sensing of rna viruses: a review of innate immune receptors

Sensing of RNA Viruses: a Review of Innate Immune ReceptorsInvolved in Recognizing RNA Virus Invasion

Søren Jensen and Allan Randrup Thomsen

Institute of International Health, Immunology, and Microbiology, University of Copenhagen, Copenhagen, Denmark

Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host re-sponse to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and themolecular mechanisms they employ to sense viral pathogen-associated molecular patterns have emerged in great detail. Thisreview presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecularstructures these receptors sense, and the involved downstream signaling pathways.

Recognition of evolutionarily conserved microbial structures,known as pathogen-associated molecular patterns (PAMPs),

is an essential function of the innate immune system. ThesePAMPs are molecular structures, such as glycoproteins, lipopoly-saccharides, proteoglycans, and nucleic acid motifs, that arebroadly shared by different microorganisms and essential to thesurvival or infectivity of the microbe. Germ line-encoded patternrecognition receptors (PRRs) are the proteins, expressed by a va-riety of cells, which are responsible for sensing the presence ofmicrobial invasion. The individual members of these receptorfamilies can be distinguished by ligand specificity, cellular local-ization, and activation of unique, but converging, downstreamsignaling pathways. The strategy of employing multiple families ofPRRs affords the host a high degree of functional redundancy andthus provides multiple mechanisms to sense and immediately re-spond to a diverse range of pathogens (3). Furthermore, activationof the innate immune system directs the specific part of the adap-tive immune system to be activated upon different threats, therebylaunching the most appropriate response to any microbial inva-sion (2).

Sensing of PAMPs by PRRs markedly upregulates the tran-scription of genes involved in inflammatory responses. Thesegenes encode proinflammatory cytokines/chemokines, type I in-terferons (IFNs), and antimicrobial proteins. The expression pat-terns of the inducible genes differ among activated PRRs (119).Production of type I IFNs (primarily alpha IFN [IFN-�] andIFN-�) plays a central role in the induction of antiviral responses,as these trigger the transcription of many IFN-inducible geneswhich influence protein synthesis, growth regulation, and apop-tosis. Type I IFNs also enhance maturation of dendritic cells(DCs), cytotoxicity of natural killer (NK) cells, and the differenti-ation of virus-specific cytotoxic T lymphocytes, thus providing animportant link between innate and adaptive immune responses(43).

This review presents an overview of the PRRs currently knownto be activated by RNA virus invasion. Toll-like receptors (TLRs)are the most extensively studied family of PRRs so far, and they areof substantial importance in the initiation of an antiviral responseupon infection. Comprising a gene family of only 10 members inhumans (12 in mice), these receptors cover an impressive range ofPAMPs involved in the recognition of parasites, fungi, bacteria,and viruses (3). Following early reports of TLR-independentmechanism of viral sensing, the retinoic acid-inducible gene I

(RIG-I)-like receptors (RLRs) were described as cytosolic sensorsof viral RNA species. These were rapidly shown to be of impor-tance in early antiviral responses to several viruses (136).

Besides the TLRs and the RLRs, different other receptors havebeen described to sense RNA virus invasion; these include thenucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs). Some of the important modulators of theinnate antiviral response will also be mentioned. These we do notconsider sensors, as they do not initiate gene transcription uponspecific recognition of viral patterns, but they have been includedin this review on the basis of their capacity to influence the finalresponse.

TOLL-LIKE RECEPTORS

Toll-like receptors (TLRs) belong to a conserved family of innateimmune recognition receptors acting as the primary sensors ofspecific PAMPs expressed by numerous pathogens. TLRs aretransmembrane glycoprotein receptors with an N-terminal extra-cellular PAMP-binding region and a C-terminal intracellular sig-naling region. The N-terminal region contains multiple leucine-rich repeats (LRRs) which form a horseshoe structure. TheC-terminal region shows similarity to the intracellular domain ofthe interleukin-1 receptor (IL-1R), and this region is referred to asthe Toll/IL-1R homology (TIR) domain, which mediates down-stream signaling events upon activation of the receptor (1, 12).Upon extracellular ligand recognition, TLR dimerization isthought to be induced, bringing together the cytoplasmic TIRdomains and subsequently recruiting adaptor molecules to initi-ate the signaling process (3, 89). The human TLR multigene familycomprises 10 members, of which TLR2, -3, -4, -7, and -8 arethought to be of importance in the recognition of structural com-ponents of RNA viruses, including viral double-stranded RNA(dsRNA), single-stranded RNA (ssRNA), and surface glycopro-teins. Table 1 contains a detailed list of RNA viruses known to betargets of TLR recognition. Among these receptors, TLR3, -7, and-8 recognize nucleic acid motifs and are preferentially confined to

Published ahead of print 18 January 2012

Address correspondence to Allan Randrup Thomsen, [email protected].

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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intracellular compartments, such as the endoplasmic reticulum(ER), endosomes, lysosomes, and endolysosomes, rather than be-ing expressed at the cell surface, which is the case for the majorityof the TLR family members (3).

TLR7. Toll-like receptor 7 (TLR7) senses ssRNA oligonucleo-tides containing guanosine- and uridine-rich sequences fromRNA viruses. This takes place in the endosomes of plasmacytoiddendritic cells (pDCs) and B cells (3, 21, 39, 61, 143). TLR7 hasbeen shown also to sense short interfering RNAs (siRNAs) (48).

pDCs comprise a subset of dendritic cells with plasmacytoidmorphology, known to secrete large amounts of type I IFN imme-diately upon viral recognition. The basis for this potent IFN re-sponse is a constitutive high expression of IRF7 in pDCs (seebelow) and a unique attribute of pDCs—retention of the TLR-bound ligands in the endosomal compartment for extended peri-ods of time (42). This increased retention of stimulating ligandsallows for a more robust downstream signaling response from theactivated TLR before the endosome fuses with lysosome and theligand is degraded.

In unstimulated cells, TLR7 is localized to the ER, and uponactivation, it rapidly traffics to the endosome in a process thatdepends on UNC93B1 and gp96 (65, 66, 117, 133).

Delivery of ssRNA to TLR7 depends on endocytosis of extra-cellular viral particles and on the cellular process of autophagy,which transports cytosolic viral replication intermediates into thelysosome (72). Notably, paramyxoviruses, which infect cells by

direct membrane fusion, effectively circumvent detection byTLR7 in the endosomes of pDCs. Consequently, the type I IFNresponse to these viruses has been shown to be independent ofTLR7 and endosome acidification (50). The IFN response wasinstead dependent on effective replication in the cytoplasm, sug-gesting RLR-mediated recognition.

TLR7 is known to recognize influenza A virus (IAV), vesicularstomatitis virus (VSV), Dengue virus, Sendai virus, and humanimmunodeficiency virus (HIV), and many unknown targets arelikely to exist in the group of RNA viruses (21, 55, 72, 76, 128).Interestingly, Kane et al. recently found that in mice from tworetrovirus-resistant strains, TLR7 recognition and the subsequentinducement of a virus-specific humoral immune response criti-cally depend upon the viral entry step. This TLR7-mediated hu-moral immune response is sufficient to impart virus clearance inthese mouse strains (55).

TLR8. Toll-like receptor 8 (TLR8) is phylogenetically andfunctionally closely related to TLR7 and recognizes ssRNA. TLR8is known to recognize HIV and likely many other RNA viruses aswell. TLR8 is preferentially expressed in myeloid DCs and mono-cytes (21, 38, 54).

Downstream signaling from TLR7 and TLR8. A common fea-ture of all TLR recognition is the activation of three major signal-ing pathways: mitogen-activated protein kinases (MAPKs), one ormore interferon regulatory factors (IRFs), and nuclear factorkappa–light-chain-enhancer of activated B cells (NF-�B). De-pending on the circumstances, IRF3 and/or IRF7 are essential forinduction of type I IFNs, while the MAPKs activates activatorprotein 1 (AP-1, a heterodimer of activating transcription factor 2with c-JUN), which together with NF-�B induce the expression ofgenes required for inflammation and adaptive immune activation,including IL-1�, IL-6, IL-18, and tumor necrosis factor (TNF) (1).

