Abstract: Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) constitute distinct families of pattern-recognition receptors that sense nucleic acids derived from viruses and trigger antiviral innate immune responses. TLR3, TLR7, and TLR9 are membrane proteins localized to the endosome that recognize viral double-stranded RNA, single-stranded RNA, and DNA, respectively, while RLRs, including RIG-I, Mda5, and LGP2, are cytoplasmic proteins that recognize viral RNA. Upon recognition of these nucleic acid species, TLRs and RLRs recruit specific intracellular adaptor proteins to initiate signaling pathways culminating in activation of NF-κB, MAP kinases, and IRFs that control the transcription of genes encoding type I interferon and other inflammatory cytokines, which are important for eliminating viruses. Here, we review recent insights into the signaling pathways initiated by TLR and RLR and their roles in innate and adaptive immune responses.

Abstract: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

Abstract: Protection against West Nile virus (WNV) infection requires rapid viral sensing and the generation of an interferon (IFN) response. Mice lacking IFN regulatory factor 3 (IRF-3) show increased vulnerability to WNV infection with enhanced viral replication and blunted IFN-stimulated gene (ISG) responses. IRF-3 functions downstream of several viral sensors, including Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Cell culture studies suggest that host recognizes WNV in part, through the cytoplasmic helicase RIG-I and to a lesser extent, MDA5, both of which activate ISG expression through IRF-3. However, the role of TLR3 in vivo in recognizing viral RNA and activating antiviral defense pathways has remained controversial. We show here that an absence of TLR3 enhances WNV mortality in mice and increases viral burden in the brain. Compared to congenic wild-type controls, TLR3(-/-) mice showed relatively modest changes in peripheral viral loads. Consistent with this, little difference in multistep viral growth kinetics or IFN-alpha/beta induction was observed between wild-type and TLR3(-/-) fibroblasts, macrophages, and dendritic cells. In contrast, a deficiency of TLR3 was associated with enhanced viral replication in primary cortical neuron cultures and greater WNV infection in central nervous system neurons after intracranial inoculation. Taken together, our data suggest that TLR3 serves a protective role against WNV in part, by restricting replication in neurons.

Abstract: TLR22 occurs exclusively in aquatic animals and its role is unknown. Herein we show that the fugu (Takifugu rubripes) (fg)TLR3 and fgTLR22 link the IFN-inducing pathway via the fg Toll-IL-1R homology domain-containing adaptor protein 1(fgTICAM-1, or TRIF) adaptor in fish cells. fgTLR3 resides in endoplasmic reticulum and recognizes relatively short-sized dsRNA, whereas fgTLR22 recognizes long-sized dsRNA on the cell surface. On poly(I:C)-stimulated fish cells, both recruit fgTICAM-1, which in turn moves from the TLR to a cytoplasmic signalosome region. Thus, fgTICAM-1 acts as a shuttling platform for IFN signaling. When fish cells expressing fgTLR22 are exposed to dsRNA or aquatic dsRNA viruses, cells induce IFN responses to acquire resistance to virus infection. Thus, fish have a novel TICAM-1-coupling TLR that is distinct from the mammalian TLR3 in cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute of human cell-surface TLR3 and serve as a surveillant for infection with dsRNA virus to alert the immune system for antiviral protection in fish.

Abstract: RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.

Abstract: Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

Abstract: Induction of antiviral innate immune responses depends on a family of innate immune receptors, the Toll-like receptors (TLR). TLR mediate the antiviral immune responses by recognizing virus infection, activating signaling pathways and inducing the production of antiviral cytokines and chemokines. ssRNA and dsRNA viruses can be recognized by TLR7/8 and TLR3, respectively. TLR receptors are also involved in the recognition of viruses containing genomes rich in CpG DNA motifs as well as envelope glycoproteins. Cytoplasmic recognition of dsRNA by RNA helicases such as RIG-I and MDA5 provides another means of recognizing viral nucleic acid. In order to counteract the innate host immune system viruses evolved mechanisms that block recognition and signaling through pattern recognition receptors, such as TLRs and RNA helicases. Recently, TLR agonists represent a promising approach for the treatment of infectious diseases. This review will focus on the current knowledge of TLR-mediated immune responses to several viral infections.

