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Microbes and Infection xx (2014) 1e11 www.elsevier.com/locate/micinf

Intracellular sensing of viral DNA by the innate immune system Daniel S. Mansur a,*,1, Geoffrey L. Smith b,1, Brian J. Ferguson b,1 a

Laboratory of Immunobiology, Department of Microbiology, Immunology and Parasitology, Universidade Federal de Santa Catarina, Floraniopolis, Brazil b Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, UK Received 6 August 2014; revised 25 September 2014; accepted 26 September 2014

Abstract Recent years have seen a great advance in knowledge of how a host senses infection. Nucleic acids, as a common denominator to all pathogens, are at the centre of several of the sensing pathways, especially those involved with the recognition of viruses. In this review we discuss the current knowledge on how intracellular DNA is sensed by the mammalian host. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: DNA sensing; Virus recognition; Immune evasion; Type one interferon; Vaccines; Autoinflammation

1. Introduction The detection of foreign DNA is important for both the simplest and most complex of living organisms. For instance, bacteria sense and destroy foreign DNA, such as that deriving from infection by bacteriophages, by the expression of restriction endonucleases that recognise, cleave and destroy DNA with non-host patterns of methylation [1,2]. But the destruction of foreign DNA is only one type of response to its presence, and this review describes how the detection of DNA inside mammalian cells induces a rapid and robust response that activates the innate immune system and results in the production of interferons (IFNs) and pro-inflammatory cytokines and chemokines. Nucleic acids have long been thought to be inducers of immune responses and some milestones in the development of our current understanding of the mechanisms involved are shown in Fig. 1. In the early 20th century Mechnikov, in his Nobel Prize speech, reported that surgeons used, amongst other things, nucleic acids to recruit phagocytes to open wounds to help * Corresponding author. E-mail addresses: [email protected] (D.S. Mansur), [email protected]. uk (G.L. Smith), [email protected] (B.J. Ferguson). 1 All the three are joint corresponding authors.

prevent subsequent infections. In 1963, a few years after the discovery of interferons (IFNs) in 1957, Isaacs showed that prior stimulation of cells with foreign nucleic acid inhibited the multiplication of different viruses in tissue culture [3]. More than 25 years later in 1989 Charles Janeway described at Cold Spring Harbour the concepts of pathogen associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in innate immunity [4]. The concept of PAMPs as inducers of inflammation and PRRs as initiators of the immune response was broadened subsequently to include self-molecules, or damage-associated molecular patterns (DAMPs), as indicators of tissue damage, and DNA itself soon became the subject of intensive investigation as a PAMP and DAMP [5]. In fact, the immune responses against self-DNA have long been associated with autoimmune diseases, such as systemic lupus erythematosus (SLE) [6]. As DNA is an extremely conserved and widespread molecule there are clear evolutionary advantages in using its detection in an abnormal location or with an unusual structure as a marker of danger, either as an alarm following microbial infection or to indicate tissue damage [7]. As our knowledge of PRRs grew and the toll-like receptors (TLRs) was discovered, TLR9 was associated with the recognition of non-methylated CpG islands from bacterial genomes [8]. Afterwards, DNA from several other sources was described to signal through TLR9, including self-DNA [9].

http://dx.doi.org/10.1016/j.micinf.2014.09.010 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Mansur DS, et al., Intracellular sensing of viral DNA by the innate immune system, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.09.010

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List of abbreviations ASC

apoptosis-associated Speck-like protein containing a CARD AIM2 absent in melanoma 2 AP-1 activator protein 1 c-GAS cyclic GMP-AMP synthase CRISPR clustered regularly interspaced short palindromic repeats DAI DNA-dependent activator of IFN regulatory factors DAMP damage associated molecular pattern DDX41 DExD/H-box helicase 41 DNA-PK DNA-dependent protein kinase DSB double strand breaks EMCV encephalomyocarditis virus HSV herpes simplex virus ICP0 infected cell polypeptide 0 IFI16 IFN gamma inducible factor 16 IFN interferon IRF interferon regulatory factor ISG interferon-stimulated gene KSHV Kaposi's sarcoma-associated herpes virus MEF murine embryonic fibroblasts MRE11 meiotic recombination 11 homologue A MVA modified virus Ankara NF-kB nuclear factor kappa B PAMP pathogen associated molecular pattern pDC plasmacytoid dendritic cells PRR pattern recognition receptor SCID severe combined immune deficiency SLE systemic lupus erythematosus STING stimulator of interferon genes TBK-1 TANK-binding kinase 1 TLR toll-like receptor TREX three prime repair exonuclease i VACV vaccinia virus YFV yellow fever virus Recognition of CpG DNA by TLR9 is mostly restricted to endosomal compartments in haematopoietic cells and so detection of exogenous DNA is restricted to this location [9]. However, several species of bacteria, parasites and DNA

viruses replicate in the cytoplasm of a range of cell types, and this prompted identification of cytosolic pathway(s) of DNA sensing. In 2006, Stetson and Medzhitov showed that cytoplasmic recognition of DNA purified from Listeria monocytogenes was dependent on IRF3 but independent on TLRs [10], indicating the existence of an unknown cytosolic DNA receptor. In that same year Ishii and colleagues showed that BDNA stimulates murine embryonic fibroblasts (MEFs) and dendritic cells (DCs) in a Toll-like receptor (TLR)-independent but TANK-binding kinase (TBK-1) and IkB kinase ε (IKKε)-dependent manner [11]. In 2008 a protein called stimulator of IFN genes (STING, also known as MITA/MYPS/ERIS), was discovered and shown to be essential for transcriptional activation downstream of cytosolic DNA sensors that triggered the transcription of type 1 IFNs [12]. This delineated what is recognised today as the canonical cytosolic DNA sensing pathway, leading to the production of type 1 IFNs and pro-inflammatory cytokines and chemokines (Fig. 2). Another important pathway that detects cytosolic DNA and activates the inflammasome was also described [13]. This pathway involves the recognition of DNA by the sensor absence in melanoma 2 (AIM2), followed by activation of caspase 1 and the proteolytic cleavage of pro-interleukin (IL)1b to IL-1b that is then released from the cell. Since then several molecules have emerged as potential sensors for the cytosolic DNA as we discuss here (Fig. 2). In chronological order of discovery these include, DNAdependent activator of IFN regulatory factors (DAI), DNAdependent RNA polymerase III (RNA-Pol III), IFN gamma inducible factor 16 (IFI16), DExD/H-box helicase 41 (DDX41), meiotic recombination 11 homologue A (MRE11), DNA protein kinase (DNA-PK) and cyclic GMP-AMP synthase (c-GAS) [14e23]. Of these, there is evidence of importance in vivo for DNA-PK and cGAS [17,24] as well as AIM2 [25,26]. The discovery that STING could sense bacterial di-nucleotides directly also suggested that a second messenger could be used as a signal produced after detection of several different DNA sources. Indeed, cyclic GMP-AMP (cGAMP) was later identified as this second messenger, being a product of the enzyme cGAS that is activated by binding DNA [22]. This review will discuss how DNA is a crucial molecule for the initiation of immune response to infection by DNA containing pathogens or self DNA, focussing specially in the

