how viruses manage to control TLR pathways

how viruses manage to control TLR pathwaysBriefly describe how viruses manage to control TLR pathways. For instance, how does a dsRNA activate the TLR pathway compared to ssRNA. Utilize multiple viruses as examples and focus on the differences between cell types that are regulated in a TLR dependent manner vs. other cells that are not.Briefly describe how viruses manage to control TLR pathways. For instance, how does a dsRNA activate the TLR pathway compared to ssRNA. Utilize multiple viruses as examples and focus on the differences between cell types that are regulated in a TLR dependent manner vs. other cells that are not.please include Tat transactivation factor for HIV and viral progression in the answer.please use only Pubmed database as a reference (5 different sources) and no other reference data base. And do in text citation.ReviewInnate immune recognition of DNA: A recent historyabstractInnate immune DNA sensing underpins many physiological and pathological responses to DNA,including anti-viral immunity to DNA viruses. Although it has been appreciated for many years thatcytosolic DNA can evoke a type I interferon response, it is only within the past decade that the cellularmechanisms responsible for such a response have been defined. Here we review the discoveries that ledto an appreciation of the existence of cytosolic DNA sensor proteins, and discuss two key such sensors,cGAS and IFI16, in detail. DNA sensors operate via STING, a protein shown to have a central role incontrolling altered gene induction in response to DNA in vivo, and as such to be central to a rapidlyexpanding list of both protective and harmful responses to DNA. We also discuss recent insights intohow and when DNA stimulates innate immunity, and highlight current outstanding questions in theDNA sensing field.& 2015 Elsevier Inc. All rights reserved.ContentsIntroduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Early studies in defining cytosolic DNA sensor signaling (2006–2009) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148The centrality of STING in controlling DNA sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Discovery of multiple putative DNA sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Mechanisms of innate DNA sensing: lessons from PYHIN proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Discovery of cGAS as a critical DNA sensor acting via STING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151IntroductionInnate immunity has undergone a revolution in the past 15 years,with the uncovering of the molecular basis underpinning CharlesJaneway’s now validated hypothesis of the existence of germlineencoded pattern recognition receptors (PRRs) capable of detectingand responding to pathogen-associated molecular patterns (PAMPs)on microbes (Janeway, 1989). With the discovery of the Toll-likereceptors (TLRs), mechanisms for pathogen detection, leading inparticular to induction of cytokines and type I interferons (IFN-Is),were revealed, and thus the innate immunity renaissance began(O’Neill et al., 2013). In terms of viral detection, cell membrane boundTLRs were shown to recognize glycoproteins on the surface of viralcapsids, while endosomally placed TLRs responded to viral nucleicacid, in particular viral RNA (O’Neill et al., 2013). Rapid progress wasmade from 2004 in understanding how PRRs also survey and protectthe cytosolic compartment from viruses, with the discovery andcharacterization of the RIG-I-like receptors (RLRs) RIG-I and MDA5,which recognize viral RNA genomes, replication intermediates and/ortranscription products (Yoneyama et al., 2004; Andrejeva et al.,2004). Further structural studies of RLRs led to an appreciation ofhow these PRRs distinguish self from non-self RNA, in that RIG-I isactivated by viral RNA with a 50triphosphate, or a 50diphosphate,moiety juxtaposed to a dsRNA panhandle, while MDA5 binds to longdsRNA (Gurtler and Bowie, 2013; Goubau et al., 2014). Both RLRs thentransduce a signal via MAVS, leading to activation of transcriptionContents lists available at ScienceDirectjournal homepage: www.elsevier.com/locate/yviroVirologyhttp://dx.doi.org/10.1016/j.virol.2015.03.0130042-6822/& 2015 Elsevier Inc. All rights reserved.n Corresponding author. Fax: þ353 1 6772400.E-mail address: agbowie@tcd.ie (A.G. Bowie).Virology 479-480 (2015) 146–152factors such as NF-κB and IFN regulatory factor 3 (IRF3) andsubsequent cytokine and