Innate recognition of alphaherpesvirus DNA.

CHAPTER TWO

Innate Recognition of Alphaherpesvirus DNA Stefanie Luecke*, Søren R. Paludan†,{,1 *Graduate School of Life Sciences, Universiteit Utrecht, Utrecht, The Netherlands † Department of Biomedicine, Aarhus University, Aarhus, Denmark { Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 Alphaherpesviruses 1.2 Immunity to alphaherpesviruses 1.3 Innate DNA sensing 2. DNA Sensors 2.1 TLR9 2.2 Discovery of intracellular DNA sensors 2.3 DAI 2.4 AIM2 2.5 IFI16 2.6 cGAS 2.7 RNA Pol III and RIG-I 3. Accessibility of Viral DNA to DNA Sensors 4. Evasion of DNA-Induced Signaling 5. Relevance for Vaccine Design 6. Conclusions and Future Perspective References

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Abstract Alphaherpesviruses include human and animal pathogens, such as herpes simplex virus type 1, which establish life-long latent infections with episodes of recurrence. The immunocompetence of the infected host is an important determinant for the outcome of infections with alphaherpesviruses. Recognition of pathogen-associated molecular patterns by pattern recognition receptors is an essential, early step in the innate immune response to pathogens. In recent years, it has been discovered that herpesvirus DNA is a strong inducer of the innate immune system. The viral genome can be recognized in endosomes by TLR9, as well as intracellularly by a variety of DNA sensors, the best documented being cGAS, RNA Pol III, IFI16, and AIM2. These DNA sensors use converging signaling pathways to activate transcription factors, such as IRF3 and NF-κB, which induce the expression of type I interferons and other inflammatory cytokines Advances in Virus Research, Volume 92 ISSN 0065-3527 http://dx.doi.org/10.1016/bs.aivir.2014.11.003

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2015 Elsevier Inc. All rights reserved.

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Stefanie Luecke and Søren R. Paludan

and activate the inflammasome. This review summarizes the recent literature on the innate sensing of alphaherpesvirus DNA, the mechanisms of activation of the different sensors, their mechanisms of signal transduction, their physiological role in defense against herpesvirus infection, and how alphaherpesviruses seek to evade these responses to allow establishment and maintenance of infection.

1. INTRODUCTION 1.1. Alphaherpesviruses The Herpesviridae family comprises more than 130 virus species, which infect mammals, birds, and reptiles. Herpesviruses are enveloped, double-stranded DNA viruses (Fig. 1A), which usually lyse productively infected cells and

Figure 1 Alphaherpesvirus structure and entry. (A) Structure of the alphaherpesvirus virion. The linear dsDNA genome is surrounded by an icosahedral capsid. Associated with the vertices of the capsid are the inner tegument proteins, which are surrounded by the outer tegument. The lipid bilayer envelope contains multiple viral glycoproteins. (B) Schematic illustration of alphaherpesviral cell entry, nuclear DNA delivery, and exposure to DNA sensors. The virion attaches to the cell surface through interaction of the glycoproteins with cellular receptors. The virus can enter the cell via two pathways: by direct fusion of the viral envelope with the plasma membrane or by endocytosis and subsequent fusion of envelope and endosomal membrane. The capsid is transported along microtubules toward the microtubule organizing center (MTOC) near the nucleus. The capsid then docks at a nuclear pore, and the viral genome is released into the nucleus. In the nucleus, the viral genome circularizes and lytic or latent infection takes place. Viral DNA is exposed to endosomal DNA sensors via endosomal entry or by autophagocytic delivery of cytosolic capsids to endosomes, to cytosolic sensors by proteasomal degradation of the capsid, and to nuclear sensors by nuclear DNA delivery.