Fig. 1. A timeline showing some of the important discoveries that have led to our current understanding of innate immunity and in particular the response to DNA. Please cite this article in press as: Mansur DS, et al., Intracellular sensing of viral DNA by the innate immune system, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.09.010

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Fig. 2. Pathways leading to the activation of NF-kB and IRF3 following detection of DNA by various DNA sensors and virus-encoded inhibitors of DNA sensing pathways. The canonical pathway by cGAS and cGAMP leading to activation of STING is central. The exact mechanisms by which other DNA sensors lead to activation of STING remain to be determined. The transcription of A:T rich DNA by RNA-dependent RNA polymerase III leading to the production of RNAs that are sensed by RNA sensors is illustrated on the right. Note that both detection of DNA by cGAS or RNA by RNA sensors leads to activation of both NF-kB and IRF3. Proteins ICP0 and UL83 from HSV-1 and HCMV, respectively, target the protein sensor IFI16. Protein C16 from VACV strain WR binds to the Ku proteins, part of the DNA-PK trimeric complex. The red bars indicate the positions at which numerous other viral inhibitors act to block these signalling pathways.

STING/IRF3 pathway. The article also describes what autoimmune diseases and infections can tell us about the relevance of this pathway in vivo and for the development of vaccines and adjuvants. 2. Mechanisms of DNA sensing Primary DNA sensing provides an anti-microbial response that is marked by the production of type one IFNs and proinflammatory cytokines and chemokines and this is certainly the case with DNA viruses (Fig. 2). These molecules are induced by several families of transcription factors that include nuclear factor kappa B (NF-kB), activator protein 1 (AP-1), and IFN regulatory factors (IRFs). Although the participation of NF-kB in the induction of pro-inflammatory cytokines is very clear after TLRs activation [27], its participation in the regulation of genes induced by cytosolic DNA receptors is still largely unknown, therefore we will focus on the STING-IRF3 axis in this review. Early experiments from Taniguchi and co-workers using herpes simplex virus type 1 (HSV-1) infection of MEFs or plasmacytoid dendritic cells (pDCs), showed that IRF7 is the main factor responsible for type one IFN production, with only a moderate role for IRF3 in MEFs [28]. IRF7/ mice are highly susceptible to

infection with encephalomyocarditis virus (EMCV) (RNA genome) or HSV-1 (DNA genome) and infection of these mice with these viruses, induced only low levels of IFNa. Infection of IRF3/ mice with EMCV, but not with HSV-1, showed markedly increased lethality compared to infection of wild type mice, and infection of IRF3/ mice with either EMCV or HSV-1, showed no difference in the IFNa serum levels compared to the wild-type animals. Also, the induction of IFN in vivo and in vitro by HSV-1 infection was shown to be myeloid differentiation primary response gene 88 (MyD88) independent [28,29]. The virus strains and inoculation routes used in studies might account for discordant results with more recent literature. For instance, MyD88/ mice were infected by HSV-1 infection via the respiratory tract these animals were highly susceptible to HSV-1-induced encephalitis [30]. It was later shown that the HSV-1 DNA genome is the most important PAMP for HSV-1 recognition, driving type I IFN and proinflammatory cytokines expression [17,24,31,32]. Poxviruses have DNA genomes and replicate exclusively in the cytoplasm and consequently have been used widely to characterise cytosolic DNA sensing pathways. Infection by vaccinia virus (VACV) activates sensing pathways in a TLR9/ IRF7-dependent and IRF3-independent manner in pDCs [33]. However, as will be discussed below, poxviruses encode

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several IRF3 inhibitors, indicating that although the type one IFN response in pDCs is IRF7-dependent, in other cells, such as fibroblasts, the IRF3 pathways is required. Indeed, IRF3/ MEFs are largely defective for transcription of mRNA encoding IFNb and other cytokines when infected with VACV strain modified virus Ankara (MVA) [17]. Also, in conventional DCs, MVA induces IFNb in an IRF3dependent manner [34]. TANK binding kinase (TBK)-1 and IKKε are the main kinases responsible for IRF3 phosphorylation [35e37]. After activation by phosphorylation, IRF3 dimerises and translocates into the nucleus to initiate gene transcription [38,39]. TBK-1 is essential for cytokine production after DNA stimulation in vitro and to induce a full immune response after DNA vaccination in vivo [17,21,40], suggesting a more pronounced role in the DNA sensing pathways than IKKε. In vitro studies show that TBK-1 is auto-phosphorylated enabling the activation of IRF3 [41]. After DNA stimulation, TBK-1 co-localises with STING at perinuclear vesicles [42]. Interestingly, TBK-1 co-localises with cytoplasmic factories that are induced following infection with VACV strain MVA and are rich in VACV DNA [17]. The mechanism by which STING activates TBK-1 is not fully understood, but it is known that after STING binds to cyclicdi-nucleotides it undergoes conformational changes, oligomerises and recruits TBK-1 [43,44]. TBK-1 is then autophosphorylated and in turn phosphorylates IRF3 causing its dimerisation and translocation into the nucleus to activate gene transcription [35]. It is less clear how IRF7 contributes to these STING-dependent pathways. STING is a central molecule in the cytosolic DNA sensing pathways, either being an adaptor or directly sensing cyclic dinucleotides. STING has been reported to interact with several of the sensors described, such as IFI16, DDX41 and Ku70 (a subunit of the heterotrimer DNA-PK) [17,21,23]. There are no reports of DAI, MRE11 or cGAS binding to STING.

stimulated gene (ISG) and is a member of the PYHIN family, a property it shares with AIM2, the DNA sensor that leads to caspase-1 activation [13]. Prior to its proposed link to DNA sensing, overexpression of IFI16 was found to cause activation of NF-kB, and hence many pro-inflammatory genes, in human umbilical vein endothelial cell (HUVEC) lines [50]. IFI16 is mainly nuclear but is redistributed to the cytoplasm after DNA stimulation and knockdown of IFI16 impairs translocation of IRF3 and the NF-kB subunit p65 to the nucleus and diminishes IFNb gene transcription in DNA stimulated THP-1, RAW and MEFs [21]. In the nucleus IFI16 is able to recognise Kaposi sarcoma herpes virus (KSHV) DNA, bind to the adaptor ASC and, via caspase-1, activate the inflammasome platform. How IFI16 is able to discriminate viral from selfDNA remains unclear [51]. IFI16 is also linked to cell death in CD4þ T cells infected with human immunodeficiency virus-1 (HIV-1). Interestingly, in the same study, the components of DNA-PK complex were also found binding HIV-Nef DNA [52]. Indeed, DNA-PK seems to be important for CD4þ T-cell death and more intriguing, and consistent with the DNA sensing function of DNA-PK [17], this is independent of the catalytic activity of DNA-PKcs [53]. 2.3. DDX41 DDX41 is a helicase that is constitutively expressed in murine myeloid DCs and THP-1 cells and was identified using a siRNA screen and was shown to bind B-DNA and initiate the IFN response to HSV-1, adenoviruses and L. monocytogenes in a STING-dependent manner [23,54]. DDX41 also binds cyclic di-nucleotides [54], a function that seems to overlap with STING, but, interestingly, in a way that is not redundant. In murine DCs, knockdown of DDX41, caused abrogation of IRF3 phosphorylation and IFNa and IFNb production, in the same way as when STING is knocked down. It will be interesting to see whether DDX41 works in parallel with STING and its relevance in vivo.