IFN-I induction.In light of these discoveries and in the context of the paradigm ofPRRs recognizing PAMPs, the questions of how, why and when DNAactivates the innate immune system has received considerableattention in the past decade. The immunostimulatory and anti-viralpotential of DNA introduced into mammalian cells had been reportedmore than 50 years ago (Isaacs et al., 1963; Rotem et al., 1963), butonly recently have the cellular mechanisms of DNA detection leadingto immune responses, and to IFN-I induction in particular, beenidentified. This has led to an understanding of the role of cytosolicDNA sensing in many protective and pathological responses to DNA,for example in detection of DNA viruses and in aberrant responses toself-DNA leading to autoimmunity respectively.Here we review the discovery of proposed cytosolic DNA sensorsover the past decade (Fig. 1), discuss the current understanding ofFig. 1. Timeline for discovery of putative cytosolic DNA sensors and associated signaling molecules. The chronology of the identification of intracellular DNA sensors isshown. DNA sensors are in red, key signaling proteins in green, and the RNA sensors Rig-I and MDA5 shown for comparison (orange). As detailed in the text, certain DNAsensors (boxed in gray here) have been confirmed to have a role in DNA- and/or DNA-virus dependent cytokine and IFN-I production in vivo, by using knockout mice.Fig. 2. Cytosolic DNA sensors and their signaling pathways. Upon recognition of dsDNA in the cytosol or nucleus, DNA sensors signal via adaptor proteins to alter geneinduction via transcription factors (IRFs and NF-κB), and to pro-IL-1β processing, via Caspase 1. DNA sensors described in the main text are shown in red.A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152 147how such sensors operate to respond to immune-stimulatory DNAto mobilize immune responses (as outlined in Fig. 2), and highlightcurrent issues that remain to be resolved in this rapidly evolving field.Early studies in defining cytosolic DNA sensor signaling(2006–2009)The first PRR implicated in detection of DNA was TLR9 (Fig. 1),shown in 2000 to be responsible for the ability of mice to respondto CpG DNA (Hemmi et al., 2000), leading to cytokine and IFN-Iinduction via NF-κB and IRF7 respectively (O’Neill et al., 2013).This provided an elegant example of self-versus non-self immunediscrimination, since TLR9 recognized under-methylated DNA(i.e. CpG DNA), which is enriched in microbial genomes comparedto mammalian cells. In contrast, it is now clear that if either self orpathogen dsDNA accumulates in the cytosol, TLR9-independentcytokine and IFN-I induction is elicited in many cell types (reviewedin (Paludan and Bowie, 2013)). TANK-binding kinase (TBK1) wasidentified in 2006 as being required for IFN-β production in responseto transfected synthetic dsDNA (poly(dA:dT)) (Ishii et al., 2006)(Fig. 1). After activation of upstream PRR signaling, TBK1 directlyphosphorylates IRF3 leading to IFN-β induction, and the TBK1-IRF3signaling axis was quickly established as fundamental to the cytosolicDNA response to other forms of synthetic dsDNA (Stetson andMedzhitov, 2006), to DNA viruses and for the immune response toDNA vaccines (reviewed in (Keating et al., 2011)). Thus by 2006the search was on to discover one or more DNA sensors that woulddirectly bind to cytosolic DNA and mediate IFN-I induction via asignaling pathway involving TBK1 and IRF3.In 2007, the first reported cytosolic DNA receptor was identifiedas DNA-dependent activator of IRFs (DAI) (Takaoka et al., 2007). DAI(or ZBP-1), encoded by an IFN-inducible gene, was found to becapable of upregulating the expression of IFN-I via NFκB and IRF3 inresponse to poly(dA:dT), and to bind to DNA (Takaoka et al., 2007).DAI knockdown in mouse L929 cells reduced HSV-1-stimulated IFN-β production, while subsequent studies showed a role for DAI in IFN-Iand cell death responses to CMV in fibroblasts (DeFilippis et al., 2010;Upton et al., 2012, 2010). However, no role for DAI in macrophageresponses to DNA was observed (Unterholzner et al., 2010), while thein vivo response to DNA vaccination was TBK1-dependent, but DAIindependent(Ishii et al., 2008). This indicated the existence of otheras of yet unidentified cytosolic DNA sensors, especially for myeloidcells. A further DNA sensing pathway was identified in 2009, which,surprisingly for a DNA response, involved RIG-I and MAVS (Ablasseret al., 2009; Chiu et al., 2009). It