Innate Recognition of Alphaherpesvirus DNA

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establish latent infections in their hosts. Eight herpesviruses are known to cause disease in humans, most notably in children and immunocompromised individuals. The family is divided into three subfamilies, alpha-, beta-, and gamma-herpesviruses. This division was originally based on biological similarities and later confirmed by genome sequencing (Pellett & Roizman, 2013). Alphaherpesviruses are characterized by a relatively broad host range and fast reproduction during lytic infection compared to other herpesviruses (Pellett & Roizman, 2013). They contain three virus species pathogenic to humans (herpes simplex virus type 1 (HSV-1, also called human herpesvirus 1), herpes simplex virus type 2 (HSV-2, also called human herpesvirus 2), and varicella zoster virus (VZV, also called human herpesvirus 3)) and many pathogens of veterinary importance, notably Marek’s disease virus (MDV, also called gallid herpesvirus 2) and pseudorabies virus (PRV, also called suid herpesvirus 1). Herpes simplex viruses are ubiquitous human pathogens, which lytically infect epithelial cells of mucosal surfaces and the skin and afterward establish latency in the cell bodies of peripheral sensory neurons innervating the infected area. During latency, the viral genome persists in the nucleus of the host while the majority of viral genes are silenced. It can be maintained for the lifetime of the host organism. Reactivation from latency can be induced by a number of factors, including tissue damage, UV radiation, immune status changes, and stress, but can also occur spontaneously. In reactivation, virus particles produced in the neuronal cell body are transported along the axons by anterograde transport and infect epithelial cells again. HSV-1 usually causes recurrent cold sores at the lips (herpes labialis), while HSV-2 is often responsible for genital herpes infections (herpes genitalis). HSV may also spread to the central nervous system, leading to herpes encephalitis, cause disseminating herpes infections in neonates and immunocompromised individuals, and infect the eyes, often leading to blindness (Roizman, Knipe, & Whitley, 2013). VZV is the causal agent of chicken pox after primary infection and of shingles (herpes zoster) upon reactivation. In primary infection, the virus usually infects epithelial cells in the upper respiratory tract followed by infection of T cells in lymphoid tissues. This allows for transport of the virus to skin areas over the whole body, where viral replications result in the characteristic rash. VZV then establishes latency in peripheral sensory neurons. The virus can reactivate to cause shingles, which may be associated with serious neurological complications (Arvin & Gilden, 2013).

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PRV first infects epithelial cells of the respiratory tract and then establishes latency in sensory neurons. The natural hosts for this virus are pigs, but it can also infect many other mammals such as cattle, sheep, and dogs. It is the causative agent of Aujeszky’s disease, which is characterized by symptoms ranging from high mortality and severe central nervous system defects in suckling piglets, via abortions and stillbirths in pregnant sows, to fever and respiratory symptoms in adult pigs (Pomeranz, Reynolds, & Hengartner, 2005). MDV establishes latency in and causes oncogenic transformation of immune cells, especially CD4+ T cells, and lytically infects epithelial cells of inner organs and the skin in chickens, leading to a variety of clinical symptoms including chronic polyneuritis and visceral lymphoma (Osterrieder, Kamil, Schumacher, Tischer, & Trapp, 2006). Herpesviruses enter their host cells by fusion of the viral lipid envelope with the plasma or endosomal membrane, which is initiated by interaction of viral glycoproteins in the virion envelope with cell surface receptors. Upon viral entry, the tegument proteins, located between the viral envelope and the capsid, are released into the cytoplasm and interact with host factors to create a favorable environment for the virus. In permissive cells, i.e., cells that allow productive replication of the virus, the icosahedral capsid containing the viral dsDNA genome moves along the microtubule network toward the nucleus, where the viral DNA is translocated through the nuclear pores. In the nucleus, the viral genome circularizes (Fig. 1). Silencing of viral gene expression can lead to the establishment of latent infection. During lytic infection, expression of viral genes takes place in three stages, immediate-early (responsible for subsequent viral gene expression and immune evasion), early (replication of viral DNA), and late (formation and release of progeny virions), and eventually leads to multiplication of the viral genome and to assembly and release of new virus particles (Arvin & Gilden, 2013; Roizman et al., 2013). However, some cells, such as macrophages in case of HSV-1, are nonpermissive for the virus, i.e., productive replication does not take place. Even in permissive cells, not all viral particles lead to productive infection. It is suspected that this is partly due to the early innate antiviral response of the cells (Paludan, Bowie, Horan, & Fitzgerald, 2011).

1.2. Immunity to alphaherpesviruses As with most infectious diseases, immunity to alphaherpesviruses relies on innate and adaptive immune responses. One of the first responses upon


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detection of a herpesvirus in an infected cell is the production and secretion of interferons (IFNs), especially type I (IFNα and IFNβ), but also type III (e.g., IFNλ), and other proinflammatory cytokines and chemokines (Ank et al., 2008; Egan, Wu, Wigdahl, & Jennings, 2013). By paracrine and autocrine signaling, IFNs mediate a number of antiviral activities in infected and neighboring cells by the induction and expression of a variety of genes (interferon-stimulated genes, ISGs) (Egan et al., 2013). IFNs induce the activation of a number of cells of the innate immune system, which are attracted to the site of infection by chemokines. Natural killer (NK) cells cause apoptotic cell death in infected cells, macrophages contribute to the inflammatory response by secretion of additional chemokines, and neutrophils clear infected dying cells by phagocytosis (Egan et al., 2013). Maturation of dendritic cells (DCs), induced upon detection of pathogens, allows for the activation of the T cell response, with the cytotoxic T cell and T helper 1 cell response being of particular importance in the attempt to achieve clearance of these intracellular pathogens and in long-term prevention of reactivation from latency (Arvin & Gilden, 2013; Roizman et al., 2013). The humoral immune response, particularly mucosal IgA production by B cells, also contributes to the immune response, albeit to a lesser extent (Cradock-Watson, Ridehalgh, & Bourne, 1979; El Falaky, Vestergaard, & Hornsleth, 1977). Herpesviruses encode a number of gene products that counteract the innate and the adaptive immune system, leading to a delicate balance between host defense and viral evasion (Arvin & Gilden, 2013; Paludan et al., 2011; Roizman et al., 2013).