2.1. DAI 2.4. DNA-PK DAI was the first described DNA sensor. It was isolated from L929 cells and had been shown previously to be a Z-DNA binding protein. Overexpression of DAI induced the transcription of IFNs in MEFs and its knockdown from L929 cells impaired cytokine response after B-DNA stimulation. However, the use of the DAI knockout mouse [17,34,40,45,46] and human cell lines lacking DAI [47] demonstrated that this sensor played no role in DNA-mediated cytokine production. Later it was demonstrated that DAI can complex with RIP3 kinase, initiating programmed necrosis as well as NF-kB activation [48,49]. 2.2. IFI16 IFI16 was isolated from THP-1 cells that had been transfected with biotinylated VACV DNA and after affinity purification of the DNA was identified by mass-spectrometry as a DNA binding protein [21]. IFI16 is a multifunctional IFN

The connection between proteins involved in the DNAdamage response and the immune system has been in the literature for some time. The hetero-trimeric complex DNAPK (composed of Ku70, Ku80 and DNA-PKcs) is strongly linked to immune responses because it plays an essential role in the generation of antibody diversity [55] and is also a component of the double stranded (ds) DNA break (DSB) repair machinery [56]. A point mutation in DNA-PKcs causes a SCID severe combined immune-deficiency (SCID) phenotype in which animals are highly susceptible to any kind of infection [55,56]. DNA-PKcs was also suggested to be the kinase that phosphorylates IRF3 [57]. However, with the generation of mice deficient for TBK-1, it was clear that TBK1 was the principal kinase responsible for IRF3 phosphorylation in vivo [36]. In addition to roles in DNA end joining and in the generation of the adaptive immune response, DNA-PK is also

Please cite this article in press as: Mansur DS, et al., Intracellular sensing of viral DNA by the innate immune system, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.09.010

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involved in innate sensing of foreign DNA. All three components of the DNA-PK complex were isolated from cytoplasmic extracts of human embryonic kidney (HEK) cells and MEFs after these cells were transfected with biotinylated DNA and the DNA was subsequently affinity purified [17]. The large adducts of cytoplasmic viral DNA in VACV replication centres provide a clear target for innate immune sensing and colocalisation of DNA-PK with this viral DNA during infection provided evidence for the association of DNA-PK with the genome of an actively replicating DNA virus. The use of cell lines lacking any one of the 3 different components of the DNA-PK complex caused diminished production of IFNb and other cytokines after stimulation with DNA. In contrast, there was no defect following stimulation with RNA. In MEFs, this pathway was dependent on STING, TBK1 and IRF3. Deletion of DNA-PKcs abrogated the translocation of IRF3 but not NFkB into the nucleus, indicating the specificity of this pathway. Interestingly, the kinase activity of DNA-PKcs was not necessary to activate the DNA-sensing pathway. Notably, infection of DNA-PKcs/ MEFs with MVA caused expression of significantly more viral proteins than parallel infection of wild type MEFs [17]. The importance of DNA-PKcs for DNA sensing was demonstrated in vivo following DNA transfection and DNA virus infection of DNA-PKcs/ mice. Transfection of DNA, but not RNA, intradermally into DNA-PKcs/ mice induced less IFNb and IL-6 locally within 1 day of transfection. Similarly, infection of DNA-PKcs/ mice with MVA reproduced the results with DNA transfection. Further evidence for the function of DNA-PK in DNA sensing was provided by a study of Ku70 in HSK293 cells. In that study, Ku70 was shown to mediate the induction of IFNl in HEK293 and mouse splenocytes by DNA in knock-down and knock-out experiments respectively in a manner mediated by IRF1 and IRF7 [58]. Early experiments from Stetson and Medzhitov on bone marrow derived monocytes (BMDMs) deficient for DNA-PK proteins showed that in these cells DNA dependent IFNb transcription was normal. This may be explained by the observation that DNA-PK components are poorly expressed or not expressed in myeloid cells [59]. 2.5. MRE11 MRE11 is another DNA damage sensor that is also linked to DNA-induced IFN transcription. In MEFs, immune stimulatory DNA (ISD) induced up-regulation of DNA damage associated genes such as ataxia telangiectasia mutated (ATM) [18]. The authors also noted the ability of DNA, but not RNA, to induce ATM phosphorylation in HEK293 cells. Knockdown of MRE11 in granulocyte macrophage colony-stimulating factor (GMeCSF)-induced-bone marrow-DCs impaired the transcription of IFNb and pro-inflammatory cytokines after DNA stimulation. Conversely, in human cells with a mutation in MRE11, the production of DNA-induced IFN was restored after overexpression of MRE11. MRE11 deficiency prevented STING from re-localising from the endoplasmic reticulum (ER) to the Golgi complex after DNA stimulation [18].