was shown that RNA polymerase III(RNA Pol III) is capable of transcribing AT-rich dsDNA, such as poly(dA:dT), into an RNA-containing 50-triphosphate moiety, whichcan then be sensed by RIG-I leading to IFNβ induction (Ablasseret al., 2009; Chiu et al., 2009). This provided an explanation for thepreviously reported involvement of MAVS in some DNA responses(Ishii et al., 2006), and certainly accounts for the ability of somehuman cell types to respond to poly(dA:dT). However the physiologicalrole of RNA Pol III in detection of pathogens has remainedcontroversial, and RNA Pol III could not account for known IFN-Iresponses to non-AT-rich dsDNA (Wilkins and Gale, 2010). Thus,given that the role of DAI is cell type-specific, and that the Pol III-RIGIpathway only detects AT-rich DNA, clearly additional cytosolic DNAPRRs remained to be discovered.The centrality of STING in controlling DNA sensingIn parallel to these early studies identifying putative cytosolic DNAsensors, a new signaling adaptor protein called STING (also calledMITA, MPYS and ERIS) was discovered in 2008 (Ishikawa and Barber,2008; Zhong et al., 2008; Jin et al., 2008; Sun et al., 2009). STING wasshown to be essential for IFN-β induction by DNA and also by HSV-1,and STING knock out mice were subsequently shown to be impairedin their ability to respond to DNA viruses (Ishikawa et al., 2009).STING is now known to have a role in vivo in responses to viral andbacterial pathogens, to self-DNA during autoimmune disease, and inDNA adjuvancy (Burdette and Vance, 2013). The mechanism wherebySTING directly engages with TBK1 to direct IRF3 activation has alsobeen determined (Tanaka and Chen, 2012) although how exactlySTING mediates NFκB activation (presumably via IKKβ) is to date stillan open question (Fig. 2) (Abe and Barber, 2014). In addition to itsadaptor function for DNA sensing pathways, STING was shown todirectly recognize and induce a type I IFN response to the bacterialsecond messenger cyclic dinucleotide (CDN) diguanylate monophosphate(c-di-GMP) (Burdette et al., 2011), and more recently to thestructurally related novel mammalian second messenger cGAMP(described below). The bacterial CDN could be being detected bySTING as a PAMP to sense the presence of a bacterial infection, oralternatively bacterial CDN stimulation of STING may be utilized as animmune escape mechanism when induction of type I IFN may benefitthe bacterial pathogen.Work since 2009 up to the present has now linked STING tomany physiological and pathological responses involving innate andadaptive immunity, and placed this innate immune adaptor at thecenter of both protective and detrimental processes in vivo. Forexample, a protective role for STING during cancer immunotherapyhas been revealed in mouse tumor models whereby STING indendritic cells is activated by the recognition of tumor cell DNA,leading to IFN-β induction which renders DCs competent to presenttumor antigens and prime CD8þ T lymphocytes (Klarquist et al.,2014; Woo et al., 2014; Deng et al., 2014). In contrast, STING has adetrimental role in driving inflammatory disease when self-DNAaccumulates due to defective or absent DNase activity (Ahn et al.,2014). Importantly, STING has been directly implicated in humandisease since a new monogenic disorder termed STING-associatedvasculopathy with onset in infancy (SAVI) has been recentlycharacterized as an autoinflammatory disease caused by gain-offunctionmutations in TMEM173 (the gene encoding STING) (Liuet al., 2014).Discovery of multiple putative DNA sensorsSince 2009 at least 10 further proteins have been proposed ascytosolic DNA sensors: AIM2, IFI16, LRRFIP1, DHX9, DHX36, DDX41,Ku70, DNA-PK, MRE11, cGAS, STING itself and Rad50 (Figs. 1 and 2).AIM2 and IFI16 are pyrin and HIN200 domain (PYHIN) proteins thathave been shown to directly bind to DNA and mediate inflammasomeand transcription factor activation respectively (discussed indetail in the next section).In 2010 an siRNA screen identified LRRFIP1 as required for IFN-βgene induction in response to Listeria monocytogenes or dsDNA inmacrophages (Yang et al., 2010). LRRFIP1 was found to not be requiredfor NF-κB nor IRF3 activation but rather promoted phosphorylation ofthe co-activator, β-catenin, leading to recruitment of p300 to the IFNβenhanceosome via IRF3 (Wathelet et al., 1998). LRRFIP1 was proposedto be a DNA sensor since it co-immunoprecipitated with dsDNA,however direct binding to DNA was not conclusively demonstrated,and