1.3. Innate DNA sensing The innate immune system is responsible for distinguishing between self and nonself during early stages of infection with pathogens. For this, it makes use of pattern recognition receptors (PRRs), which recognize pathogenassociated molecular pattern (PAMP). PAMPs are pathogen-specific molecules or molecules with aberrant localization. Toll-like receptors (TLRs) recognize a variety of pathogen-derived proteins and lipids at the cell surface (e.g., TLR2 recognizes herpesviral glycoproteins) and foreign nucleic acids in endosomes (e.g., TLR3 recognizes dsRNA, which is a product of herpesviral replication, and TLR9 recognizes viral dsDNA). Recently, intracellular nucleic acids are emerging as potent PAMPs. RIG-I (retinoic acid-inducible gene 1) and MDA5 (melanoma differentiation-associated protein 5) recognize viral RNAs produced during HSV-1 replication and

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signal via the adaptor protein MAVS (mitochondrial antiviral signaling protein) to induce expression of inflammatory cytokines and type I IFNs. Over 10 intracellular DNA sensors have been proposed in recent years. They include DAI (DNA-dependent activator of IFN-regulatory factors), RNA Pol III (RNA polymerase III), AIM2 (absent in melanoma 2), IFI16 (interferon-inducible protein 16), cGAS (cyclic GMP–AMP synthase), DDX41, DHX9, DHX36, LRRFIP1 (leucine-rich repeat flightless-interacting protein 1), and two proteins involved in the DNA damage response, Mre11 and Ku70/DNA-PK (DNA-dependent protein kinase; Atianand & Fitzgerald, 2013; Goubau, Deddouche, & Reis e Sousa, 2013; Nie & Wang, 2013; Paludan et al., 2011). This review focuses on TLR9, AIM2, IFI16, RNA Pol III, and cGAS, which are best documented as DNA sensors (Fig. 2). As alphaherpesviruses contain a DNA genome, DNA sensing can take place soon after infection, once the viral DNA is exposed to sensors and without the need for viral replication taking place. Therefore, it is of particular interest to understand the early innate immune response to these viruses. TLR9, the first recognition system for herpesvirus DNA discovered (Lund, Sato, Akira, Medzhitov, & Iwasaki, 2003), senses viral DNA in endosomes and signals via the adaptor MyD88 (myeloid differentiation primary response gene 88) to activate the transcription factors NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) via the IKK (IκB kinase) complex and IRF7 (interferon regulatory factor 7) via IKKα (Kawai & Akira, 2011). IFI16 and cGAS activate the signaling adaptor STING (stimulator of interferon genes), which is central to DNA-activated innate immune responses. The details of STING signaling have been reviewed in Ran, Shu, and Wang (2014) and Burdette and Vance (2013). Upon DNA binding, cGAS produces the secondary messenger, cyclic GMP– AMP (cGAMP), a cyclic dinucleotide (CDN) which binds to achieve STING activation (Sun, Wu, Du, Chen, & Chen, 2013; Wu et al., 2013), while IFI16 activates STING via an unknown mechanism. STING then interacts with the signaling kinase TBK1 (TANK-binding kinase 1) to induce the activation of IRF3 and NF-κB; activation of the latter also includes the IKK complex (Abe & Barber, 2014; Atianand & Fitzgerald, 2013; Tanaka & Chen, 2012; Zhong et al., 2008). Upon DNA stimulation, RNA Pol III synthesizes a 50 -ppp RNA product, which can bind to RIG-I to induce the MAVS adaptor-dependent signaling pathway, also induced by cytosolic RNA. MAVS mediates the activation of IRF3 and NF-κB via TBK1 and the IKK complex, respectively (Wu & Chen, 2014). While IFNα