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2.6. cGAS The discovery that STING could directly sense dinucleotides from bacteria [60] suggested that STING might also detect di-nucleotides derived from DNA sensing and these di-nucleotides might function as a second messenger. Chen and co-workers demonstrated that the cytosolic extracts from cells stimulated with DNA could, when added to permeabilized recipient cells, activate IRF3 phosphorylation. The factor responsible was shown to be heat stable and protease resistant and was identified as cGAMP [22]. 20 30 -cGAMP is a cyclic dinucleotide with noncanonical bonds between the 20 OH of GMP and the 50 phosphate of AMP and from the 30 OH of the AMP to the 50 phosphate of GMP [15,61e63]. The enzyme responsible for the synthesis of cGAMP is cGAS [15,19]. Until recently cGAS, originally named C6orf150 or Mb21d1, was a putative nucleotidyltransferase [64] and had been identified using a high throughput screen as an ISG and its overexpression led to impairment of the replication of several RNA viruses, including yellow fever virus (YFV) and West Nile virus (WNV) [65]. Later it was shown that purified cGAS was able to synthesise cGAMP in response to DNA [19], and the crystal structure of cGAS alone and in complex with DNA has provided a molecular mechanism for DNA binding activates enzymatic activity [43,66,67]. The ability of cGAMP to activate an innate immune response via activation of STING is not limited to the cell in which cGAS senses DNA, for cGAMP is small enough to diffuse from the stimulated cell via gap junctions to bystander cells, which then also activate STING and produce IFNs [68]. Cells that are deficient for cGAS have an impaired production of IFN and chemokines when infected with HIV and other retroviruses, and the reverse transcribed genome is the PAMP that triggers this response [69]. Lung fibroblasts and DCs from cGAS/ mice fail to induce IFNb to several different DNA sources including infection with HSV-1 and VACV strain Western Reserve (WR) [24]. Infection of cGAS/ mice showed increased mortality compared to wild type mice when infected intravenously with HSV-1 [24], by intranasal route with VACV and subcutaneous route with WNV [70]. The latter observation is particularly interesting because cGAS is unable to interact with RNA and cGAS deficient cells do not have defects in the IFN response to Sendai virus [19]. It could be argued that overexpression of cGAS automatically triggers the STING pathway [19] and therefore the induction of the anti-viral state, however, the in vivo studies with the knockout mice provide evidence that cGAS plays a significant role in fighting the infection of RNA viruses. It is notable that in an unbiased phenotype screen, the International Mouse Phenotype Consortium characterised a cGAS/ mouse that was generated independently and reported metabolic and respiratory disorders. It remains unclear if these phenotypes are linked to the susceptibility to infection with DNA and RNA viruses of these mice (https://www.mousephenotype.org/data/search? q¼mb21d1). For a cell to be ready to sense a pathogen or damage requires that the given sensor be constitutively expressed so that

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a response may be made rapidly. Also, sensors should exist in any cell that can be infected including those not directly related to the immune system (i.e. non-haematopoietic cells) and hence such cells also play a role in the initiation of the anti-viral immune response. Accordingly, the STING/IRF3 pathway is functional in adult fibroblasts, endothelial cells and other cell types. DNA-PK and DDX41 are constitutively expressed in non-myeloid and myeloid cells, respectively [17,23], but DAI and IFI16 are either not present or present at low levels in resting cells and are up-regulated upon IFN or DNA stimulation [20,23]. Despite DNA-PK not being expressed in myeloid cells [59], in vivo transfection of DNA and intradermal infection with MVA (the route used for vaccination against variola virus) yields a defective IFN and IL-6 response in DNA-PKcs/ mice. However, this response is not completely abrogated, indicating a level of redundancy in vivo, which should be expected because the initiation of an immune response against viruses is crucial to survival. For cGAS, there is expression in both fibroblasts and myeloid cells, and being an enzyme is effective at inducing a rapid response and robust even when expressed at low levels [19]. The initial response to infection is subsequently amplified by sensors in myeloid cells, or even other sensors in nonmyeloid cells, and these certainly play a major role in disease outcome, as shown by several studies with DCs, neutrophils and the participation of TLR in the models studied [5,9,28,29,71]. To improve our understanding of the physiological function of the known PRRs and transcription factors in the context of infection, more attention should be focused onto the biology of the pathogen being studied and its natural infection route. 3. Virus evasion of DNA sensing The innate immune response to infection provides strong evolutionary pressure for the emergence of microbial countermeasures. This is illustrated clearly with mammalian viruses that have evolved a myriad of mechanisms to respond to the antiviral action of IFNs. They may do this by preventing the induction of IFNs, blocking the binding of IFNs to IFN receptors, blocking the JAK/STAT signal transduction pathway leading to transcription of ISGs or blocking the antiviral activity of the IFN-induced proteins [72]. The blockade of the pathways leading from the detection of foreign nucleic acid by PRRs is a frequent strategy to achieve this goal, and there are many examples of proteins that block the innate response following detection of RNA by PRRs. Indeed the RNA sensor Mda-5 was discovered by the ability of a paramyxovirus protein to bind to it and inhibit its function [73]. There are literally scores of virus proteins that inhibit signalling pathways leading to the activation of IRF3 or NF-kB and the large DNA viruses, such as herpes viruses and poxviruses, have many proteins devoted to this task. For instance, VACV expresses at least 10 proteins that prevent the activation of NF-kB and several others that block IRF3 activation [74]. Although there are several known inhibitors of sensing of RNA by PRRs [72], there are very few described direct

inhibitors of DNA sensing, at least acting at the level of the DNA sensor itself. This is likely to be related to the fact that DNA sensors and the cGAS e cGAMP pathway (Fig. 2) were identified only recently. But there are already a few examples. One of these is protein C16 from VACV strain WR. C16 was shown to be an intracellular virulence factor that affected the innate immune response to infection by an unknown mechanism [75]. To address its mechanism of action, C16 was tandem affinity purification (TAP)-tagged and used to identify binding partners in cells. This revealed that C16 bound to the Ku proteins, part of the DNA-PK complex, which in a parallel study was shown to bind cytosolic DNA and trigger the STING/TBK-1/IRF3 pathway [17]. Expression of C16 in cells diminished the production of IFNs and chemokines in response to DNA stimulation and infection by a VACV strain engineered to lack the C16L gene resulted in reduced virulence and a stronger innate response to infection [75,76]. The C16 protein is also encoded by variola virus, the causative agent of smallpox, and the variola virus C16 protein also binds the Ku proteins. In contrast, the comparable protein from VACV strain MVA, has a 5 amino acid deletion in the region of C16 required for interaction with Ku binding and has greatly reduced Ku binding [76]. IFI-16 is a member of the haematopoietic IFN-inducible nuclear antigens with 200 amino acid repeats (HIN-200) family and contains a DNA binding domain, a transcriptional regulatory domain, and a protein domain associated with IFN response called DAPIN/PAAD [77]. IFI16 is predominantly nuclear and has been reported to interact with many proteins including BRCA1 via which it is involved in p53-mediated transmission of DNA damage signals, apoptosis and control of the cell cycle [77]. IFI16 has also been reported to bind to STING [21], ASC and pro-caspase 1 [51] and DNA from several viruses including VACV [21], HCMV [78,79], KSHV [51] and HSV-1 [80]. Although there is not yet evidence that IFI16 is important for DNA sensing in vivo, two proteins from herpes virus target IFI16 during infection. HCMV protein UL83 binds to IFI16 [78] and knockdown of IFI16 expression from human embryonic lung fibroblasts enhanced HCMV infection [81]. UL83 is a 65-kDa protein that is packaged into the tegument of HCMV particles and is therefore introduced into infected cells immediately after infection. UL83 antagonises the expression of ISGs, but it is also a transactivator of the HCMV immediate early promoter [78]. HSV-1 also antagonises IFI16 function by inducing its degradation via protein ICP0 [82]. If the cGAS pathway of DNA sensing proves as important as indicated, it may be predicted with confidence that other viral or microbial inhibitors of this pathway exist and are yet to be discovered. Microbial counter-strategies might include i) proteins that bind to or inactivate cGAS, ii) proteins that bind DNA and prevent DNA-mediated activation of cGAS, iii) phosphodiesterases that cleave cGAMP to prevent activation of STING, iv) proteins that bind to STING to block its activation by cGAS, prevent its relocation from the ER post binding to cGAMP, or block its ability to recruit and activate TBK-1, and v) miRNAs that downregulate activators of the