Specifically, upon stimulation, TLR7 and TLR8 recruit a TIR-containing adaptor named myeloid differentiation primary re-sponse gene 88 (MyD88) to the cytoplasmic TIR domain of thereceptor (82) (Fig. 1). MyD88 consists of a TLR-binding TIR do-main in the C-terminal portion and a death domain in theN-terminal part, and through the latter it forms a complex withtwo interleukin-1 receptor-associated kinases (IRAKs), IRAK-4and IRAK-1. Upon activation, IRAK-4 phosphorylates IRAK-1;activated IRAK-1 then binds the C-terminal domain of TNFreceptor-associated factor 6 (TRAF6), and this IRAK-1/TRAF6complex then dissociates from the TLR. Upon activation, TRAF6performs K63-linked polyubiquitination of tumor growth factorbeta (TGF-�) activated kinase 1 (TAK1) in addition to I�B kinasegamma (IKK�), also known as NEMO (NF-�B essential modula-tor) (126). IKK� subsequently associates with IKK� and IKK�.IKK� is phosphorylated by the activated TAK1 associated with theTAK1 binding protein 1 (TAB1), TAB2, and TAB3. This leads tothe IKK-mediated phosphorylation and subsequent degradationof I�B, which in the unphosphorylated state is coupled to NF-�B.NF-�B, formerly sequestered in the cytosol, is now free to enterthe nucleus to induce gene expression. TAK1 in association withTAB1, TAB2, and TAB3 also triggers a MAPK pathway leading tothe formation of AP-1. Similar to NF-�B, AP-1 enters the nucleus,and together, NF-�B and AP-1 induce the expression of proin-flammatory genes (1).

IRF5 and IRF7 also interact with the complex of IRAKs andTRAF6. This leads to IRAK1-dependent phosphorylation andsubsequent nuclear translocation of both molecules (118, 122).

TABLE 1 Individual TLRs and the RNA viruses recognized by thema

Receptor Virus Ligand Reference(s)

TLR7 Influenza A virus ssRNA 21Vesicular stomatitis virus ssRNA 76Human immunodeficiency

virusssRNA 9, 36

Dengue virus ssRNA 128Sendai virus ssRNA 72Lactate dehydrogenase-

elevating virusssRNA 6

Mouse mammary tumor virus ssRNA 55Murine leukemia virus ssRNA 55

TLR8 Human immunodeficiencyvirus

ssRNA 21, 54

TLR3 Reoviridae dsRNA 62, 129Respiratory syncytial virus dsRNA 62, 129West Nile virus dsRNA 17, 62, 129Coxsackievirus B3 dsRNA 88Poliovirus dsRNA 91Influenza A virus dsRNA 34Punta Toro virus dsRNA 33

TLR2 Measles virus HA 11Lymphocytic

choriomeningitis virus? 141, 142

Hepatitis C virus Core protein/NS3 22

TLR4 Respiratory syncytial virus Fusion protein 70Coxsackievirus B4 ? 121Mouse mammary tumor virus Envelope protein 97Murine leukemia virus Envelope protein 97

a ?, ligand unknown.

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While IRF5 is involved primarily in regulating the induction ofproinflammatory cytokines (e.g., IL-6 and IL-12p40) (118), thismediator is also essential for in vivo resistance to infection withVSV and herpes simplex virus (HSV). Thus, while infected mouseembryonic fibroblasts from IRF5-deficient mice produce normalamounts of type I IFN, virus-challenged mice exhibit increasedmortality and reduced serum levels of IL-6 and type I IFN (IFN-�and IFN-�) (132). Unlike IRF5, IRF7 is a key mediator in TLR7/TLR8-dependent type I IFN production (43, 45), and mice defi-cient in the expression of IRF7 in hematopoietic cells fail to pro-

duce significant amounts of IFN-� systemically and are oftenmore susceptible to systemic viral infection (71, 114). The consti-tutively high expression of IRF7, required for complex formation,is a unique feature of the pDCs and is not seen for other cells (44,45, 63); however, in conventional DCs (cDCs), it is possible thatIRF1 may take over as the mediator of type I IFN production(107).

TLR3. Toll-like receptor 3 (TLR3) recognizes dsRNA, whichconstitutes the genome of some RNA viruses, e.g., rotavirus (areovirus), and is a viral replication intermediate of ssRNA viruses

FIG 1 Signaling cascade following TLR2/TLR4, TLR7/TLR8, and TLR3 activation with envelope glycoprotein, ssRNA, and dsRNA, respectively. MyD88 andTRIF are the adaptor proteins that initiate the different pathways activating IRF3, IRF7 (pDCs only), NF-�B, and AP-1 in order to induce the antiviral response.NF-�B and AP-1 combine to induce the transcription of inflammatory cytokines. IRF3/IRF7 in association with both NF-�B and AP-1 form the enhanceosomefor the transcription of IFN-�/IFN-�, respectively. In addition, IRF7 also has some IFN-�-stimulatory effect (not shown in the figure). (. . .), pathway outlinedelsewhere in the illustration.

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(4, 10, 16, 130). TLR3 is localized to the intracellular compartmentin macrophages, B lymphocytes, and cDCs and is found both in-tracellularly and on the surfaces of NK cells, epithelial cells, andfibroblasts (49, 62, 80, 92).

RNA virus invasion upregulates type I IFN expression viaTLR7 and TLR8 and via constitutively expressed TLR3 (see be-low). This enhanced IFN expression induces TLR3 gene transcrip-tion in many cell types (84). TLR3 function depends on UNC93B(117), but this dependence is not related to the trafficking of TLR3from the ER to the endosome as is seen for TLR7 and TLR9 (adsDNA sensor) (65).

Downstream signaling from TLR3 occurs in much the sameway as TLR7 and -8 signaling. Upon ligand binding to the TLR3ectodomain, TLR3 dimerizes (3, 74, 89). This brings together theTIR domains of TLR3s and causes the recruitment of TIR domain-containing adaptor protein inducing IFN-� (TRIF), representinga specific adaptor shared only with TLR4 among the TLRs (131).

As outlined in Fig. 1, TRIF recruits TRAF6 for the activationand translocation of NF-�B and AP-1 by following the same pathof activation via TAK1 as that seen for TLR7 and TLR8 signal-ing described above. In addition to this pathway, a TAK1-independent pathway of NF-�B activation is also triggered. Thispathway is initiated when receptor-interacting protein 1 (RIP1)binds to the non-TIR region of TRIF and subsequently convergeson IKK�, which is also used by the TAK1-dependent route (83).

TRIF also recruits TRAF3 for association with TRAF familymember-associated NF-�B activator (TANK)-binding kinase 1(TBK1), and IKK�. TBK1/IKK� subsequently phosphorylatesIRF3 (24, 112); upon phosphorylation, IRF3 dimerizes and trans-locates to the nucleus to initiate transcription of type I IFNs(IFN-� and IFN-�4). In a positive feedback system, these type IIFNs, among many other effects, upregulate the level of IRF7 ex-pression in responding cells. IRF7, when upregulated, is phos-phorylated by TBK1/IKK�, as is IRF3. Dimerized IRF7 then stim-ulates further type I IFN release (entire range of IFN-� species)(61). Furthermore, as type I IFN release stimulates the expressionof TLR3 in cells that were initially TLR3 negative, this adds to thepositive feedback loop, enhancing the capacity to provide the an-tiviral response. In addition to the TLR3-mediated expression oftype I IFNs and inflammatory cytokines, TLR3 activation alsoprovides a link between the innate immune system and the adap-tive immune system. TLR3 activation in CD8�� DCs enhancesthe efficiency of cross-presentation of peptide fragments fromapoptotic bodies, derived from virally infected cells. These aretaken up by the CD8�� DCs and presented to CD8� T lympho-cytes (108).

Interestingly, a recent study by Negishi et al. suggests a noveltarget of TLR3-TRIF signaling, in which TLR3 signaling via TRIFdirectly induces a type II IFN (i.e., IFN-�) response. This IFN-�response was shown to be critical in establishing protectionagainst coxsackievirus B3 (CVB3) infection (88).