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Figure 2 Model of sensing of alphaherpesvirus DNA. TLR9 senses herpesviral DNA in endosomes and signals via MyD88, which induces the activation of the IKK complex, consisting of IKKα, IKKβ, and the regulatory subunit NEMO. The IKK complex induces the degradation of IκB. Thus, NF-κB is released from inhibition by IκB and translocates to the nucleus. In pDCs, MyD88 additionally induces IKKα to activate IRF7. RNA Pol III produces a 50 ppp RNA when bound to AT-rich DNA. This RNA activates RIG-I, which signals via MAVS to activate the NF-κB and the IRF3 pathways. IRF3 homodimerizes or heterodimerizes with IRF7 and translocates to the nucleus. Upon cytosolic DNA stimulation, cGAS produces the cyclic dinucleotide cGAMP, which binds to STING to activate it. IFI16 senses DNA in the cytosol and in the nucleus and activates STING via an unknown mechanism that involves colocalization. Activated STING acts as a scaffold for TBK1 and IRF3 to induce IRF3 activation and it also activates the NF-κB pathway. While IFNα expression mostly requires the IRF pathway, IFNβ requires both the IRF and the NF-κB pathway. Many of the other cytokines are mainly under the control of NF-κB. IFI16 also induces inflammasome activation by interaction with ASC, which converts procaspase-1 to caspase-1, resulting in the maturation of the cytokines IL-1β and IL-18.

expression mostly requires the IRF pathway, IFNβ requires both the IRF and the NF-κB pathway. Many of the other cytokines are mainly under the control of NF-κB (Paludan et al., 2011). In addition to the induction of gene expression, AIM2 and IFI16 stimulate the formation of the inflammasome, a protein complex that mediates the proteolytic cleavage and maturation of the cytokines IL-1β (interleukin 1β) and IL-18, which have a broad proinflammatory effect and activate NK cells, respectively

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(Atianand & Fitzgerald, 2013; Egan et al., 2013; Strowig, Henao-Mejia, Elinav, & Flavell, 2012). Although the availability of mouse strains deficient in the proposed DNA sensors is still not complete, clear experimental evidence already exists showing that cytokine production based on endosomal and intracellular DNA sensing has a strong, nonredundant influence on the activation of the immune system in response to alphaherpesvirus infection (Ishii et al., 2008; Ishikawa, Ma, & Barber, 2009; Kis-Toth, Szanto, Thai, & Tsokos, 2011; Sorensen et al., 2008; Wuest et al., 2006). This review summarizes the recent literature on the innate recognition of alphaherpesvirus DNA, the mechanisms of activation of the different sensors, their methods of signal transduction, and their physiological role in defense against herpesviral infection. We discuss how the viral DNA becomes accessible to the sensors, how these recognition systems are evaded by the viruses, how DNA sensing can be applied to vaccine design, and what challenges await this field of research in the future.

2. DNA SENSORS 2.1. TLR9 TLR9 is the first discovered PRR recognizing DNA (Hemmi et al., 2000). Binding of TLR9 to DNA was confirmed among others in quantitative ligand-binding assays (Latz et al., 2007). It preferentially recognizes unmethylated CpG motifs in DNA (Kawai & Akira, 2011), which is abundant in the HSV-1 genome (Lund et al., 2003). TLR9 sensing takes place in the endosome (Ahmad-Nejad et al., 2002), where the sensor binds the DNA with its luminal ligand-binding domain containing leucine-rich repeats and signals via the cytoplasmic TIR (Toll/interleukin-1 receptor homology domain) signaling domain. Binding of DNA to TLR9 homodimers induces a conformational change in the cytosolic domain, resulting in signaling (Latz et al., 2007). In humans, TLR9 is expressed abundantly in plasmacytoid DCs (pDCs) and B cells, especially when the latter are activated, and in corneal endothelial cells (Bourke, Bosisio, Golay, Polentarutti, & Mantovani, 2003; Kadowaki et al., 2001; Takeda et al., 2011; Wagner et al., 2004). In mice, TLR9 expression is more ubiquitous, with TLR9 present in many cell types such as conventional DCs (cDCs), macrophages, B cells, and corneal epithelial cells (Sarangi, Kim, Kurt-Jones, & Rouse, 2007; Wagner, 2004). When activated by DNA binding, TLR9 signals via the adaptor protein MyD88 (Hacker et al., 2000; Schnare, Takeda, Akira, & Medzhitov, 2000) to induce a type I IFN, type III IFN, and inflammatory cytokine response