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DNA sensing pathway or upregulate negative regulators of the pathway. The opportunities for pathogen interference are legion. Against this background it is notable that hitherto the inhibitors of DNA sensing by pathogens are limited to i) a protein that binds to components of DNA-PK (e.g. VACV protein C16), ii) proteins from HCMV and HSV-1 that bind to IFI16, and iii) proteins that inhibit activation or function of IRF3 and NF-kB (Fig. 2). In the case of proteins that bind to IFI16, it remains unclear if the primary purpose of binding or degrading IFI16 is to inhibit DNA sensing by IFI16 rather than the numerous other activities reportedly attributable to this protein. 4. DNA sensing in auto-immunity Aside from its function as a PAMP, there is strong evidence that self-DNA can act as a damage-associated molecular pattern (DAMP) driving a sterile inflammatory response [83]. Several lines of evidence in humans and mice indicate that the accumulation of self-DNA can induce IFN-associated, inflammatory pathologies. The compartmentalisation and packaging of genomic DNA as chromatin in the nucleus and mitochondrion allows it to remain physically separated from endosomal and cytoplasmic DNA sensors. Under homoeostatic conditions DNA that escapes these compartments is digested by cytoplasmic and serum nucleases such as DNase I and II and three prime repair exonuclease I (TREX1) [83e85]. Strikingly, DNase II deletion is embryonic lethal in mice [85] and a causal link between innate immune DNA sensing and this lethality has been established by an elegant series of genetic crosses. Embryonic livers lacking DNase II contain high levels of IFNb, IFNg and ISGs, and macrophages that have engulfed apoptotic cells but have failed to digest the DNA of these cellular fragments are the source of this IFN. Crossing the DNase II/ mice with IFNR1/ mice rescues this phenotype and ascribes the pathology to the production of type I IFNs [85]. Confirmation that DNA is the causative pathological agent in this context and that cytoplasmic innate immune DNA sensing of undigested self-DNA drives the IFN production came from observations that deletion of STING also restores the viability of the DNase II/ mice [86]. In a similar vein TREX1/ mice have type I IFN driven autoinflammatory pathology [84] that can also be rescued by crossing with IFNR1/ and STING/ mice, indicating a common molecular mechanism underlying the pathologies caused by deletion of either TREX or DNase II [87]. However, genetic crosses combined with analysis of bone marrow chimeras confirmed that DNA drives IFN production from nonhaematopoietic cells in TREX/ mice [87], which is strikingly different from the DNase II/mouse where the source of IFN is macrophages [85]. Such studies highlight how essential it is to understand cell-type specific roles for sensors and regulators of innate immune sensing in the pathological context to which they are relevant. Further analysis of TREX/ fibroblasts (a source of IFN in TREX/ mice) has indicated that the spontaneous production of IFN by these cells is cGAS dependent [88].

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Together these data have served to highlight the importance of cytoplasmic DNA sensing in self-DNA driven sterile inflammation and this concept is further enhanced by the discovery of patients with mutations in these same genes that suffer from auto-inflammatory and autoimmune disorders. The clearest data in relation to such discoveries comes from Aicardi-Goutieres syndrome (AGS) [89], an autoinflammatory disorder characterised by severe, IFNassociated pathology [90,91]. Many patients with AGS have TREX1 mutations [92] and indicating that accumulation (i.e. a lack of clearance) of self-DNA in humans also results in an auto-inflammatory phenotype [90]. Notably, however AGS manifests as an early-onset encephalopathy whereas TREX1deficient mice develop an often-lethal inflammatory myocarditis without a cerebral phenotype. Self-DNA has now been linked to a wide variety of other autoimmune or autoinflammatory disorders, either directly or indirectly. There is a clear link between SLE and DNA sensing. SLE is an IFNdriven pathology associated with the presence of anti-DNA and anti-nuclear protein antibodies [83]. Although the aetiology of SLE is polygenic and initiated by a number of possible environmental triggers, there is clear evidence that DNA and innate DNA sensing plays a role in a proportion of cases. One of the strongest links between DNA and SLE is the association with an accumulation of neutrophil extracellular traps (NETs). NETs are long anti-microbial strands of genomic DNA coated with proteins that are extruded from neutrophils in response to a number of environmental stimuli, including virus infection [93]. Accumulation, or lack of clearance, of NET DNA is strongly linked to SLE [94,95]. Intriguingly, many SLE patients also have autoantibodies targetting DNA sensors, including Ku [96], DNA-PKcs [97], IFI16 [98], MRE11 [97], RNA-polymerase III [99] and these, in the form of immunocomplexes, may stimulate DCs to produce IFNs via endosomal TLRs contributing to SLE pathologies [83,100]. Although causal mechanistic links between cytoplasmic nucleic acid sensing and SLE are not yet available, there is a clear understanding that accumulation of selfDNA, whether in the form of NETs or otherwise is a contributory factor to this autoimmune disease. In a similar way, there are several reports that self-DNA can contribute to a raft of further autoimmune and auto-inflammatory pathologies, including deep vein thrombosis [101], atherosclerosis [102], and type 1 diabetes [103]. However, in these cases it remains unclear what are the molecular signalling events downstream of DNA, which receptors are involved, and perhaps equally pertinently, what is the precise nature of the DNA ligand that is responsible for these pathologies. Further efforts are required in this area to provide more detailed molecular understanding of the role of DNA in autoimmune disorders. 5. DNA sensing in vaccine development An understanding of innate immune DNA sensing is crucial for the success of DNA-plasmid based vaccines and also those based on DNA virus vectors. The establishment of an appropriate cytokine milieu at the site of vaccination is critical for