Abstract: Type I interferon (IFN-alpha/beta) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7(-/-) mice Compared to congenic wild-type mice, IRF-7(-/-) mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice Consistent with this, IFN-alpha gene expression and protein production were reduced and viral titers were increased in IRF-7(-/-) primary macrophages, fibroblasts, dendritic cells, and cortical neurons In contrast, in these cells the IFN-beta response remained largely intact Our data suggest that the early protective IFN-alpha response against WNV occurs through an IRF-7-dependent transcriptional signal

Abstract: Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.

Abstract: Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-alpha, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-kappaB-, and TANK (TNF receptor-associated NF-kappaB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-beta and TNF-alpha upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.

Abstract: Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia ΔE3L mutant virus triggers the production of beta interferon (IFN-β), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. ΔE3L induction of IFN-β, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with ΔE3L, an event that depends on MAVS. The response of keratinocytes to ΔE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.

Abstract: Mitochondria are known to be semi‐autonomous organelles that are responsible for energy production and cellular respiration. Mitochondria break down glucose to release energy in the form of ATP through oxidative phosphorylation. However, as a by‐product, a steady stream of reactive oxygen species (ROS) is released from these cellular powerhouses. ROS can potentially cause damage to cellular components, and are therefore closely linked to diseases such as Alzheimer disease and cancer. Recently, mitochondria were also found to have a crucial role in innate immunity. Innate immune responses against invading viruses rely on the detection of viral pathogen‐associated molecular patterns (PAMPs) and the subsequent production of antiviral cytokines such as type I interferons (IFNs). One prototypical viral PAMP is double‐stranded (ds)RNA, which can be detected by Toll‐like receptor 3 (TLR3) in endosomes (Akira & Takeda, 2004). TLR3 was the first reported dsRNA receptor able to signal to interferon regulatory factor (IRF) and NF‐κB, which are essential transcription factors that regulate type I IFN production (Fig 1). Since then, two homologous RNA helicases—retinoic‐acid‐inducible gene I (RIG‐I) and melanoma‐differentiation‐associated protein 5 (MDA5)—have been identified as cytoplasmic sensors of viral‐derived RNA (Yoneyama et al , 2004). Figure 1. Mitochondria as anti‐pathogen platforms. Through its mitochondrial anchor (MA) sequence, Cardif is targeted to the outer membrane of mitochondria, where it orchestrates RLH‐dependent antiviral responses through the recruitment of both viral RNA sensors (RIG‐I or MDA5) and effector proteins (IKK). Several cellular (SIKE, PIN1) but also viral (for example, the protease of HCV) proteins tightly regulate the antiviral response. NLRX1, another mitochondria‐targeted protein (MT), might act as a negative regulator of Cardif signalling, diminishing virally induced Cardif–RLH interactions. However, NLRX1 also promotes ROS production at the mitochondria, which consequently helps to fight bacteria and viruses. How NLRX1 performs these functions, and how it becomes activated, remain unanswered questions. CARD, …

Abstract: Inosine residues may be introduced into long dsRNA (double-stranded RNA) molecules by the action of a family of editing enzymes, ADARs (adenosine deaminases that act on RNA). Furthermore, hyperediting of dsRNA by ADARs may result in up to 50% of adenosine residues being converted into inosine. While the effect of hyperediting has traditionally been thought to be limited to the edited dsRNA, we have recently shown that hyperedited dsRNA [I-dsRNA (inosine-containing dsRNA)] is able to down-regulate the expression of both reporter and endogenous mRNAs in cells, in trans. Down-regulation by I-dsRNA occurs both by reducing mRNA levels and by inhibiting of translation. This finding has important functional consequences for hyperediting by ADARs.

Abstract: Double stranded RNA(dsRNA) is a trigger of cellular responses to viral infections.An essential prerequisite is that dsRNA acts on enzymes and functional proteins which can specifically recognize dsRNA and elicit cellular response.Some data indicated that dsRNA-binding domain(DRBD) not only binds with dsRNA,but also interacts with DNA or RNA,or only acts as a regulator without binding any nucleic acid.In addition,DRBD can interact with each other in the same protein.Among different proteins,DRBD can also interact with each other.These interactions result in complicated protein-protein complexes,which participate in multiple cell metabolic pathways.Therefore,DRBD has diverse functions.