In addition to CVB3, the known RNA viruses targeted byTLR3 include reoviruses (dsRNA viruses), respiratory syncytialvirus (RSV; ssRNA virus), West Nile virus (WNV; ssRNA vi-rus), poliovirus (PV; ssRNA virus), IAV, and a phlebovirus(Punta Toro virus [PTV]; ssRNA virus) (17, 33, 34, 62, 91).Notably, in the case of WNV, RSV, IAV, and PTV, some studiessuggest that the cytokine expression induced by TLR3 (partic-ularly IL-6) may have a detrimental rather than a protectiveeffect on the host (33, 73, 101, 129).

TLR2. Toll-like receptor 2 (TLR2) differs from the majority ofPRRs recognizing viral invasion, as it does not sense nucleic acidmotifs; instead, TLR2 recognizes viral glycoproteins. TLR2 is ex-pressed in particular on the cell surface of most immune cells (85,119). It is believed that TLR2 forms a heterodimer with corecep-tors TLR1 or TLR6 and recognizes virion components, such aspeptide sequences of envelope glycoproteins (106). Thus, TLR2has been shown to recognize the hemagglutinin (HA) protein ofmeasles virus (11) and to initiate a response to infection withlymphocytic choriomeningitis virus (LCMV; a murine arenavi-rus) (141, 142). TLR2 has also been associated with hepatitis Cvirus (HCV)-induced inflammation via detection of HCV coreand nonstructural 3 (NS3) proteins (15, 22). However, a recentstudy has questioned the in vivo impact of this association, asHoffmann et al. concluded that only monomeric HCV core pro-tein but not core protein as part of intact nucleocapsid and subvi-ral HCV particles is sensed by TLR2 (41). Therefore, TLR2 ex-pressed at the plasma membrane will be able to bind its relevantligand only when monomeric core protein is released from disin-tegrated infected cells containing degraded nucleocapsids. How-ever, as some studies indicate that TLR2 is also expressed in theintracellular compartment, this could support a role for TLR2 inthe sensing of HCV core protein (67, 124).

TLR2 signaling involves the recruitment to the TIR domainsof the TLR2/TLR1 or TLR2/TLR6 complex of an adaptor namedTIR domain-containing adaptor protein/MyD88-adaptor-like(TIRAP/Mal) (Fig. 1) (25, 46). TIRAP/Mal binds MyD88 to initi-ate the same signaling pathway as that outlined previously forTLR7 and TLR8, culminating in a proinflammatory cytokines re-sponse through NF-�B (127), but TLR2 activation may also exertsome effect on the expression of type I IFNs (8).

TLR4. Toll-like receptor 4 (TLR4) has been found to partici-pate in antiviral defense to RNA viruses in human cells by recog-nizing the fusion protein of respiratory syncytial virus (RSV) (70),and the induction of cytokines in response to coxsackievirus B4(CVB4) depends on TLR4 (121). In addition, TLR4 is known torecognize the envelope (Env) proteins of both mouse mammarytumor virus (MMTV; a murine retrovirus) and murine leukemiavirus (MuLV; a murine retrovirus), and the subsequent triggeringof DC maturation and proinflammatory cytokine expression de-pends on TLR4 functionality (13, 97). Upon activation anddimerization of TLR4, two adaptors are known to be recruited tothe TIR domains (as shown in Fig. 1). TIRAP, which was describedabove, is one, while the other, TRIF-related adaptor molecule(TRAM), is unique to the TLR4 signaling pathway and recruitsTRIF, also described previously. Consequently, TLR4 ligationleads to signaling through both the MyD88-dependent pathway asutilized by TLR7, TLR8, and TLR2 and the MyD88-independentpathway shared only with TLR3 (120).

RIG-I-LIKE RECEPTORS AND OTHER CYTOSOLICNUCLEOTIDE RECEPTORS

The RIG-I-like receptors (RLRs) are cytosolic proteins recogniz-ing viral RNA species. Expressed by most cells of the human or-ganism, though most thoroughly studied for fibroblasts andcDCs, RLRs belong to the family of aspartate-glutamate-anyamino acid-aspartate/histidine (DExD/H)-box helicases and in-clude three members of relevance: the retinoic acid-induciblegene I product (RIG-I), melanoma differentiation-associated an-tigen 5 (MDA5), and laboratory of genetics and physiology 2

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(LGP2) (57, 134–136). Studies of gene-deficient mice indicate thatRLRs are critical sensors of viral infection in most cell types exceptpDCs, which preferentially employ TLRs for detection of RNAvirus infection (59). However, as mentioned above, in response toviruses that circumvent the endosomal TLRs of the pDCs bydirect membrane fusion (e.g., paramyxoviruses), cytosolic rec-ognition by RLRs is assumed to be of great importance also inpDCs. RLRs are expressed at low concentrations in the restingcell, and these concentrations are greatly increased upon stim-ulation (56, 135, 136).

RIG-I. Retinoic acid-inducible gene I (RIG-I) contains anssRNA/dsRNA (ss/dsRNA)-binding C-terminal domain (CTD)which, when unbound, functions as a repressor domain (RD)(103). Upon binding to viral RNA structures produced duringviral replication, two repeats of a cysteine-aspartic protease(caspase)-recruiting domain (CARD)-like region at the N termi-nus are exposed. These are then able to interact with other CARD-containing proteins to trigger downstream signaling events. Themiddle portion of the RIG-I protein contains the DExD/H heli-case domain with an ATP-binding motif. In the inactive state,RIG-I adopts a closed structure with unexposed CARD. The RDspecifically recognizes virus-associated RNA species, includingdsRNA and 5=-triphosphate ssRNA (60), which differ from self-ssRNA, such as mRNA and tRNA, by having an exposed 5=-triphosphate. Unlike some viral RNA, self-mRNA is inevitablycapped posttranscriptionally with a 5=-7-methylguanosine cap,which is subject to 2=-O methylation. While the N-7-methylationis important for stability and translation of the RNA, the 2=-Omethylation is suggested to be introduced solely to distinguishself- from nonself (e.g., viral)-5=-capped RNA (18). Self-tRNAundergoes 5= cleavage and a series of nucleotide base modifica-tions. Association with the ribosome also protects tRNA frombeing recognized by RIG-I (47). Recent studies suggest a require-ment for virus-associated polyuridine-rich sequences in thessRNA to be recognized by RIG-I in addition to the unmasked5=-triphosphate (104). Research also suggests that a 3=-phosphoryl group can replace the 5=-triphosphate for recognitionby RIG-I (see below) (78).

Ligand binding combined with ATP binding by the helicasedomain unmask the RIG-I CARD, allowing it to interact with theCARD of beta interferon promoter stimulator 1 (IPS-1). IPS-1 islocated at the cytosolic face of the outer mitochondrial mem-brane, and this mitochondrial association is necessary for initiat-ing further signaling events (64, 110). RIG-I is important in rec-ognizing and initiating cytokine production in response to a widerange of viruses from many different families, including Flaviviri-dae (27, 60, 75, 103, 115), Paramyxoviridae (35, 59, 75, 94, 135),Rhabdoviridae (59, 135), Orthomyxoviridae (60, 75), Arenaviridae(35, 140), and a bunyavirus (35) and in the recognition of Ebolavirus (a filovirus). The latter recognition is antagonized by theEbola virus V35 protein, as this secreted protein binds the dsRNAligand, preventing activation of RIG-I-mediated signaling (14,109). Table 2 contains a detailed list of known viral targets ofRIG-I. Many unknown targets are likely to exist.

MDA5. Melanoma differentiation-associated antigen 5(MDA5) is very homologous to RIG-I, and they exhibit the sameoverall domains. Upon binding of long dsRNA fragments (at least2 kbp), MDA5 exposes a CARD and initiates cytokine and type IIFN production via IPS-1 similarly to RIG-I (see below) (57). Inuninfected cells, long dsRNA is not normally present and there-

fore represent an effective PAMP signifying viral invasion. MDA5is crucial for triggering a cytokine response to an invasion withpicornaviruses, such as encephalomyocarditis virus (EMCV),Theiler’s virus, and Mengo virus (31, 60). MDA5 has also beenshown to be of importance in the antiviral response to Sendai virus(32) and in cooperation with RIG-I as well as WNV, rabies virus,Dengue virus, and rotavirus (23, 27, 75, 109). MDA5 is also re-sponsible for the recognition of murine hepatitis virus (a corona-virus) and murine norovirus 1 (a calicivirus) (81, 99). Table 2contains a list of known viral targets of MDA5.