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the provision of an optimal environment for leucocyte recruitment and activation, antigen presentation and the induction long-lived, effective memory lymphocytes [104,105]. For a DNA plasmid vector that expresses an antigen and acts at its own adjuvant, the ability to stimulate cytoplasmic DNA sensing pathways has been shown to be crucial for antigen specific memory development [40,105]. The absence of TBK1 results in a complete loss of DNA vaccine-driven T and B cell memory in mice, but the loss of TLR signalling has no effect on this process [40]. However, DNA vaccination is a nonoptimal system that generally leads to poor immunological memory and is mainly used (to date) in prime-boost regimes [104,106]. Further work is needed to understand which DNA receptors are implicated in which cell types during DNA vaccination and how to optimise their formulation and delivery to gain optimal access to the appropriate compartments within the appropriate cells. Furthermore, optimising the sequences and structures of DNA vaccine vectors may allow maximised adjuvant capacity whilst avoiding degradation by DNases. Once this is achieved, DNA vaccines have the potential to be used more extensively as stand-alone vaccines away from complex and empirically designed prime-boost regimes. DNA sensing is also important to DNA virus-based vaccine vectors since the viral DNA in such vectors is a potent stimulator of innate responses. The use of VACV strain MVA, adenoviruses, HCMV adeno-associated viruses (AAVs) and other DNA virus vaccine vectors is now commonplace in vaccine development [107]. Since it is known that IFN has an important function not only in anti-viral responses but also in immunological memory development [105,108], the detection of virus vector genomes by DNA sensors is crucial for generation of the optimal cytokine environment to promote memory development. The innate sensing of some DNA vaccine vectors, in particular MVA DNA, has been shown to be detected by DNA-PK in fibroblasts [17] and cGAS in pDCs [34] and the whole virus can activate Mda5, TLR2 and TLR6 and the NALP3 inflammasome in macrophages [109]. However, there are likely to be cell-type differences in these responses and so exactly how MVA activates an innate immune response following vaccination remains poorly understood, particularly if different vaccination routes are used (e.g. subcutaneous versus intranasal) such that different cell types are infected. This situation is complicated further by the production of immune evasion molecules by these viruses (Fig. 2). The impact of immune evasion of DNA sensing apparatus by, for example, UL83 [79] or C16 [76], on the adaptive immune system is not well understood. There is clear evidence, however, that the removal of intracellular viral immune evasion molecules does have impact on immunological memory development. For example, removal of the TBK1 inhibitor C6 from VACV strains MVA or WR, leads to enhanced immunogenicity and a less virulent (and hence safer) virus [110,111], consistent with the crucial role of TBK1 in DNAdriven adaptive immunity [40]. It is likely that the future optimisation of DNA virus-based vaccine vectors will involve more detailed understanding of the how viral proteins interfere

with innate immune responses and how that impacts on TH1 memory development. 6. Conclusions  DNA is a central molecule in pathogen sensing  DNA can be sensed directly or processed as the second messenger c-GAMP  The axis of cytosolic DNA sensing signalling pathways is STING/TBK-1/IRF3/type I IFN  DNA viruses developed several molecules to counteract DNA sensing mechanisms, either targeting directly the sensors or its signalling pathways  Dysregulation of intracellular DNA sensing leads to autoinflammatory and auto-immune disease

Conflict of interest We declare no conflict of interests. Acknowledgements The work in the authors' laboratories has been supported by grants from the Medical Research Council (Grant No. G1000207) and the Wellcome Trust Principal Research Fellowship (Grant No. 090315), G.L.S. is a Wellcome Trust Principal Research Fellow. References [1] Barrangou R, Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell 2014;54:234e44. [2] Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol 2010;8:317e27. [3] Rotem Z, Cox RA, Isaacs A. Inhibition of virus multiplication by foreign nucleic acid. Nature 1963;197:564e6. [4] Janeway Jr CA. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 1989;54(Pt 1):1e13. [5] Medzhitov R. Origin and physiological roles of inflammation. Nature 2008;454:428e35. [6] Blatt NB, Glick GD. Anti-DNA autoantibodies and systemic lupus erythematosus. Pharmacol Ther 1999;83:125e39. [7] Menezes GB, Mansur DS, McDonald B, Kubes P, Teixeira MM. Sensing sterile injury: opportunities for pharmacological control. Pharmacol Ther 2011;132:204e14. [8] Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A toll-like receptor recognizes bacterial DNA. Nature 2000;408:740e5. [9] Barton GM, Kagan JC, Medzhitov R. Intracellular localization of tolllike receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 2006;7:49e56. [10] Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 2006;24:93e103. [11] Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, et al. A toll-like receptor-independent antiviral response induced by doublestranded B-form DNA. Nat Immunol 2006;7:40e8. [12] Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 2008;455:674e8. [13] Bauernfeind F, Hornung V. Of inflammasomes and pathogensesensing of microbes by the inflammasome. EMBO Mol Med 2013;5:814e26.

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D.S. Mansur et al. / Microbes and Infection xx (2014) 1e11 [14] Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 2009;10:1065e72. [15] Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Rohl I, et al. cGAS produces a 20 -50 -linked cyclic dinucleotide second messenger that activates STING. Nature 2013;498:380e4. [16] Chiu YH, Macmillan JB, Chen ZJ. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009;138:576e91. [17] Ferguson BJ, Mansur DS, Peters NE, Ren H, Smith GL. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. Elife 2012;1:e00047. [18] Kondo T, Kobayashi J, Saitoh T, Maruyama K, Ishii KJ, Barber GN, et al. DNA damage sensor MRE11 recognizes cytosolic double-stranded DNA and induces type I interferon by regulating STING trafficking. Proc Natl Acad Sci U S A 2013;110:2969e74. [19] Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 2013;339:786e91. [20] Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007;448:501e5. [21] Unterholzner L, Keating SE, Baran M, Horan KA, Jensen SB, Sharma S, et al. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 2010;11:997e1004. [22] Wu J, Sun L, Chen X, Du F, Shi H, Chen C, et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 2013;339:826e30. [23] Zhang Z, Yuan B, Bao M, Lu N, Kim T, Liu YJ. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol 2011;12:959e65. [24] Li XD, Wu J, Gao D, Wang H, Sun L, Chen ZJ. Pivotal roles of cGAScGAMP signaling in antiviral defense and immune adjuvant effects. Science 2013;341:1390e4. [25] Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J, et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat Immunol 2010;11:385e93. [26] Rathinam VA, Jiang Z, Waggoner SN, Sharma S, Cole LE, Waggoner L, et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol 2010;11:395e402. [27] Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011;34:637e50. [28] Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 2005;434:772e7. [29] Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 2005;434:1035e40. [30] Mansur DS, Kroon EG, Nogueira ML, Arantes RM, Rodrigues SC, Akira S, et al. Lethal encephalitis in myeloid differentiation factor 88deficient mice infected with herpes simplex virus 1. Am J Pathol 2005;166:1419e26. [31] Krug A, Luker GD, Barchet W, Leib DA, Akira S, Colonna M. Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. Blood 2004;103:1433e7. [32] Lima GK, Zolini GP, Mansur DS, Freire Lima BH, Wischhoff U, Astigarraga RG, et al. Toll-like receptor (TLR) 2 and TLR9 expressed in trigeminal ganglia are critical to viral control during herpes simplex virus 1 infection. Am J Pathol 2010;177:2433e45. [33] Dai P, Cao H, Merghoub T, Avogadri F, Wang W, Parikh T, et al. Myxoma virus induces type I interferon production in murine plasmacytoid dendritic cells via a TLR9/MyD88-, IRF5/IRF7-, and IFNARdependent pathway. J Virol 2011;85:10814e25. [34] Dai P, Wang W, Cao H, Avogadri F, Dai L, Drexler I, et al. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNAsensing pathway. PLoS Pathog 2014;10:e1003989.

