNA via three distinct pathways: (i) a TRIF-dependent pathway in macrophages via TLRs 3 and 4; (ii) a MyD88-dependent pathway in plasmacytoid dendritic cells (pDCs) via TLRs 7/8 and 9; and (iii) an intracellular recognition pathway utilizing the cytoplasmic receptors RIG-I/MDA5. Interestingly, these viral recognition pathways converge on TRAF3, which induces interferon through the activation of IRF3 or IRF7 by the TBK-1 and IKKi complexes. While type I IFN has been traditionally associated with antiviral responses, recent studies have demonstrated that many bacteria also induce type I interferon responses. The mechanisms of type I IFN induction and its role in host defense, however, are largely unclear. Studies with the Gram-positive intracellular bacterium Listeria monocytogenes indicated that it may trigger type I IFN induction through novel TLR-independent intracellular receptors and type I IFN may play a detrimental role to host response against listerial infection. In this article, we summarize some of these findings and discuss the functional differences of type I IFNs in bacterial and viral infections.

Abstract: Recognition of viral nucleic acids with pattern recognition receptors (PRRs) is the first step to induce innate immune system. Type I interferons (IFNs), central mediators in the antiviral innate immunity, are responsible for induction of cytokines and chemokines that disrupt virus replication. Recent studies indicated that there are at least two distinct pathways for the induction of type I IFN by viral infection. Toll-like receptors (TLRs) are extracellular and endosomal PRRs for microbial pathogens whereas retinoic acid inducible gene-I(RIG-I) and melanoma differentiation-associated gene 5(MDA5) are novel intracellular PRRs for viral dsRNA. In this report we describe the distinct mechanisms inducing type I IFNs through TLRs and RIG-I/MDA5 pathways.

Abstract: Type I interferons (IFNalpha/beta) are central mediators for antiviral responses. Using a functional cloning strategy, we have identified a molecule designated IPS-1. IPS-1 overexpression caused antiviral responses by producing type I IFN and IFN-inducible genes through activation of IRF3, IRF7 and NF-kappaB. TBK1 and IKKi protein kinases were required for the IPS-1-mediated IFN induction. IPS-1 contains an N-terminal caspase recruiting domain (CARD)-like structure that mediates interaction with the CARD of RIG-I and Mda5, cytoplasmic RNA helicases sensing RNA viruses. Reduction of IPS-1 by siRNA blocked IFN induction by virus infection. Thus, IPS-1 is an adapter that mediates RIG-I- and Mda5-dependent antiviral responses.

Abstract: Antiviral innate immune responses can be triggered by accumulation of intracellular nucleic acids resulting from virus infections. Double-stranded RNA (dsRNA) can be detected by the cytoplasmic RNA helicase proteins RIG-I and MDA5, two proteins that share sequence similarities within a caspase recruitment domain (CARD) and a DExD/H box RNA helicase domain. These proteins are considered dsRNA sensors and are thought to transmit the signal to the mitochondrial adapter, IPS-1 (also known as MAVS, VISA, or CARDIF) via CARD interactions. IPS-1 coordinates the activity of protein kinases that activate transcription factors needed to induce beta interferon (IFN-β) gene transcription. Another helicase protein, LGP2, lacks the CARD region and does not activate IFN-β gene expression. LGP2 mRNA is induced by interferon, dsRNA treatments, or Sendai virus infection and acts as a feedback inhibitor for antiviral signaling. Results indicate that LGP2 can inhibit antiviral signaling independently of dsRNA or virus infection intermediates by engaging in a protein complex with IPS-1. Experiments suggest that LGP2 can compete with the kinase IKKi (also known as IKKe) for a common interaction site on IPS-1. These results provide the first demonstration of protein interaction as an element of negative-feedback regulation of intracellular antiviral signaling by LGP2.

Abstract: Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

Abstract: Mice lacking the adaptor protein that initiates an antiviral response downstream of the RNA helicases retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) have recently been described. These studies highlight the essential and nonredundant role of nucleic acid recognition in the induction of type I interferon production and raise important questions regarding the nature of cell-autonomous virus detection in coordinating the antiviral response.

Abstract: Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.

Abstract: Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.