in fact LRRFIP1 also had a role in dsRNA-stimulated IFN-βinduction. Hence, by promoting β-catenin phosphorylation followingnucleic acid detection, LRRFIP1 is capable of indirectly up regulatingIFN-β (Yang et al., 2010).Many DEAD/H-box helicases, such as RIG-I and MDA5, havebeen implicated in innate nucleic acid sensing, but DHX9 andDHX36 were the first such proteins proposed to sense cytosolicDNA. These two helicases were identified in plasmacytoid DCs148 A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152(pDCs) by mass spectroscopy as CpG binding proteins, andsurprisingly, shown to signal to altered gene expression via theTLR adaptor MyD88 (Kim et al., 2010). DHX36 was associated withIFN-α production and IRF7 nuclear translocation in response toCpG-A DNA, while DHX9 was found to be important for TNF-α andIL-6 production and NF-κB activation in response to CpG-B DNA(Kim et al., 2010). Knockdown of TLR9 in these cells was also foundto almost completely abrogate CpG DNA-dependent responses(Kim et al., 2010), consistent with the possibility that DHX9 andDHX36 may be required for the TLR9 pathway in pDCs, rather thanacting as DNA sensors themselves. DHX9 has also been proposedas a dsRNA sensor in myeloid DCs since knockdown of DHX9 byshRNA showed DHX9 to be required for the production of type IIFNs and pro-inflammatory cytokines in response to poly(I:C),influenza A and reovirus (Zhang et al., 2011). DHX9 was shown tobind poly(I:C) via its dsRNA-binding domain and signal via MAVSto trigger activation of NF-κB and IRF3 (Zhang et al., 2011). Howexactly DHX9 might activate MyD88 and MAVS for signaling afterDNA and RNA sensing respectively has not been resolved to date.In order to identify the potential involvement of other DExD/Hfamily members in innate immunity, Yong-Jun Liu’s group carriedout a siRNA screen with 59 members of the family. In 2011 theyidentified DDX41 as being required for NF-κB and IRF3 activation,and for cytokine induction, following stimulation of mDCs or humanmonocytes with dsDNA or HSV-1 (Zhang et al., 2011). DDX41 wasshown to bind DNA and to also associate with both STING and TBK1,in keeping with its role as a proposed DNA sensor (Zhang et al.,2011). More recently, DDX41 was reported to directly bind thebacterial CDNs, cyclic-di-GMP and cyclic di-AMP (Parvatiyar et al.,2012). Upon binding to CDNs, DDX41 was shown to induce an IFN-Iresponse via STING, TBK1 and IRF3 (Parvatiyar et al., 2012). AsDDX41 was shown to bind bacterial CDNs, it may also have a role incGAMP sensing (see below) which could contribute to the mountingof an effective response to DNA virus infection.Unlike other PRRs such as TLRs and RIG-I, DExD/H-box proteinsimplicated in DNA sensing do not have clearly defined signalingdomains that are distinct from their DNA-binding domains. Thismakes it difficult to explain how the binding of dsDNA (and ofCDNs in the case of DDX41) would lead to the recruitment ofsignaling adapters such as STING, and there have been no followonstudies to date addressing this issue or providing clarity on thesignaling mechanism. Further, in vivo studies demonstrating anessential role for DExD/H-box proteins in cytosolic dsDNA sensinghave yet to emerge.Apart from the DExD/H-box family of proteins, proteins knownpreviously to have a role in responding to DNA damage in the nucleushave also been implicated as cytosolic DNA sensors. Such proteinsmight act independently of their role in the DNA damage response(DDR) as direct sensors of viral DNA, and/or they may be mobilized tostimulate innate immune signaling in response to virus-inducedcellular DNA damage in the nucleus (discussed in (Paludan andBowie, 2013)). DNA dependent protein kinase (DNA-PK) is a heterotrimericprotein complex consisting of Ku70, Ku80 and the catalyticsubunit DNA-PKcs. Ku70 is mainly known for its involvement in theDNA repair process (Walker et al., 2001). However, in 2011 it was alsodescribed as having a role in the cytoplasmic detection of dsDNA ofgreater than 500 bp in length, triggering the production of type III IFN(IFNλ1), rather than IFN-I, via IRF1 and IRF7 activation (Zhang et al.,2011). A year after identification of Ku70 as a DNA sensor (Zhang et al.,2011), the DNA-PK complex was identified as a DNA sensor with aparticular role in fibroblasts, where it associated with DNA in thecytoplasm and signaled via the STING-TBK1-IRF3 axis to IFN-I induction(Ferguson et al., 2012). DNA-PK colocalised with sites of vacciniavirus