9

[35] Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 2003;4:491e6. [36] McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Maniatis T. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci U S A 2004;101:233e8. [37] Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway. Science 2003;300:1148e51. [38] Iwamura T, Yoneyama M, Yamaguchi K, Suhara W, Mori W, Shiota K, et al. Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways. Genes Cells 2001;6:375e88. [39] Tamura T, Yanai H, Savitsky D, Taniguchi T. The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol 2008;26:535e84. [40] Ishii KJ, Kawagoe T, Koyama S, Matsui K, Kumar H, Kawai T, et al. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 2008;451:725e9. [41] Shu C, Sankaran B, Chaton CT, Herr AB, Mishra A, Peng J, et al. Structural insights into the functions of TBK1 in innate antimicrobial immunity. Structure 2013;21:1137e48. [42] Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNAmediated, type I interferon-dependent innate immunity. Nature 2009;461:788e92. [43] Gao P, Ascano M, Zillinger T, Wang W, Dai P, Serganov AA, et al. Structure-function analysis of STING activation by c[G(20 ,50 )pA(30 ,50 ) p] and targeting by antiviral DMXAA. Cell 2013;154:748e62. [44] Tanaka Y, Chen ZJ. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal 2012;5:ra20. [45] Pham TH, Kwon KM, Kim YE, Kim KK, Ahn JH. DNA sensingindependent inhibition of herpes simplex virus 1 replication by DAI/ ZBP1. J Virol 2013;87:3076e86. [46] Sharma S, DeOliveira RB, Kalantari P, Parroche P, Goutagny N, Jiang Z, et al. Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome. Immunity 2011;35:194e207. [47] Lippmann J, Rothenburg S, Deigendesch N, Eitel J, Meixenberger K, van Laak V, et al. IFNbeta responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/ DAI). Cell Microbiol 2008;10:2579e88. [48] Rebsamen M, Heinz LX, Meylan E, Michallet MC, Schroder K, Hofmann K, et al. DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB. EMBO Rep 2009;10:916e22. [49] Upton JW, Kaiser WJ, Mocarski ES. DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe 2012;11:290e7. [50] Caposio P, Gugliesi F, Zannetti C, Sponza S, Mondini M, Medico E, et al. A novel role of the interferon-inducible protein IFI16 as inducer of proinflammatory molecules in endothelial cells. J Biol Chem 2007;282:33515e29. [51] Kerur N, Veettil MV, Sharma-Walia N, Bottero V, Sadagopan S, Otageri P, et al. IFI16 acts as a nuclear pathogen sensor to induce the inflammasome in response to Kaposi Sarcoma-associated herpesvirus infection. Cell Host Microbe 2011;9:363e75. [52] Monroe KM, Yang Z, Johnson JR, Geng X, Doitsh G, Krogan NJ, et al. IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV. Science 2013;343:428e32. [53] Cooper A, Garcia M, Petrovas C, Yamamoto T, Koup RA, Nabel GJ. HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration. Nature 2013;498:376e9. [54] Parvatiyar K, Zhang Z, Teles RM, Ouyang S, Jiang Y, Iyer SS, et al. The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response. Nat Immunol 2012;13:1155e61.

Please cite this article in press as: Mansur DS, et al., Intracellular sensing of viral DNA by the innate immune system, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.09.010

10

D.S. Mansur et al. / Microbes and Infection xx (2014) 1e11

[55] Blunt T, Finnie NJ, Taccioli GE, Smith GC, Demengeot J, Gottlieb TM, et al. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995;80:813e23. [56] Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 2010;79:181e211. [57] Karpova AY, Trost M, Murray JM, Cantley LC, Howley PM. Interferon regulatory factor-3 is an in vivo target of DNA-PK. Proc Natl Acad Sci U S A 2002;99:2818e23. [58] Zhang X, Brann TW, Zhou M, Yang J, Oguariri RM, Lidie KB, et al. Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN. J Immunol 2011;186:4541e5. [59] Moll U, Lau R, Sypes MA, Gupta MM, Anderson CW. DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues. Oncogene 1999;18:3114e26. [60] Burdette DL, Monroe KM, Sotelo-Troha K, Iwig JS, Eckert B, Hyodo M, et al. STING is a direct innate immune sensor of cyclic diGMP. Nature 2011;478:515e8. [61] Gao P, Ascano M, Wu Y, Barchet W, Gaffney BL, Zillinger T, et al. Cyclic [G(20 ,50 )pA(30 ,50 )p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 2013;153:1094e107. [62] Zhang X, Shi H, Wu J, Zhang X, Sun L, Chen C, et al. Cyclic GMPAMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell 2013;51:226e35. [63] Diner EJ, Burdette DL, Wilson SC, Monroe KM, Kellenberger CA, Hyodo M, et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep 2013;3:1355e61. [64] Kuchta K, Knizewski L, Wyrwicz LS, Rychlewski L, Ginalski K. Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human. Nucleic Acids Res 2009;37:7701e14. [65] Schoggins JW, Wilson SJ, Panis M, Murphy MY, Jones CT, Bieniasz P, et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature 2011;472:481e5. [66] Zhang X, Wu J, Du F, Xu H, Sun L, Chen Z, et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Rep 2014;6:421e30. [67] Civril F, Deimling T, de Oliveira Mann CC, Ablasser A, Moldt M, Witte G, et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 2013;498:332e7. [68] Ablasser A, Schmid-Burgk JL, Hemmerling I, Horvath GL, Schmidt T, Latz E, et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 2013;503:530e4. [69] Gao D, Wu J, Wu YT, Du F, Aroh C, Yan N, et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 2013;341:903e6. [70] Schoggins JW, MacDuff DA, Imanaka N, Gainey MD, Shrestha B, Eitson JL, et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 2013;505:691e5. [71] Diebold SS, Montoya M, Unger H, Alexopoulou L, Roy P, Haswell LE, et al. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 2003;424:324e8. [72] Randall RE, Goodbourn S. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 2008;89:1e47. [73] Andrejeva J, Childs KS, Young DF, Carlos TS, Stock N, Goodbourn S, et al. The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter. Proc Natl Acad Sci U S A 2004;101:17264e9. [74] Smith GL, Benfield CT, Maluquer de Motes C, Mazzon M, Ember SW, Ferguson BJ, et al. Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity. J Gen Virol 2013;94:2367e92. [75] Fahy AS, Clark RH, Glyde EF, Smith GL. Vaccinia virus protein C16 acts intracellularly to modulate the host response and promote virulence. J Gen Virol 2008;89:2377e87.