LGP2. Laboratory of genetics and physiology 2 (LGP2) is thethird member of the RLRs and is less well-characterized. TheLGP2 gene lacks the region encoding CARD in RIG-I and MDA5.Since this region is responsible for the association with IPS-1 andtherefore further signaling events, LGP2 is thought to be a nega-tive regulator of RLR signaling via interaction between the RD ofLGP2 and that of RIG-I (100, 135). Studies with mice show in-creased responses to VSV in LGP2-deficient cells (125). VSV isknown to be specifically recognized by RIG-I rather than byMDA5. In contrast, some evidence suggests that LGP2 deficiencyreduces the response to infection with EMCV, which is known tobe recognized preferentially by MDA5 (125). This correlates witha positive regulatory effect on the MDA5 response. Thus, bothpositive and negative effects have been observed, depending onthe virus studied and the RLR involved in recognizing this virus(125). LGP2 is therefore assumed to be a modulator of the innateimmune response to a viral infection and not a sensor of PAMPs inthat LGP2 does not initiate antiviral gene expression. A study by

TABLE 2 Individual RLRs and the RNA viruses recognized by them

Receptor Virus Ligand Reference

RIG-I Sendai virus ss/dsRNA 59, 135Newcastle disease virus ss/dsRNA 59Respiratory syncytial virus ss/dsRNA 75Measles virus ss/dsRNA 94Nipah virus ss/dsRNA 35Vesicular stomatitis virus ss/dsRNA 59, 135Rabies virus ss/dsRNA 23Influenza A virus ss/dsRNA 60Influenza B virus ss/dsRNA 75Ebola virus ss/dsRNA 14, 35Lassa virus ss/dsRNA 35Lymphocytic choriomeningitis

virusss/dsRNA 140

Rift Valley fever virus ss/dsRNA 35Japanese encephalitis virus ss/dsRNA 60Hepatitis C virus ss/dsRNA 103, 115West Nile virus ss/dsRNA 27Dengue virus ss/dsRNA 75Rotavirus ss/dsRNA 109

MDA5 Encephalomyocarditis virus dsRNA 31, 60Theiler’s virus dsRNA 60Mengo virus dsRNA 60Rabies virus dsRNA 23West Nile virus dsRNA 27Sendai virus dsRNA 32Dengue virus dsRNA 75Rotavirus dsRNA 109Murine hepatitis virus dsRNA 99Murine norovirus 1 dsRNA 81

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Pippig et al. (93) showed that LGP2 binds dsRNA independent of5=-triphosphates. The authors suggest that LGP2 mediates themodulator role by inhibiting RIG-I signaling via competitive in-teraction with viral dsRNA species in one scenario, while in an-other, enhancing the ability of MDA5 to sense long dsRNA struc-tures by complexing with MDA5 (93).

RNase L. RNase L is an endoribonuclease that has been asso-ciated with antiviral defense (77). It is known that RNaseL-deficient mice are compromised in their response to infectionwith EMCV, WNV, and CVB4 (26, 105, 139). Furthermore, anincrease in prostate infections with xenotropic murine leukemiavirus-related virus (XMRV, a retrovirus) is seen in humans ho-mozygotic for a variant of RNase L with reduced activity (123).Viral dsRNA activates 2=-5=-oligoadenylate synthetase (OAS) toincrease the concentration of 2=,5=-linked oligoadenylate (2-5A)from cytosolic ATP, and this activates RNase L (113). Malathi etal. propose a model for the antiviral effect of RNase L, in whichRNase L, upon activation, cleaves single-stranded regions of RNAto form small pieces of 5=-hydroxylated, 3=-phosphorylated RNAs(78). Substrates for the RNase cleavage process include, in addi-tion to viral RNAs, cellular self-RNA (77). RNase L produces smallRNAs, often in the form of duplexes, which are shown to activateRIG-I and MDA5, and via IPS-1 result in an IRF3-mediatedIFN-� response (see below). The authors show that the 3=-phosphoryl groups of the RNA products are important to thedownstream antiviral activity.

According to this model, by recruiting and cleaving host RNA,RNase L via OAS amplifies the antiviral response to virally derivedRNA by increasing the amount of ligand involved in RIG-I andMDA5 recognition. OAS is induced by type I IFNs, which uponbinding to type I IFN receptors on surrounding cells triggers anassociated JAK kinase and via STAT induces the expression ofIFN-inducible antiviral gene products, such as protein kinase R(PKR) and OAS (119).

Downstream signaling from RIG-I and MDA5. Both RIG-Iand MDA5 interact with the adaptor IPS-1 through CARD re-peats, and IPS-1-deficient mice are impaired in the production ofproinflammatory cytokines and type I IFN in response to all RNAviruses recognized by RIG-I and MDA5 (69, 116), indicating a keyrole of IPS-1 in downstream signaling from RIG-I and MDA5.IPS-1 itself is probably not directly involved in the signaling pro-cess but serves as a platform to orchestrate the molecular interac-tions which subsequently lead to the activation of IRF3 and NF-�B(136). Recently, another adaptor, stimulator of IFN genes(STING, also called MITA) was described (52, 138). STING is alsofound in the mitochondrial membrane but resides predominantlyin the endoplasmic reticulum. Considering that STING interactswith RIG-I and IPS-1 in the mitochondrial membrane, this po-tentially opens the possibility for cross talk between the two or-ganelles in viral sensing (7). This is of considerable interest in viewof the fact that many viruses replicate in the membranous webconnecting these organelles (98). However, the precise impor-tance of such interactions is not clear at this moment.

As shown in Fig. 2, IPS-1 coordinates the activation of two ofthe same pathways as those activated by TRIF during downstreamsignaling from TLR3 (cf. Fig. 1), namely, the activation of RIP1leading to NF-�B nuclear translocation and the activation of IKKs,mediating phosphorylation of IRF3 (40). Central in the initiationof both pathways is the TNF receptor-associated death domain(TRADD), which is recruited to IPS-1 and coordinates interac-

tions with relevant downstream molecules. Thus, the IPS-1/TRADD complex recruits TRAF3, which together with TANK andIKK�/NEMO initiates the activation of IKKs. Similarly, this com-plex recruits RIP1 (in a complex with the Fas-associated deathdomain [FADD]) for the initiation of the NF-�B pathway. Fol-lowing IRF3-mediated type I IFN release, IRF7 gene expressionmay be induced in cell types not constitutively expressing the IRF7gene (i.e., all cell types except pDCs), leading to enhancement ofthe antiviral IFN response.

As RLR signaling converges on pathways also utilized by theTLRs, the induced gene expression is also similar, leading to syn-thesis and release of type I IFNs and proinflammatory cytokines inorder to launch an antiviral inflammatory response (61, 64).

RLR signaling is modified by ubiquitination, direct protein in-teractions, and caspase activity in an elaborate network of bothpositive and negative regulation. Numerous ubiquitinating agentshave been discovered, of which the RING finger protein, Riplet,and the E3 ubiquitin ligase, tripartite motif 25 (TRIM25), are no-tably in activating RIG-I through K63 ubiquitin ligation. Themodifications imparted by TRIM25 are crucial to the interactionbetween RIG-I and IPS-1 (29, 30, 90). Zinc finger antiviral proteinshorter isoform (ZAPS) has recently been suggested to be a keyregulator of RIG-I function by direct protein interaction with thereceptor to promote oligomerization and ATPase activity (37).Negative regulation by direct protein interaction is found both atthe level of IPS-1, e.g., the proteasomal degradation of IPS-1 fol-lowing binding of PSMA7(�4), and also of individual receptors,e.g., LGP2 inhibition of RIG-I (see above) and DAK inhibition ofMDA5 (20, 37, 53). Caspase-8 has recently been shown to nega-tively regulate RLR signaling by associating with the assembledIPS-1 signalosome and cleaving RIP1 (96).