replication and both cells and mice lacking DNA-PKcs showedattenuated cytokine responses to both DNA and DNA viruses, butnot to RNA or RNA viruses. Interestingly, although crucial for IRF3activation in response to immunostimulatory DNA or vaccinia virusinfection, DNA-PK was not required for DNA-dependent NF-κBactivation (Ferguson et al., 2012). Further investigations on howDNA-PK signals via STING for IRF3 activation without NF-κB activationmay shed light on differential mechanisms for DNA-stimulatedtranscription factor activation.Yet another DNA damage sensor protein, Mre11 has also beenshown to mediate STING-dependent cytosolic responses to dsDNA(but not to DNA virus) (Kondo et al., 2013). Most recently, Rad50,which is part of a complex together with Mre11 and Nbs1 that sensesand responds to double-strand breaks in nuclear DNA, has also beenimplicated in innate immune DNA sensing (Roth, 2014). In that case,after vaccinia virus infection, Rad50 translocated to the cytosol,engaged with viral DNA and coupled to a STING-independent signalingaxis involving CARD9 and Bcl10. This led to NF-κB activation, andin particular induction of pro-IL-1β mRNA. Thus in the context whereDNA viruses cause inflammasome activation, the Rad50-CARD9-Bcl10-NF-κB pathway contributes to virus-stimulated IL-1β release(Roth, 2014). The discovery of the role of Rad50 in DNA sensingsuggests that in certain contexts STING-independent DNA responsepathways to gene induction exist.Apart from its key role as a signaling adaptor in cytosolic DNAsensing, it has recently been reported that STING binds directly toDNA both in vitro and in intact cells, possibly leading to directactivation of the STING signaling pathway (Abe et al., 2013). However,further work will need to be carried out in order to establish whetherSTING can directly sense foreign DNA without the aid of other DNAsensors in certain contexts, especially since stimulation of HEK293cells stably expressing STING can induce IFNI in response to CDNs butnot DNA (Burdette et al., 2011).Mechanisms of innate DNA sensing: lessons from PYHINproteinsAs well as triggering altered gene induction via transcription factoractivation, cytosolic DNA is also capable of stimulating inflammasomeactivity leading to production of the mature active forms of IL-1β andIL-18. Many inflammasome activators work via stimulation of anNLRP3-containing inflammasome complex that involves ASC recruitmentand subsequent caspase 1 activation, however cytosolic DNAwas shown to stimulate an NLRP3-independent inflammasome complex.In 2009, the human PYHIN family member absent in melanoma2 (AIM2) was shown to detect viral dsDNA in the cytoplasm by directbinding via the AIM2 HIN200 domain. This facilitated recruitment ofASC to AIM2, presumably via homotypic PYRIN domain interactions,and subsequent caspase 1 activation (Roberts et al., 2009; Hornunget al., 2009; Burckstummer et al., 2009; Fernandes-Alnemri et al.,2009). Importantly, since there is an ortholog of human AIM2 in themouse, the generation of AIM2 knock out mice allowed demonstrationof a role for AIM2 in DNA- and DNA virus-stimulated IL-1β andIL-18 production in vivo (Fernandes-Alnemri et al., 2010; Rathinamet al., 2010).After the discovery of the role of AIM2 in DNA sensing, a secondhuman PYHIN protein was implicated as a PRR for intracellularDNA. Interferon-γ-inducible protein 16 (IFI16) was identified as anovel PRR for DNA based on its association with, and requirementfor IFN-I responses to, a 70 base pair dsDNA derived from thevaccinia virus genome (Unterholzner et al., 2010). Upon DNAstimulation (Unterholzner et al., 2010) or infection of cells withherpes viruses (Horan et al., 2013), IFI16 associated with STINGwhile siRNA knockdown of IFI16 or its presumed mouse counterpart,p204 (IFI204), inhibited gene induction induced by DNA orHSV-1, but not by RNA or RNA virus (Unterholzner et al., 2010).The structure of the PYHIN proteins is consistent with theirproposed role as cytosolic DNA sensors, as they possess a definedA. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152 149signaling (PYRIN) and DNA binding (HIN200) domain. IFI16, p204and AIM2 form a new family of PRRs, termed the AIM2-likereceptors (ALRs) (Keating et al., 2011), and whether other PYHINprotein family members also function as ALRs remains to bedetermined. Ongoing study of the ALRs has revealed structural andmechanistic insights into how innate immune DNA sensing operates.The crystal structure of the AIM2 and IFI16 HIN200 domains boundto dsDNA provided a rationale for sequence-independent sensing ofDNA, since the contacts between the protein receptors andthe DNA were primarily electrostatic interactions with thephosphate-sugar DNA backbone (Jin et al., 2012). This is consistentwith the fact that DNA of any sequence, when transfected intomonocytic cells, is capable of eliciting cell death and IFN-I responses.Thus self-DNA is not discriminated from pathogen DNA based onnucleotide composition.Alternatively, self:non-self discrimination might be based on theaberrant appearance of DNA in the cytosol, and hence the nucleuswould be ‘immune privileged’. However this is also not the case sinceIFI16 is mainly nuclear in its expression, and has now been demonstratedto detect multiple nuclear herpes viruses (reviewed in (Orzalliand Knipe, 2014)). Thus innate DNA sensing can also initiate from thenucleus, and not just the cytosol. In fact IFI16 has been shown toshuttle between the cytosol and nucleus for DNA sensing in bothcompartments, depending on the acetylation status of its nuclearlocalization sequence (Li et al., 2012). The mechanism by which IFI16discriminates between pathogen and cellular DNA in the nucleus isnot yet clear. However insights into this puzzle come from recent workshowing that in vitro IFI16 assembles on naked dsDNA strands in acooperative manner, which is dependent on homotypic pyrin domaininteractions (Morrone et al., 2014). This leads to the formation ofoligomeric IFI16 foci, reminiscent of the assembly of inflammasomes,which could be expected to be signaling-competent. Optimal IFI16polymerization required approximately 150 bp of dsDNA. Althoughthese observations remain to be shown in intact cells, this workpotentially provides an elegant mechanism for IFI16 to discriminatebetween self and non-self-DNA based on ‘measuring’ the length ofnaked DNA, since host nuclear DNA should normally be complexedwith histones. Thus the true ligand for DNA sensing by PYHIN proteinsis likely long naked DNA.IFI16 has also been identified as having a role in the detection ofHIV DNA (Jakobsen et al., 2013; Monroe et al., 2014). In HIV-infectedmacrophages, IFI16 bound HIV ssDNA in duplex structure due tolong stem and terminal loop regions that form during the HIVlentiviral life cycle (Jakobsen et al., 2013). Thus in macrophages IFI16contributed to the early control of HIV. IFI16 also has a HIV detectionrole in abortively infected CD4þ T cells. In that case, the accumulationof viral DNA replication intermediates caused IFI16-dependentpyroptosis (Monroe et al., 2014), leading to depletion of T cells. Thussimilar to AIM2, in certain contexts IFI16 can promote inflammasomeactivation. This is also true for IFI16 sensing of the herpesviruses KSHV, HSV-1 and EBV (Kerur et al., 2011; Johnson et al.,2013; Ansari et al., 2013).Currently the molecular details as to how in response to virusesIFI16 signals to the inflammasome in some contexts, and also toSTING for IFN-I induction remain unclear. Activated AIM2, likeNLRP3, has recently been shown to mediate inflammasome signalingby nucleating pyrin domain filaments of ASC, leading toclustering of the ASC CARD domains and activation of Caspase-1(Lu et al., 2014; Cai et al., 2014). Whether IFI16-containing inflammasomeswould function in a similar manner remains to be tested.Notwithstanding the lack of information as to how IFI16transmits a downstream signal, the importance of IFI16 in viralDNA sensing is underscored by the fact that viral immune evasionstrategies have already been identified that target IFI16 function.Specifically, the HSV-1 protein ICP0 targets IFI16 for proteasomaldegradation in order to suppress IRF3 activation (Orzalli et al.,2012), while the HCMV protein pUL83 interacts with the pyrindomain of IFI16 in order to prevent it forming an oligomer, likelyblocking receptor activation (Li et al., 2013).Discovery of cGAS as a critical DNA sensor acting via STINGIn 2013, the discovery of a novel signaling pathway upstream ofSTING involving cGAMP synthase (cGAS) represented a significantadvance in our understanding of the signaling mechanisms underpinninginnate DNA sensing. Zhijian Chen’s group identified a factorsynthesized by mammalian cells following stimulation with DNA thatwas capable of activating STING (Wu et al., 2013). This factor wascyclic-GMP-AMP (cGAMP) and it was shown to directly bind STINGand lead to IRF3 activation (Wu et al., 2013; Gao