[76] Peters NE, Ferguson BJ, Mazzon M, Fahy AS, Krysztofinska E, Arribas-Bosacoma R, et al. A mechanism for the inhibition of DNAPK-mediated DNA sensing by a virus. PLoS Pathog 2013;9:e1003649. [77] Aglipay JA, Lee SW, Okada S, Fujiuchi N, Ohtsuka T, Kwak JC, et al. A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in the p53-mediated apoptosis pathway. Oncogene 2003;22:8931e8. [78] Cristea IM, Moorman NJ, Terhune SS, Cuevas CD, O'Keefe ES, Rout MP, et al. Human cytomegalovirus pUL83 stimulates activity of the viral immediate-early promoter through its interaction with the cellular IFI16 protein. J Virol 2010;84:7803e14. [79] Li T, Chen J, Cristea IM. Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion. Cell Host Microbe 2013;14:591e9. [80] Li T, Diner BA, Chen J, Cristea IM. Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc Natl Acad Sci U S A 2012;109:10558e63. [81] Gariano GR, Dell'Oste V, Bronzini M, Gatti D, Luganini A, De Andrea M, et al. The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication. PLoS Pathog 2012;8:e1002498. [82] Orzalli MH, DeLuca NA, Knipe DM. Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein. Proc Natl Acad Sci U S A 2012;109:E3008e17. [83] Ablasser A, Hertrich C, Wassermann R, Hornung V. Nucleic acid driven sterile inflammation. Clin Immunol 2013;147:207e15. [84] Stetson DB, Ko JS, Heidmann T, Medzhitov R. Trex1 prevents cellintrinsic initiation of autoimmunity. Cell 2008;134:587e98. [85] Yoshida H, Okabe Y, Kawane K, Fukuyama H, Nagata S. Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA. Nat Immunol 2005;6:49e56. [86] Ahn J, Gutman D, Saijo S, Barber GN. STING manifests self DNAdependent inflammatory disease. Proc Natl Acad Sci U S A 2012;109:19386e91. [87] Gall A, Treuting P, Elkon KB, Loo YM, Gale Jr M, Barber GN, et al. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 2012;36:120e31. [88] Ablasser A, Hemmerling I, Schmid-Burgk JL, Behrendt R, Roers A, Hornung V. TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner. J Immunol 2014. [89] Aicardi J, Goutieres F. A progressive familial encephalopathy in infancy with calcifications of the basal ganglia and chronic cerebrospinal fluid lymphocytosis. Ann Neurol 1984;15:49e54. [90] Behrendt R, Roers A. Mouse models for Aicardi-Goutieres syndrome provide clues to the molecular pathogenesis of systemic autoimmunity. Clin Exp Immunol 2014;175:9e16. [91] Rice GI, Forte GM, Szynkiewicz M, Chase DS, Aeby A, AbdelHamid MS, et al. Assessment of interferon-related biomarkers in Aicardi-Goutieres syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study. Lancet Neurol 2013;12:1159e69. [92] Crow YJ, Hayward BE, Parmar R, Robins P, Leitch A, Ali M, et al. Mutations in the gene encoding the 30 -50 DNA exonuclease TREX1 cause Aicardi-Goutieres syndrome at the AGS1 locus. Nat Genet 2006;38:917e20. [93] Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 2007;5:577e82. [94] Hakkim A, Furnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V, et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A 2010;107:9813e8. [95] Villanueva E, Yalavarthi S, Berthier CC, Hodgin JB, Khandpur R, Lin AM, et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. J Immunol 2011;187:538e52.

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D.S. Mansur et al. / Microbes and Infection xx (2014) 1e11 [96] Tojo T, Kaburaki J, Hayakawa M, Okamoto T, Tomii M, Homma M. Precipitating antibody to a soluble nuclear antigen “Ki” with specificity for systemic lupus erythematosus. Ryumachi 1981;21(Suppl.):129e40. [97] Schild-Poulter C, Su A, Shih A, Kelly OP, Fritzler MJ, Goldstein R, et al. Association of autoantibodies with Ku and DNA repair proteins in connective tissue diseases. Rheumatology 2008;47:165e71. [98] Caneparo V, Cena T, De Andrea M, Dell'oste V, Stratta P, Quaglia M, et al. Anti-IFI16 antibodies and their relation to disease characteristics in systemic lupus erythematosus. Lupus 2013;22:607e13. [99] Cavazzana I, Ceribelli A, Airo P, Zingarelli S, Tincani A, Franceschini F. Anti-RNA polymerase III antibodies: a marker of systemic sclerosis with rapid onset and skin thickening progression. Autoimmun Rev 2009;8:580e4. [100] Means TK, Latz E, Hayashi F, Murali MR, Golenbock DT, Luster AD. Human lupus autoantibody-DNA complexes activate DCs through cooperation of CD32 and TLR9. J Clin Invest 2005;115:407e17. [101] Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012;10:136e44. [102] Oka T, Hikoso S, Yamaguchi O, Taneike M, Takeda T, Tamai T, et al. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 2012;485:251e5. [103] Diana J, Simoni Y, Furio L, Beaudoin L, Agerberth B, Barrat F, et al. Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes. Nat Med 2013;19:65e73.

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[104] Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate immunity to work. Immunity 2010;33:492e503. [105] Desmet CJ, Ishii KJ. Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination. Nat Rev Immunol 2012;12:479e91. [106] Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011;12:509e17. [107] Liu MA. Immunologic basis of vaccine vectors. Immunity 2010;33:504e15. [108] Gonzalez-Navajas JM, Lee J, David M, Raz E. Immunomodulatory functions of type I interferons. Nat Rev Immunol 2012;12:125e35. [109] Delaloye J, Roger T, Steiner-Tardivel QG, Le Roy D, Knaup Reymond M, Akira S, et al. Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome. PLoS Pathog 2009;5:e1000480. [110] Sumner RP, Ren H, Smith GL. Deletion of immunomodulator C6 from vaccinia virus strain Western Reserve enhances virus immunogenicity and vaccine efficacy. J Gen Virol 2013;94:1121e6. [111] Unterholzner L, Sumner RP, Baran M, Ren H, Mansur DS, Bourke NM, et al. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. PLoS Pathog 2011;7:e1002247.

Please cite this article in press as: Mansur DS, et al., Intracellular sensing of viral DNA by the innate immune system, Microbes and Infection (2014), http:// dx.doi.org/10.1016/j.micinf.2014.09.010

Intracellular sensing of viral DNA by the innate immune system.

Recent years have seen a great advance in knowledge of how a host senses infection. Nucleic acids, as a common denominator to all pathogens, are at th...
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