In addition to the signaling pathway described above, researchalso suggests that RIG-I activation can trigger inflammasome for-mation and cysteine-aspartic protease 1 (caspase-1) activity, lead-ing to the maturation of proinflammatory cytokines such asinterleukin-1� (IL-1�) (95). This IPS-1-independent pathway isalso used by NLRP3 as described next.

NOD-LIKE RECEPTORS AND INFLAMMASOME FORMATION

Nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLR)— or, according to newer nomenclature,nucleotide-binding domain, leucine-rich repeat-containing(NLR) proteins—are cytosolic proteins regulating inflammatoryand apoptotic responses, and recent studies point to the impor-tance of these receptors in the antiviral defense. Table 3 contains alist of known viral targets. As the new nomenclature implies, theseproteins contain an LRR motif located at the C terminus. The LRRdomain is considered to be the sensor region of the NLRs. Cen-trally located is a NACHT (NAIP, CIITA, HET-E, TP-1) domainthat mediates oligomerization and activation. In the N terminus,an effector-binding domain, most often a CARD or a pyrin do-main (PYD), signals downstream following induced proximityupon activation and oligomerization of the NLRs (28, 68).

NLRP3. NOD-like receptor family, pyrin domain-containing3 (NLRP3) contains a pyrin domain (PYD) which can interactwith the N-terminal PYD of apoptosis-associated speck-like pro-tein containing a CARD (ASC). As the name implies, ASC con-tains a CARD domain in the C terminus (19, 58, 79, 86).

NLRP3 oligomerizes upon activation and recruits ASC andprocaspase-1 to form an inflammasome complex. This activates

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caspase-1, which subsequently mediates the conversion of pro-IL-1� and pro-IL-18 to fully functional IL-1� and IL-18 (Fig. 2)(137). Activation is observed to occur upon infection with adeno-virus (dsDNA virus), Sendai virus (ssRNA virus), and IAV (ssRNAvirus) (5, 51, 58, 86). The details concerning the specific stimuliinitiating aggregation of the NLRP3 inflammasome are still unre-solved. Furthermore, the response depends on lysosomal matura-tion and generation of reactive oxygen species (ROS). This is thebasis for the hypothesis that NLRP3 is activated through commonintracellular changes, such as lysosomal disintegration, mem-brane disruption, or generation of ROS, caused either by viral

FIG 2 Left half, signaling via IPS-1 following dsRNA/ssRNA recognition by RIG-I and MDA5. IPS-1 serves as a platform to coordinate the activation of two ofthe signaling pathways also utilized by the TLRs (cf. Fig. 1), that is, the activation of RIP1 for NF-�B nuclear translocation and the activation of IKKs for IRF3phosphorylation and translocation, leading to the expression of proinflammatory cytokines and IFN-�, respectively. Right half, formation of inflammasomes byNLRP3. Inactive NLRP3 oligomerizes upon stimulation by an unknown mechanism. ASC becomes associated with the complex. This leads to the recruitment ofprocaspase-1, which dimerizes and autoactivates by proteolytic cleavage, generating active caspase-1. Caspase-1 mediates the activation of IL-18 and IL-1� frominactive pro-IL-18 and pro-IL-1�, respectively. (. . .), the continuation of this pathway is outlined in Fig. 1.

TABLE 3 Individual NLRs and the RNA viruses recognized by thema

Receptor Virus Ligand Reference

NLRP3 Influenza A virus Virus¡cell stress? 58Sendai virus Virus¡cell stress? 58

NLRC2 Respiratory syncytial virus ssRNA 102Influenza A virus ssRNA 102Parainfluenza virus ssRNA 102

a Virus¡cell stress?, virus-induced cell stress.

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PAMPs or endogenous danger-associated molecular patterns(DAMPs), rather than through a direct interaction of NLRP3 withthese ligands. NLRP3 is therefore most likely an indirect sensor ofviral invasion (5).

NLRC2. NOD-like receptor family, CARD-containing 2(NLRC2) has recently been shown to recognize ssRNA speciesderived from RSV, influenza A virus, and parainfluenza virus,making NLRC2 the only NLR reported to directly sense viral com-ponents (102, 111). Upon recognition, NLRC2 associates withIPS-1 through an interaction dependent upon the LRR andnucleotide-binding domains (NBDs) of NLRC2. This initiates theIPS-1-dependent pathway previously described for the RLRs andculminates in type I IFN and proinflammatory cytokine release. Incontrast to the RLRs, the NLRC2 interaction with IPS-1 does notinvolve CARD-CARD binding (102).

NLRC5. NOD-like receptor family, CARD-containing 5(NLRC5) participates in the response to RNA viruses but has notbeen reported as having a direct sensor function (68, 87). NLRC5is highly expressed in hematopoietic cells and is further induced inmany cell types following viral invasion. NLRC5 contains a CARDdomain and a NACHT domain but differs from other members ofthe NLR family by having a longer LRR domain that consequentlyadopts a different structure from the conventional horseshoeshape, perhaps forming a helical shape as suggested by Neerincx etal. (68, 87).

Kuenzal et al. (68) showed that NLRC5 gene expression ismarkedly increased upon infection with human cytomegalovirus(HCMV) and that this response depends on the IFN-�-inducedJAK-STAT (Janus kinase-signal transducer and activator of tran-scription) pathway. Apart from being a target of antiviral signal-ing, NLRC5 has also been identified as an important activator ofantiviral signaling pathways. This suggests a role of NLRC5 as anendogenous amplifier of antiviral responses without sensor func-tion (68, 87).

CONCLUSION

Nucleic acid motifs are the main virus-derived PAMPs to be rec-ognized by the innate immune system. Viral ssRNA is distin-guished from the capped mRNA of the host by an exposed 5=-triphosphate moiety in a subset of viral RNA and is sensed byTLR7 and TLR8 in the endosomes of pDCs and myeloid DCs andby RIG-I in the cytosol of many cell types. Viral dsRNA is sensedby RIG-I and MDA5 in the cytosol, by TLR3 in the endosomecompartment in innate immune cells, and by certain NLRs. Inaddition to these first-line sensors, RNase L amplifies the RLRresponse, and IFN-inducible antiviral proteins such as PKR andOAS are upregulated upon viral recognition to control the infec-tion. Autophagy is utilized to bring cytosolic RNA species to theendosomal compartment. The potential importance of under-standing innate recognition of viral invasion cannot be overstated.The role of the innate antiviral response in protecting the hostduring the early phase of a viral infection has been known for along time. More recently, it has also become apparent that theinnate response greatly influences the formation of the subse-quent adaptive response. Consequently, clarifying the underlyingprocesses and the many ways in which viruses have evolved to tryand evade innate immunity is extremely important with regard toour general understanding of viral pathogenesis, and is the onlyway in which we will stand reasonably prepared for the nextemerging virus disease.

REFERENCES1. Akira S, Takeda K. 2004. Toll-like receptor signalling. Nat. Rev. Immu-

nol. 4:499 –511.2. Akira S, Takeda K, Kaisho T. 2001. Toll-like receptors: critical proteins

linking innate and acquired immunity. Nat. Immunol. 2:675– 680.3. Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and

innate immunity. Cell 124:783– 801.4. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. 2001. Recognition

of double-stranded RNA and activation of NF-kappa B by Toll-like re-ceptor 3. Nature 413:732–738.

5. Allen IC, et al. 2009. The NLRP3 inflammasome mediates in vivo innateimmunity to influenza A virus through recognition of viral RNA. Immu-nity 30:556 –565.

6. Ammann CG, Messer RJ, Peterson KE, Hasenkrug KJ. 2009. Lactatedehydrogenase-elevating virus induces systemic lymphocyte activationvia TLR7-dependent IFNalpha responses by plasmacytoid dendriticcells. PLoS One 4:e6105.

7. Arnoult D, Carneiro L, Tattoli I, Girardin SE. 2009. The role ofmitochondria in cellular defense against microbial infection. Semin. Im-munol. 21:223–232.