Abstract: When a cell gets infected with a virus, the innate immune system swings into action within minutes. The rapid production of pro-inflammatory cytokines and antivirally active type-I Interferons (IFN-a/p) is the most significant mechanism to limit virus spread. Two conceptually different pathogen recognition mechanisms are known that lead to antiviral responses through production of IFN-a/p: Specialised immune cells possess Toll-like receptors (TLRs), which sense incoming viruses in endosomes. Most other cells rely on the cytoplasmic RNA-helicases RIG-I and MDA5 that sense the presence of viruses within the cell. However, although proteins and signalling networks involved in innate recognition of viruses are well known, the exact molecular details of their interactions with the virus are only marginally understood. During my PHD thesis I dedicated myself to aid our understanding of virus recognition. I could show that recombinant lentiviruses are weak inducers of IFN-ct/p* in murine immune cells. Standard preparations of lentiviral vectors, however, are strong activators of the innate immune system. This activity is contained in tubulo-vesicular structures that are present within standard lentiviral preparations and have the ability to activate TLR9. Tubulo-vesicular structures can serve as adjuvant to facilitate adaptive immune responses and may therefore be important when considering lentiviral vectors for clinical applications. In my second project I focused on cytoplasmic virus recognition. Surprisingly, viral genomic single-stranded RNA from influenza virus can activate the cytoplasmic virus recognition receptor RIG-I. Unlike most cellular RNA species, single-stranded RNA from influenza and other viruses bear a 5' triphosphate group, which marks this RNA as 'foreign' and thereby induces interferon responses. Importantly, influenza virus codes for an interferon antagonist, the non-structural protein 1 (NS1), which forms a complex with RIG-I, suggesting that influenza virus specifically interferes with this pathway. In conclusion, the innate immune system employs diverse mechanisms to sense the presence of a virus through recognising diverse forms of viral nucleic acid.

Abstract: Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5−/− dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5−/− DCs as well as in MDA5−/− mice in vivo. Interestingly, TLR3−/− DCs do not have a defect in vitro, but TLR3−/− mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.

Abstract: A conundrum of innate antiviral immunity is how nucleic acid-sensing Toll-like receptors (TLRs) and RIG-I/MDA5 receptors cooperate during virus infection. The conventional wisdom has been that the activation of these receptor pathways evokes type I IFN (IFN) responses. Here, we provide evidence for a critical role of a Toll-like receptor 3 (TLR3)-dependent type II IFN signaling pathway in antiviral innate immune response against Coxsackievirus group B serotype 3 (CVB3), a member of the positive-stranded RNA virus family picornaviridae and most prevalent virus associated with chronic dilated cardiomyopathy. TLR3-deficient mice show a vulnerability to CVB3, accompanied by acute myocarditis, whereas transgenic expression of TLR3 endows even type I IFN signal-deficient mice resistance to CVB3 and other types of viruses, provided that type II IFN signaling remains intact. Taken together, our results indicate a critical cooperation of the RIG-I/MDA5-type I IFN and the TLR3-type II IFN signaling axes for efficient innate antiviral immune responses.

Abstract: Vertebrate cells activate multiple signaling modules upon virus infection to eliminate the invading pathogen and to prevent the establishment of a persistent infection. A major immediate response pathway is controlled by the RNA helicases RIG-I and MDA5, which, after recognition of viral nucleic acids, signal induction of the interferon (IFN)-alpha/beta cytokine family that upregulates numerous antiviral effector proteins. Virulent viruses, in contrast, have learned during co-evolution with their hosts to manipulate or avoid this response in order to prevail in a repulsive environment. Focusing on the influenza viruses and their IFN-antagonistic NS1 proteins, we summarize recent progress in this rapidly evolving field at the intersection of virology and immunobiology involving studies of how viral pathogens induce and sabotage cellular defenses.

Abstract: Double-stranded RNA, polyriboinosinic-polyribocytidylic acid (poly IC), acts as an adjuvant that enhances adaptive immune responses. The recognition of poly IC is mediated by endosomal TLR3 and cytoplasmic RNA helicase melanoma differentiation-associated gene 5 (Mda5), which signal through the adaptors Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) and IFN-β promoter stimulator-1 (IPS-1), respectively. However, the contribution of these pathways to the adjuvant effects of poly IC remains unclear. In this study, we found that poly IC-enhanced, Ag-specific Ab production was severely decreased in IPS-1-deficient mice but not in TRIF-deficient mice. However, the double deficiency resulted in a complete loss of Ab production. Furthermore, Ag-specific CD8 + T cell expansion was reduced in both IPS-1-deficient and TRIF-deficient mice and entirely abrogated in the doubly deficient mice. Taken together, these results demonstrate that the adjuvant effects of poly IC require a cooperative activation of TLR and cytoplasmic RNA helicase pathways.