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(Fig. 2). In most cells, TLR9-induced MyD88 signaling leads to activation of TRAF6 (TNF-associated factor 6), which results in phosphorylation of IκB by the IKK complex, consisting of IKKα, IKKβ, and NEMO (NF-κB essential modulator)/IKKγ. Upon subsequent degradation of IκB, the uninhibited NF-κB can translocate to the nucleus to modulate the gene expression of inflammatory cytokines (Kawai & Akira, 2011; Takeuchi & Akira, 2010). In pDCs, TLR9 signals via an additional MyD88-dependent pathway which activates the type I IFN response by phosphorylation and activation of the transcription factor IRF7 via association of MyD88, TRAF6, TRAF3, IRAK1 (interleukin-1 receptor-associated kinase 1), and IKKα (Honda et al., 2005; Kawai & Akira, 2011; Takeuchi & Akira, 2010). While TLR9-DNA binding in early endosomes results in NF-κB signaling, later signaling from lysosome-related organelles preferentially results in IRF activation (Kawai & Akira, 2011). TLR9 mediates cytokine expression in response to HSV in a cell typeand tissue-dependent manner. pDCs are an important source of IFNα in response to DNA stimulation and HSV infection (Ank et al., 2008; Hochrein et al., 2004; Lund, Linehan, Iijima, & Iwasaki, 2006). Therefore, the first study that revealed a role for TLR9 in sensing of alphaherpesviruses focused on HSV-2-infected, bone marrow-derived pDCs. Cells derived from TLR9- or MyD88-deficient mice were incapable of secreting IFNα in response to UV-inactivated HSV-2 or CpG DNA (Lund et al., 2003). IFNα and IL-12 secretion in response to HSV-1 in pDCs was also TLR9 dependent (Krug et al., 2004). An intact endosomal pathway and viral DNA in the capsid were essential for the response, confirming the role of TLR9. Viral replication was not required, indicating that the incoming viral DNA was triggering the response (Krug et al., 2004; Lund et al., 2003; Rasmussen et al., 2007). While many cell types require multiple PRRs to induce the full response to HSV, TLR9 is sufficient in pDCs to induce IFNα/β, IL-6, IL-12, and RANTES (CCL5/chemokine (C–C) motif ligand 5) (Sato, Linehan, & Iwasaki, 2006; Sorensen et al., 2008). Additionally, pDCs induce expression of the inflammatory cytokines TNFα (tumor necrosis factor α), CCL2, CCL3, CCL4, and CXCL10 (chemokine (C–X–C) motif ligand 10) in response to HSV-1 (Megjugorac, Young, Amrute, Olshalsky, & Fitzgerald-Bocarsly, 2004). There are indications that the TLR9 dependency of the IFNα response in pDCs is time dependent, with later responses being TLR9 independent (Rasmussen et al., 2007), and dependent on the tissue from which the pDCs are derived, with bone marrowderived pDCs showing less dependency than spleen-derived pDCs

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(Hochrein et al., 2004). In vitro chemotaxis assays revealed that the chemokines produced by pDCs in a TLR9-dependent fashion in response to HSV-1 attract NK cells (Megjugorac et al., 2004). Although pDCs and their TLR9-dependent type I IFN secretion are very important for the innate immune response, a normal Th1 response was shown to develop in response to HSV-2 in the absence of this particular DC subset (Lund et al., 2006). However, activated T cells are attracted by TLR9-dependent chemokine secretion by HSV-1-infected pDC (Megjugorac et al., 2004). In murine cDCs, which are also important producers of IFN upon HSV infection (Ank et al., 2008), the production of type I and type III IFN in response to HSV infection is independent of TLR9 (Ank et al., 2008; Hochrein et al., 2004; Rasmussen et al., 2007; Sato et al., 2006). However, HSV-1-induced expression of TNFα, IL-6, and IL-12 is reduced in the absence of TLR9 (Hochrein et al., 2004; Sato et al., 2006), while HSV2-induced cytokine expression (CCL5, IL-6) in cDCs is independent of TLR9 (Sorensen et al., 2008). Similarly, in murine macrophage cell lines or primary macrophages, IFNα expression upon HSV-1 and HSV-2 is independent of TLR9, while the expression of inflammatory cytokines (TNFα, CCL5, IL-6, IL-12) in response to the viruses is partially TLR9 dependent (Hochrein et al., 2004; Lima et al., 2010; Malmgaard, Melchjorsen, Bowie, Mogensen, & Paludan, 2004; Rasmussen et al., 2007, 2009; Sorensen et al., 2008). In primary human macrophages, early sensing of HSV is TLR9 independent (Melchjorsen et al., 2010). Although B cells express TLR9, they are not major producers of IFNα in response to HSV-1 (Hochrein et al., 2004). In a human corneal endothelial cell line, a vast array of inflammatory cytokines, including CCL5, IL-6, and CXCL10, are expressed in a TLR9-dependent fashion via the NF-κB pathway in response to HSV-1 infection (Takeda et al., 2011). The production of the chemokines CXCL9 and CXCL10 in the cornea of TLR9-deficient mice infected with HSV-1 was reduced compared to wild-type mice. This was related to reduced infiltration of neutrophils into the cornea of these mice; however, macrophage infiltration was unaltered (Wuest et al., 2006). In human primary vaginal epithelial cells, a major target of HSV-2 infection, both IFNβ and IL-6 expression in response to live HSV-2 or purified HSV-2 DNA were reduced by TLR9 RNA interference (Triantafilou, Eryilmazlar, & Triantafilou, 2014). No reduction in expression of CXCL9 and CXCL10 was observed in the vaginal tissue of HSV-2-infected TLR9-deficient mice (Wuest et al., 2006).