8. Barbalat R, Lau L, Locksley RM, Barton GM. 2009. Toll-like receptor2 on inflammatory monocytes induces type I interferon in response toviral but not bacterial ligands. Nat. Immunol. 10:U1200 –U1287.

9. Beignon AS, et al. 2005. Endocytosis of HIV-1 activates plasmacytoiddendritic cells via toll-like receptor-viral RNA interactions. J. Clin. In-vest. 115:3265–3275.

10. Bell JK, Askins J, Hall PR, Davies DR, Segal DM. 2006. The dsRNAbinding site of human toll-like receptor 3. Proc. Natl. Acad. Sci. U. S. A.103:8792– 8797.

11. Bieback K, et al. 2002. Hemagglutinin protein of wild-type measles virusactivates Toll-like receptor 2 signaling. J. Virol. 76:8729 – 8736.

12. Bowie A, O’Neill LAJ. 2000. The interleukin-1 receptor/Toll-like recep-tor superfamily: signal generators for pro-inflammatory interleukins andmicrobial products. J. Leukoc. Biol. 67:508 –514.

13. Burzyn D, et al. 2004. Toll-like receptor 4-dependent activation ofdendritic cells by a retrovirus. J. Virol. 78:576 –584.

14. Cardenas WB, et al. 2006. Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced byRIG-I signaling. J. Virol. 80:5168 –5178.

15. Chang S, Dolganiuc A, Szabo G. 2007. Toll-like receptors 1 and 6 areinvolved in TLR2-mediated macrophage activation by hepatitis C viruscore and NS3 proteins. J. Leukoc. Biol. 82:479 – 487.

16. Choe J, Kelker MS, Wilson IA. 2005. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309:581–585.

17. Daffis S, Samuel MA, Suthar MS, Gale M, Diamond MS. 2008. Toll-like receptor 3 has a protective role against West Nile virus infection. J.Virol. 82:10349 –10358.

18. Daffis S, et al. 2010. 2=-O methylation of the viral mRNA cap evades hostrestriction by IFIT family members. Nature 468:452– 456.

19. Delaloye J, et al. 2009. Innate immune sensing of modified vacciniavirus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and theNALP3 inflammasome. PLoS Pathog. 5:e1000480.

20. Diao F, et al. 2007. Negative regulation of MDA5- but not RIG-I-mediated innate antiviral signaling by the dihydroxyacetone kinase.Proc. Natl. Acad. Sci. U. S. A. 104:11706 –11711.

21. Diebold SS, Kaisho T, Hemmi H, Akira S, Sousa CRE. 2004. Innateantiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303:1529 –1531.

22. Dolganiuc A, et al. 2004. Hepatitis C core and nonstructural 3 proteinstrigger toll-like receptor 2-mediated pathways and inflammatory activa-tion. Gastroenterology 127:1513–1524.

23. Faul EJ, et al. 2010. Rabies virus infection induces type I interferonproduction in an IPS-1 dependent manner while dendritic cell activationrelies on IFNAR signaling. PLoS Pathog. 6:e1001016.

24. Fitzgerald KA, et al. 2003. IKKepsilon and TBK1 are essential compo-nents of the IRF3 signaling pathway. Nat. Immunol. 4:491– 496.

25. Fitzgerald KA, et al. 2001. Mal (MyD88-adapter-like) is required forToll-like receptor-4 signal transduction. Nature 413:78 – 83.

26. Flodstrom-Tullberg M, et al. 2005. RNase L and double-stranded RNA-dependent protein kinase exert complementary roles in islet cell defenseduring coxsackievirus infection. J. Immunol. 174:1171–1177.

27. Fredericksen BL, Keller BC, Fornek J, Katze MG, Gale M, Jr. 2008.

Minireview



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org

http://jvi.asm.org/

Establishment and maintenance of the innate antiviral response to WestNile virus involves both RIG-I and MDA5 signaling through IPS-1. J.Virol. 82:609 – 616.

28. Fritz JH, Ferrero RL, Philpott DJ, Girardin SE. 2006. Nod-like proteinsin immunity, inflammation and disease. Nat. Immunol. 7:1250 –1257.

29. Gack MU, et al. 2007. TRIM25 RING-finger E3 ubiquitin ligase is es-sential for RIG-I-mediated antiviral activity. Nature 446:U916 –U912.

30. Gao D, et al. 2009. REUL is a novel E3 ubiquitin ligase and stimulator ofretinoic-acid-inducible gene-I. PLoS One 4:e5760.

31. Gitlin L, et al. 2006. Essential role of mda-5 in type IIFN responses topolyriboinosinic:polyribocytidylic acid and encephalomyocarditis pi-cornavirus. Proc. Natl. Acad. Sci. U. S. A. 103:8459 – 8464.

32. Gitlin L, et al. 2010. Melanoma differentiation-associated gene 5(MDA5) is involved in the innate immune response to Paramyxoviridaeinfection in vivo. PLoS Pathog. 6:e1000734.

33. Gowen BB, et al. 2006. TLR3 deletion limits mortality and diseaseseverity due to phlebovirus infection. J. Immunol. 177:6301– 6307.

34. Guillot L, et al. 2005. Involvement of toll-like receptor 3 in the immuneresponse of lung epithelial cells to double-stranded RNA and influenza Avirus. J. Biol. Chem. 280:5571–5580.

35. Habjan M, et al. 2008. Processing of genome 5= termini as a strategy ofnegative-strand RNA viruses to avoid RIG-I-dependent interferon in-duction. PLoS One 3:e2032.

36. Hardy AW, Graham DR, Shearer GM, Herbeuval J-P. 2007. HIV turnsplasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDCand down-regulates HIV coreceptors by Toll-like receptor 7-inducedIFN-alpha. Proc. Natl. Acad. Sci. U. S. A. 104:17453–17458.

37. Hayakawa S, et al. 2011. ZAPS is a potent stimulator of signaling medi-ated by the RNA helicase RIG-I during antiviral responses. Nat. Immu-nol. 12:U37–U56.

38. Heil F, et al. 2004. Species-specific recognition of single-stranded RNAvia toll-like receptor 7 and 8. Science 303:1526 –1529.

39. Hemmi H, et al. 2002. Small anti-viral compounds activate immunecells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol.3:196 –200.

40. Hemmi H, et al. 2004. The roles of two I kappa B kinase-related kinasesin lipopolysaccharide and double stranded RNA signaling and viral in-fection. J. Exp. Med. 199:1641–1650.

41. Hoffmann M, et al. 2009. Toll-like receptor 2 senses hepatitis C viruscore protein but not infectious viral particles. J. Innate Immun. 1:446 –454.

42. Honda K, et al. 2005. Spatiotemporal regulation of MyD88-IRF-7 sig-nalling for robust type-I interferon induction. Nature 434:1035–1040.

43. Honda K, Takaoka A, Taniguchi T. 2006. Type I interferon gene induc-tion by the interferon regulatory factor family of transcription factors.Immunity 25:349 –360.

44. Honda K, et al. 2004. Role of a transductional-transcriptional processorcomplex involving MyD88 and IRF-7 in Toll-like receptor signaling.Proc. Natl. Acad. Sci. U. S. A. 101:15416 –15421.

45. Honda K, et al. 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434:772–777.

46. Horng T, Barton GM, Medzhitov R. 2001. TIRAP: an adapter moleculein the Toll signaling pathway. Nat. Immunol. 2:835– 841.

47. Hornung V, et al. 2006. 5=-Triphosphate RNA is the ligand for RIG-I.Science 314:994 –997.

48. Hornung V, et al. 2005. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells throughTLR7. Nat. Med. 11:263–270.

49. Hornung V, et al. 2002. Quantitative expression of Toll-like receptor1-10 mRNA in cellular subsets of human peripheral blood mononuclearcells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531– 4537.

50. Hornung V, et al. 2004. Replication-dependent potent IFN-� inductionin human plasmacytoid dendritic cells by a single-stranded RNA virus. J.Immunol. 173:5935–5943.

51. Ichinohe T, Lee HK, Ogura Y, Flavell R, Iwasaki A. 2009. Inflam-masome recognition of influenza virus is essential for adaptive immuneresponses. J. Exp. Med. 206:79 – 87.