et al., 2013). cGAMP isvery similar in structure to the bacterial CDNs that can bind andactivate STING, suggesting that such CDNs are actually mimicking hostcGAMP as a means of stimulating STING-dependent type I IFNInductionto benefit the bacteria (Gurtler and Bowie, 2013; Burdetteet al., 2011). Chen’s group also identified the enzyme that generatescGAMP upon DNA stimulation of cells, from the substrates ATP andGTP, as cGAS (Sun et al., 2013). This novel cGAS-cGAMP signalingpathway is reminiscent of the pathway whereby adenylate cyclasegenerates the second messenger cAMP from ATP in response to Gprotein-coupled receptors. The crystal structures of both free cGAS andof cGAS bound to DNA have already been solved. This revealed thatcGAS resembles the dsRNA binding protein OAS1 in overall structure,with modifications in the nucleotide binding region that specifyaffinity for dsDNA rather than dsRNA (Gao et al., 2013; Civril et al.,2013; Kranzusch et al., 2013). In the case of cGAS, enzyme activationoccurs in response to direct binding of DNA to cGAS, which causes aconformational change allowing access of the nucleotide substratesinto the active site, and subsequent synthesis of cGAMP (reviewed in(Cai et al., 2014)). Similar to the case for DNA recognition by AIM2 andIFI16, cGAS contacts dsDNA exclusively via the DNA phosphate backbone,again leading to nucleotide sequence-independent DNA sensing(Cai et al., 2014).Importantly, the role of cGAS in DNA sensing in vivo has beenconfirmed since cGAS knock out mice are now available. Cells fromsuch mice (fibroblasts and BMDMs) were unable to induce IFN andcytokines in response to DNA or live DNA viruses (vaccinia virus,HSV-1 and MHV68), while cGAS-/- mice were more susceptible tolethal infection with HSV-1 or vaccinia virus compared to wildtype mice (Li et al., 2013; Schoggins et al., 2014). In addition,similar to IFI16, cGAS has been shown to have a role in sensingHIV-1 since HIV-1-infected cells produced cGAMP, and HIV-1induced IFN was cGAS-dependent (Gao et al., 2013).DNA detection by cGAS also provides a mechanism for thespread of intrinsic innate immunity to DNA viruses from infectedcells into neighboring cells, since Ablasser et al. (2013) recentlyshowed that cGAMP produced in virally-infected cells can betransferred into uninfected neighboring cells through gap junctions,leading to direct activation of STING and induction of IFNs .Thus antiviral immunity can be rapidly conferred on surroundinguninfected cells independent of the need for the infected cell tofirst produce IFNs.ConclusionA vast array of cytosolic DNA sensors have been described in thepast decade, leading to many new insights into when, how and whyinnate immune responses to DNA occur. In particular in the case ofIFI16 and cGAS, many studies have now demonstrated key roles forthese PRRs in sensing multiple types of DNA viruses and of retroviruses,while structural studies with these proteins have confirmed150 A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152that innate DNA sensors respond to dsDNA in a sequence-independentmanner. Many questions remain to be answered and given the rapidrate of progress in this field we can expect further clarification onmany of these in the near future. A key issue is why so many DNAsensors exist, and what is their relative role in DNA sensing in vivo,and in different cell types? In particular it is interesting that IFI16 andcGAS have been shown to both be required for detection of HIV-1 andHSV-1 so whether these sensors actually cooperate to signal to STINGin a given cell type, and/or operate in distinct cell types is unclear.Interestingly, Storek et al. (2015) recently showed an essential role forboth IFI16 (p204) and cGAS in the same cell type for IFN I inductionfollowing detection of Francisella novicida DNA during a bacterialinfection, by using the CRISPR/Cas9 system to generate cGAS andp204 single- and double-knock out cells . In the case of HIV-1 sensing,the role of IFI16 and cGAS in different cells examined to date correlateswell with the relative expression of these proteins in such cells.Furthermore, how cytosolic cGAS might be so crucial for sensingnuclear HSV-1 and for protection against HSV-1 in vivo is unclear, asare the mechanisms whereby nuclear IFI16 would signal to the STINGTBK1-IRF3signaling axis in the cytosol.What is clear to date is that innate immune DNA sensing underpinsmany physiological and pathological responses and so further i