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There are several studies that emphasize the importance of TLR9 in HSV-induced cytokine expression not only after local infection of cell lines, primary cells, and tissues but also after systemic infections. Upon intravenous injection of HSV-2, no IFNα could be detected in the serum of TLR9deficient mice (Lund et al., 2003). IFNα and CCL5 levels in the serum upon intraperitoneal infection with HSV-1 or HSV-2 were partially dependent on TLR9 at early time points after infection, while TNFα and IL-6 levels were not (Rasmussen et al., 2007; Sorensen et al., 2008). In the brain, TLR9-dependent TNFα and CXCL9 expression was detected after vaginal infection of mice with HSV-2, while type I IFN expression could not be detected (Sorensen et al., 2008). TLR9-deficient mice had altered cytokine profiles upon intranasal HSV-1 infection, both in brains and in trigeminal ganglia, with the expression of some cytokines increased and others decreased (Lima et al., 2010). Despite the important role of TLR9 in cytokine expression after HSV infection, studies on viral replication and disease outcomes in TLR9-deficient mice had mixed results. A first study using HSV-1 footpad injections showed no differences in viral replication and disease between TLR9-deficient and wild-type mice (Krug et al., 2004). The viral titers in the brains of mice infected intraperitoneally with HSV-2 were not altered by TLR9 deficiency (Sorensen et al., 2008). HSV-2-infected, TLR9- and TLR2-double-deficient mice showed impaired recruitment of NK cells to the spleen, but activation (measured by surface marker expression) and cytotoxic activity were unaltered (Sorensen et al., 2008). While TLR9deficient mice infected intraperitoneally with HSV-2 showed higher viral titers in the spleen, the survival was not affected (Rasmussen et al., 2007). TLR9-deficient mice were much more likely to succumb to lethal encephalitis caused by intranasal HSV-1 infection, and in this model, the viral titers in the brain were greatly increased (Lima et al., 2010). Ocular infection of MyD88 and TRIF (TIR-domain-containing adaptor-inducing IFNβ)double-deficient mice, which are incapable of any TLR signaling, did not result in higher viral titers in the cornea (Conrady, Zheng, Fitzgerald, Liu, & Carr, 2012). TLR9-deficient animals showed milder corneal lesions in response to HSV-1, but increased herpetic ocular disease scores. In this study, TLR9-deficient mice were not more susceptible to lethal infections, although they showed higher viral titers in brains and trigeminal ganglia (Sarangi et al., 2007). TLR9 deficiency also caused increased HSV-1 shedding in tears (Wuest et al., 2006). Interestingly, HSV-1-induced angiogenesis in a corneal micropocket assay and a murine macrophage cell line

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produced vascular endothelial growth factor upon CpG DNA stimulation. This process is speculated to contribute to the corneal scarring typical for HSV-1 infection (Zheng, Klinman, Gierynska, & Rouse, 2002). Interestingly, TLR9 signaling to induce NF-κB activation not only limits viral replication by activation of the immune system but is simultaneously required to some extent for initiation of HSV-1 gene expression, as the immediate-early infected cell protein (ICP)0 promoter contains NF-κB-responsive elements (Takeda et al., 2011). Less research has been performed on the involvement of TLR9 in the innate immune response to VZV. pDCs were shown to be responsible for IFNα production in a TLR9-dependent fashion during VZV infection, while IL-12 was not produced by human peripheral blood mononuclear cells in response to the virus. UV-inactivated virus led to less IFNα expression than infectious virus, indicating that an additional, TLR9-independent, replication-dependent process exists. In VZV infection, NF-κB was shown to be important in IFNα induction (Yu et al., 2011). In general, the TLR9 dependency of HSV recognition and the exact nature of downstream gene expression seems to depend very much on the cell and tissue type, the time course after infection, and the virus strain. However, it is likely that some of the observed differences are due to differences in experimental conditions and detection methods. Clearly, TLR9 has an important role in innate PAMP recognition, but several other mechanisms are in place that prevent too severe effects in animals lacking TLR9. For example, there are several indications that TLR9 acts in synergy with TLR2 in murine macrophages, in human vaginal epithelial cells, in the trigeminal ganglia, and in the brain (Lima et al., 2010; Sorensen et al., 2008; Triantafilou et al., 2014; Zolini et al., 2014). In humans, deficiency in IRAK4, which is required for TLR9 (and TLR7 and TLR8) signaling (Mogensen, 2009), does not lead to an increased susceptibility to alphaherpesvirus infections (Ku et al., 2007). Several attempts have been made to modulate the immune response to herpesviruses with synthetic TLR9 agonists and antagonists, for example, by Boivin and colleagues, who showed application of TLR9 agonists before infection could reduce the amount of virus in the brain, the production of inflammatory cytokines, and subsequently the mortality due to encephalitis (Boivin, Menasria, Piret, & Boivin, 2012). In the future, modulation of the TLR9 response may be used in combination with antiviral drugs to control HSV infection and resulting harmful inflammation.