52. Ishikawa H, Barber GN. 2008. STING is an endoplasmic reticulumadaptor that facilitates innate immune signalling. Nature 455:674 – 678.

53. Jia Y, et al. 2009. Negative regulation of MAVS-mediated innate im-mune response by PSMA7. J. Immunol. 183:4241– 4248.

54. Jurk M, et al. 2002. Human TLR7 or TLR8 independently confer re-sponsiveness to the antiviral compound R-848. Nat. Immunol. 3:499.

55. Kane M, et al. 2011. Innate immune sensing of retroviral infection viaToll-like receptor 7 occurs upon viral entry. Immunity 35:135–145.

56. Kang DC, et al. 2004. Expression analysis and genomic characterizationof human melanoma differentiation associated gene-5, mda-5: a noveltype I interferon-responsive apoptosis-inducing gene. Oncogene 23:1789 –1800.

57. Kang DC, et al. 2002. mda-5: an interferon-inducible putative RNAhelicase with double-stranded RNA-dependent ATPase activity and mel-anoma growth-suppressive properties. Proc. Natl. Acad. Sci. U. S. A.99:637– 642.

58. Kanneganti TD, et al. 2006. Critical role for cryopyrin/Nalp3 in activa-tion of caspase-1 in response to viral infection and double-strandedRNA. J. Biol. Chem. 281:36560 –36568.

59. Kato H, et al. 2005. Cell type-specific involvement of RIG-I in antiviralresponse. Immunity 23:19 –28.

60. Kato H, et al. 2006. Differential roles of MDA5 and RIG-I helicases in therecognition of RNA viruses. Nature 441:101–105.

61. Kawai T, Akira S. 2006. Innate immune recognition of viral infection.Nat. Immunol. 7:131–137.

62. Kawai T, Akira S. 2008. Toll-like receptor and RIG-1-like receptorsignaling. Ann. N. Y. Acad. Sci. 1143:1–20.

63. Kawai T, et al. 2004. Interferon-alpha induction through Toll-like re-ceptors involves a direct interaction of IRF7 with MyD88 and TRAF6.Nat. Immunol. 5:1061–1068.

64. Kawai T, et al. 2005. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6:981–988.

65. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL. 2008. UNC93B1delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature452:U234 –U280.

66. Kiyokawa T, et al. 2008. A single base mutation in the PRAT4A genereveals differential interaction of PRAT4A with Toll-like receptors. Int.Immunol. 20:1407–1415.

67. Kobayashi M, et al. 2009. Expression of toll-like receptor 2, NOD2 anddectin-1 and stimulatory effects of their ligands and histamine in normalhuman keratinocytes. Br. J. Dermatol. 160:297–304.

68. Kuenzel S, et al. 2010. The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immuneresponses. J. Immunol. 184:1990 –2000.

69. Kumar H, et al. 2006. Essential role of IPS-1 in innate immune responsesagainst RNA viruses. J. Exp. Med. 203:1795–1803.

70. Kurt-Jones EA, et al. 2000. Pattern recognition receptors TLR4 andCD14 mediate response to respiratory syncytial virus. Nat. Immunol.1:398 – 401.

71. Lang PA, et al. 2009. Hematopoietic cell-derived interferon controlsviral replication and virus-induced disease. Blood 113:1045–1052.

72. Lee HK, Lund JM, Ramanathan B, Mizushima N, Iwasaki A. 2007.Autophagy-dependent viral recognition by plasmacytoid dendritic cells.Science 315:1398 –1401.

73. Le Goffic R, et al. 2006. Detrimental contribution of the Toll-like recep-tor (TLR)3 to influenza A virus-induced acute pneumonia. PLoS Pathog.2:e53.

74. Liu L, et al. 2008. Structural basis of toll-like receptor 3 signaling withdouble-stranded RNA. Science 320:379 –381.

75. Loo Y-M, et al. 2008. Distinct RIG-I and MDA5 signaling by RNAviruses in innate immunity. J. Virol. 82:335–345.

76. Lund JM, et al. 2004. Recognition of single-stranded RNA viruses byToll-like receptor 7. Proc. Natl. Acad. Sci. U. S. A. 101:5598 –5603.

77. Malathi K, Dong B, Gale M, Jr, Silverman RH. 2007. Small self-RNAgenerated by RNase L amplifies antiviral innate immunity. Nature 448:U816 –U819.

78. Malathi K, et al. 2010. RNase L releases a small RNA from HCV RNAthat refolds into a potent PAMP. RNA 16:2108 –2119.

79. Masumoto J, et al. 1999. ASC, a novel 22-kDa protein, aggregates duringapoptosis of human promyelocytic leukemia HL-60 cells. J. Biol. Chem.274:33835–33838.

80. Matsumoto M, et al. 2003. Subcellular localization of toll-like receptor3 in human dendritic cells. J. Immunol. 171:3154 –3162.

81. McCartney SA, et al. 2008. MDA-5 recognition of a murine norovirus.PLoS Pathog. 4:e1000108.

82. Medzhitov R, et al. 1998. MyD88 is an adaptor protein in the hToll/IL-1receptor family signaling pathways. Mol. Cell 2:253–258.

Minireview



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org

http://jvi.asm.org/

83. Meylan E, et al. 2004. RIP1 is an essential mediator of Toll-like receptor3-induced NF-kappa B activation. Nat. Immunol. 5:503–507.

84. Miettinen M, Sareneva T, Julkunen I, Matikainen S. 2001. IFNs acti-vate toll-like receptor gene expression in viral infections. Genes Immun.2:349 –355.

85. Mogensen TH. 2009. Pathogen recognition and inflammatory signalingin innate immune defenses. Clin. Microbiol. Rev. 22:240 –273.

86. Muruve DA, et al. 2008. The inflammasome recognizes cytosolic mi-crobial and host DNA and triggers an innate immune response. Nature452:103–107.

87. Neerincx A, et al. 2010. A role for the human nucleotide-binding do-main, leucine-rich repeat-containing family member NLRC5 in antiviralresponses. J. Biol. Chem. 285:26223–26232.

88. Negishi H, et al. 2008. A critical link between Toll-like receptor 3 andtype II interferon signaling pathways in antiviral innate immunity. Proc.Natl. Acad. Sci. U. S. A. 105:20446 –20451.

89. O’Neill LAJ. 2006. How Toll-like receptors signal: what we know andwhat we don’t know. Curr. Opin. Immunol. 18:3–9.

90. Oshiumi H, et al. 2010. The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell HostMicrobe 8:496 –509.

91. Oshiumi H, et al. 2011. The TLR3/TICAM-1 pathway is mandatory forinnate immune responses to poliovirus infection. J. Immunol. 187:5320 –5327.

92. Paludan SR, Bowie AG, Horan KA, Fitzgerald KA. 2011. Recognitionof herpesviruses by the innate immune system. Nat. Rev. Immunol. 11:143–154.

93. Pippig DA, et al. 2009. The regulatory domain of the RIG-I familyATPase LGP2 senses double-stranded RNA. Nucleic Acids Res. 37:2014 –2025.

94. Plumet S, et al. 2007. Cytosolic 5=-triphosphate ended viral leader tran-script of measles virus as activator of the RIG I-mediated interferon re-sponse. PLoS One 2:e279.

95. Poeck H, et al. 2010. Recognition of RNA virus by RIG-I results inactivation of CARD9 and inflammasome signaling for interleukin1[beta] production. Nat. Immunol. 11:63– 69.

96. Rajput A, et al. 2011. RIG-I RNA helicase activation of IRF3 transcrip-tion factor is negatively regulated by caspase-8-mediated cleavage of theRIP1 protein. Immunity 34:340 –351.

97. Rassa JC, Meyers JL, Zhang YM, Kudaravalli R, Ross SR. 2002. Murineretroviruses activate B cells via interaction with Toll-like receptor 4. Proc.Natl. Acad. Sci. U. S. A. 99:2281–2286.

98. Rehermann B, Nascimbeni M. 2005. Immunology of hepatitis B virusand hepatitis C virus infection. Nat. Rev. Immunol. 5:215–229.

99. Roth-Cross JK, Bender SJ, Weiss SR. 2008. Murine coronavirus mousehepatitis virus is recognized by MDA5 and induces type I interferon inbrain macrophages/microglia. J. Virol. 82:9829 –9838.

100. Rothenfusser S, et al. 2005. The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-induciblegene-I. J. Immunol. 175:5260 –5268.

101. Rudd BD, Burstein E, Duckett CS, Li XX, Lukacs NW. 2005. Differ-ential role for TLR3 in respiratory syncytial virus-induced chemokineexpression. J. Virol. 79:3350 –3357.

102. Sabbah A, et al. 2009. Activation of innate immune antiviral responsesby Nod2. Nat. Immunol. 10:1073–1080.

103. Saito T, et al. 2007. Regulation of innate antiviral defenses through ashared repressor domain in RIG-I and LGP2. Proc. Natl. Acad. Sci.U. S. A. 104:582–587.

104. Saito T, Owen DM, Jiang F, Marcotrigiano J, Gale M, Jr. 2008. Innateimmunity induced by composition-dependent RIG-I recognition ofhepatitis C virus RNA. Nature 454:523–527.

105. Samuel MA, et al. 2006. PKR and RNase L contribute to protectionagainst lethal West Nile virus infection by controlling early viral spread inthe periphery and replication in neurons. J. Virol. 80:7009 –7019.

106. Sandor F, et al. 2003. Importance of extra- and intracellular domains ofTLR1 and TLR2 in NF kappa B signaling. J. Cell Biol. 162:1099 –1110.

107. Schmitz F, et al. 2007. Interferon-regulatory-factor 1 controls Toll-likereceptor 9-mediated IFN-beta production in myeloid dendritic cells.Eur. J. Immunol. 37:315–327.

108. Schulz O, et al. 2005. Toll-like receptor 3 promotes cross-priming tovirus-infected cells. Nature 433:887– 892.

109. Sen A, Pruijssers AJ, Dermody TS, Garcia-Sastre A, Greenberg HB.2011. The early interferon response to rotavirus is regulated by PKR and

depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3. J. Virol. 85:3717–3732.

110. Seth RB, Sun LJ, Ea CK, Chen ZJJ. 2005. Identification and character-ization of MAVS, a mitochondrial antiviral signaling protein that acti-vates NF-kappa B and IRF3. Cell 122:669 – 682.

111. Shapira SD, et al. 2009. A physical and regulatory map of host-influenzainteractions reveals pathways in H1N1 infection. Cell 139:1255–1267.

112. Sharma S, et al. 2003. Triggering the interferon antiviral responsethrough an IKK-related pathway. Science 300:1148 –1151.

113. Silverman RH. 2007. Viral encounters with 2 =,5=-oligoadenylate synthe-tase and RNase L during the interferon antiviral response. J. Virol. 81:12720 –12729.

114. Steinberg C, et al. 2009. The IFN regulatory factor 7-dependent type IIFN response is not essential for early resistance against murine cytomeg-alovirus infection. Eur. J. Immunol. 39:1007–1018.

115. Sumpter R, Jr, et al. 2005. Regulating intracellular antiviral defense andpermissiveness to hepatitis C virus RNA replication through a cellularRNA helicase, RIG-I. J. Virol. 79:2689 –2699.

116. Sun Q, et al. 2006. The specific and essential role of MAVS in antiviralinnate immune responses. Immunity 24:633– 642.

117. Tabeta K, et al. 2006. The Unc93b1 mutation 3d disrupts exogenousantigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat.Immunol. 7:156 –164.

118. Takaoka A, et al. 2005. Integral role of IRF-5 in the gene inductionprogramme activated by Toll-like receptors. Nature 434:243–249.

119. Takeuchi O, Akira S. 2010. Pattern recognition receptors and inflam-mation. Cell 140:805– 820.

120. Thompson MR, Kaminski JJ, Kurt-Jones EA, Fitzgerald KA. 2011.Pattern recognition receptors and the innate immune response to viralinfection. Viruses 3:920 –940.

121. Triantafilou K, Triantafilou M. 2004. Coxsackievirus B4-induced cyto-kine production in pancreatic cells is mediated through Toll-like recep-tor 4. J. Virol. 78:11313–11320.

122. Uematsu S, et al. 2005. Interleukin-1 receptor-associated kinase-1 playsan essential role for Toll-like receptor (TLR)7- and TLR9-mediatedinterferon-alpha induction. J. Exp. Med. 201:915–923.

123. Urisman A, et al. 2006. Identification of a novel gammaretrovirus inprostate tumors of patients homozygous for R462Q RNASEL variant.PLoS Pathog. 2:e25.

124. Uronen-Hansson H, et al. 2004. Toll-like receptor 2 (TLR2) and TLR4are present inside human dendritic cells, associated with microtubulesand the Golgi apparatus but are not detectable on the cell surface: integ-rity of microtubules is required for interleukin-12 production in re-sponse to internalized bacteria. Immunology 111:173–178.

125. Venkataraman T, et al. 2007. Loss of DExD/H box RNA helicase LGP2manifests disparate antiviral responses. J. Immunol. 178:6444 – 6455.

126. Wang C, et al. 2001. TAK1 is a ubiquitin-dependent kinase of MKK andIKK. Nature 412:346 –351.

127. Wang JP, et al. 2005. Varicella-zoster virus activates inflammatory cy-tokines in human monocytes and macrophages via Toll-like receptor 2. J.Virol. 79:12658 –12666.

128. Wang JP, et al. 2006. Flavivirus activation of plasmacytoid dendritic cellsdelineates key elements of TLR7 signaling beyond endosomal recogni-tion. J. Immunol. 177:7114 –7121.

129. Wang T, et al. 2004. Toll-like receptor 3 mediates West Nile virus entryinto the brain causing lethal encephalitis. Nat. Med. 10:1366 –1373.

130. Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR. 2006.Double-stranded RNA is produced by positive-strand RNA viruses andDNA viruses but not in detectable amounts by negative-strand RNAviruses. J. Virol. 80:5059 –5064.

131. Yamamoto M, et al. 2003. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301:640 – 643.

132. Yanai H, et al. 2007. Role of IFN regulatory factor 5 transcription factorin antiviral immunity and tumor suppression. Proc. Natl. Acad. Sci.U. S. A. 104:3402–3407.

133. Yang Y, et al. 2007. Heat shock protein gp96 is a master chaperone fortoll-like receptors and is important in the innate function of macro-phages. Immunity 26:215–226.

134. Yoneyama M, Fujita T. 2007. Function of RIG-I-like receptors in anti-viral innate immunity. J. Biol. Chem. 282:15315–15318.

135. Yoneyama M, et al. 2005. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J.Immunol. 175:2851–2858.

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http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org

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136. Yoneyama M, et al. 2004. The RNA helicase RIG-I has an essentialfunction in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 5:730 –737.

137. Yu JW, et al. 2006. Cryopyrin and pyrin activate caspase-1, but notNF-kappa B, via ASC oligomerization. Cell Death Differ. 13:236 –249.

138. Zhong B, et al. 2008. The adaptor protein MITA links virus-sensingreceptors to IRF3 transcription factor activation. Immunity 29:538 –550.

139. Zhou AM, et al. 1997. Interferon action and apoptosis are defective in micedevoid of 2=,5=-oligoadenylate-dependent RNase L. EMBO J. 16:6355–6363.

140. Zhou S, et al. 2010. Induction and inhibition of type I interferon re-

sponses by distinct components of lymphocytic choriomeningitis virus.J. Virol. 84:9452–9462.

141. Zhou S, et al. 2008. Lymphocytic choriomeningitis virus (LCMV) in-fection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflam-matory responses. J. Neuroimmunol. 194:70 – 82.

142. Zhou SH, et al. 2005. MyD88 is critical for the development of innateand adaptive immunity during acute lymphocytic choriomeningitis vi-rus infection. Eur. J. Immunol. 35:822– 830.

143. Zucchini N, et al. 2008. Cutting edge: overlapping functions of TLR7and TLR9 for innate defense against a herpesvirus infection. J. Immunol.180:5799 –5803.

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sensing of rna viruses: a review of innate immune receptors

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