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2.2. Discovery of intracellular DNA sensors Herpesvirus DNA is not only found in the lumen of endosomes. During the cause of the replication cycle, the virus migrates through the cytosol to the nucleus (Fig. 1B). Thus, the viral DNA is abundantly present in the cytoplasm and the nucleus. Soon after the discovery of TLR9 as a sensor of herpesvirus DNA, several studies demonstrated TLR-independent pathways for intracellular nucleic acid recognition. IFNα/β expression in murine macrophage-like cells in response to HSV-2 is maintained in the absence of MyD88 and under TLR9-inhibiting conditions (Malmgaard et al., 2004). Murine cDCs, macrophages, and bone marrow-derived pDCs were shown to produce IFNα and other cytokines largely independent of TLR9 upon HSV-1 stimulation (Hochrein et al., 2004). Ishii and colleagues were the first to stringently show that cytosolic DNA induces innate immune responses. Transfected genomic DNA derived from mammals, bacteria, and viruses, including HSV-1 and HSV-2, induced TLR- and RIG-Iindependent expression of IFNβ and inflammatory cytokines in MEFs (murine embryonic fibroblasts) (Ishii et al., 2005). It was then demonstrated that transfected DNA can induce a TLR-independent type I IFN response in a number of cell types, including murine pDCs, cDCs, macrophages, and MEFs. The sequence of the DNA was irrelevant to the response; however, a native DNA backbone composition was found to be essential (Stetson & Medzhitov, 2006). IRF3 is a key transcription factor in this DNA-induced type I IFN response (Ishii et al., 2005; Stetson & Medzhitov, 2006). The type I IFN expression in murine cDCs upon HSV-1 infection was then also shown to be independent of TLR9, to require viral entry and the presence of the viral genome in the virion, but to not require viral replication (Rasmussen et al., 2007). This indicated that recognition of intracellular DNA, rather than RNA, is taking place. Together, these studies suggested that additional, intracellular receptors exist for herpesviral DNA.

2.3. DAI In the search for the factor responsible for the innate signaling upon cytosolic DNA stimulation, the ISG DAI was the first protein proposed to bind cytosolic DNA and to initiate the innate immune response to it (Takaoka et al., 2007). DNA binding by DAI was demonstrated by fluorescence resonance energy transfer (FRET) and by coprecipitation of purified DAI with biotinlabeled DNA (Takaoka et al., 2007; Wang et al., 2008). DAI can bind both

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B- and Z- forms of DNA, independent of sequence (Kim et al., 2011; Takaoka et al., 2007; Wang et al., 2008). In fibroblasts, IRF3 was reported to be needed for DAI-mediated IFNβ response, while both IRF3 and IRF7 were needed for IFNα induction. Upon stimulation with cytosolic DNA, DAI associated with TBK1 and IRF3 (Takaoka et al., 2007). DAI was also found to be phosphorylated at multiple sites by TBK1, and this phosphorylation was necessary for IRF3-mediated gene expression (Wang et al., 2008). In addition to signaling to IRF3, an essential role for DAI in DNA-induced NF-κB activation was shown (Takaoka et al., 2007). DAI-induced activation of NF-κB was shown to depend on the interaction of DAI with RIP1 (receptor-interacting protein kinase 1) and to further increase upon interaction with RIP3 (Kaiser, Upton, & Mocarski, 2008). An initial study showed that overexpression of DAI in a mouse fibroblast cell line, L929 cells, increased the type I IFN response to transfected DNA species, including viral DNA. The innate response to HSV-1 infection of L929 cells was modestly reduced by RNA interference of DAI (Takaoka et al., 2007). With a focus on the neurotropism of HSV-1, it was shown that the innate response of microglia and astrocyte cells to B-DNA and HSV-1 was reduced by DAI knockdown (Furr, Chauhan, MoerdykSchauwecker, & Marriott, 2011). In vaginal cells with reduced DAI expression, the response to HSV-2 infection or HSV-2 DNA stimulation was found to be impaired (Triantafilou et al., 2014). However, soon after the proposal of DAI as a DNA sensor, several studies challenged the notion that DAI was an universal DNA sensor. While its role in the fibroblast-like L929 cell line was confirmed, MEFs and human alveolar epithelial cells did not require DAI for their innate response to DNA as shown by RNA interference (Lippmann et al., 2008; Wang et al., 2008). Additionally, MEFs, bone marrow-derived DCs, and immortalized bone marrow-derived macrophages from DAI-deficient mice responded normally to DNA, and the mice were not impaired in their immune response to a DNA vaccine (Ishii et al., 2008; Unterholzner et al., 2010). In vivo RNA interference of DAI in the cornea of mice did not result in increased viral titers after ocular HSV-1 infection (Conrady et al., 2012). Recently, a DNA sensing-independent function of DAI on HSV-1 replication in a hepatocellular carcinoma cell line was shown based on its ability to prevent expression of the immediate-early ICP0 gene. The cytokine response in these cells was not affected by modulation of DAI expression (Pham, Kwon, Kim, Kim, & Ahn, 2013).


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In conclusion, although DAI was convincingly shown to bind DNA, it is unlikely to be a central cytosolic sensor for the recognition of alphaherpesvirus DNA. However, it may be involved in DNA-induced signaling in some cell types, especially in fibroblasts. It is conceivable that DAI has a redundant function as a PRR for DNA and/or as a signaling element, which is more prominent in certain cell types. Independent of its DNAsensing properties, it is a restriction factor for HSV by targeting ICP0 expression.

2.4. AIM2 Transfected DNA of bacterial, viral, and mammalian origin causes formation of the inflammasome in murine macrophages and in the human monocyte cell line THP-1 (Muruve et al., 2008). The inflammasome is a protein complex formed around one of multiple sensor proteins, member of the NOD-like receptor (NLR) or PYHIN (pyrin and HIN domain-containing) protein families, which together recognize various viral PAMPs. The adaptor protein ASC (apoptosis-associated speck-like protein containing CARD), consisting of a pyrin and a CARD (caspase recruitment) domain, binds to the sensor proteins via its pyrin domain and to procaspase 1 via its CARD domain through homotypic interaction, resulting in caspase activation by autolytic cleavage. Caspase-1 mediates the maturation and secretion of the cytokines IL-1β and IL-18 (Strowig et al., 2012). These cytokines are thus regulated at a posttranslational level, in contrast to many other cytokines which are regulated at a transcriptional level. The search for the protein that induces inflammasome formation upon stimulation with cytosolic DNA yielded the ISG AIM2 as a cytosolic DNA sensor (B€ urckst€ ummer et al., 2009; Fernandes-Alnemri, Yu, Datta, Wu, & Alnemri, 2009; Hornung et al., 2009; Roberts et al., 2009). In studies with AIM2 RNA interference and AIM2-deficient mice, AIM2 was shown to be essential for inflammasome formation, caspase-1 activation, and processing of IL-1β and IL-18, but also for pyroptosis, which is an inflammasomemediated form of cell death (B€ urckst€ ummer et al., 2009; FernandesAlnemri et al., 2009; Hornung et al., 2009; Rathinam et al., 2010; Roberts et al., 2009; Sagulenko et al., 2013). Studies with AIM2-deficient mice showed that, in macrophages and DCs, AIM2 is the inflammasome formation-mediating DNA sensor in response to infection with various pathogens, including intracellular bacteria, vaccinia virus, and the betaherpesvirus murine cytomegalovirus

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(Hornung et al., 2009; Rathinam et al., 2010). HSV-1 induces strong caspase-1 activation in human and mouse macrophages and primary human fibroblasts ( Johnson, Chikoti, & Chandran, 2013; Muruve et al., 2008; Nour et al., 2011; Rathinam et al., 2010). However, this is independent of AIM2 ( Johnson et al., 2013; Rathinam et al., 2010). VZV also induces formation of the inflammasome complex in THP-1 cells and primary human lung fibroblasts. However, it also causes inflammasome activation in melanoma cells, which lack AIM2. Thus, the VZV-induced inflammasome is AIM2 independent; instead, it is dependent on NLRP3 (Nour et al., 2011). Since inflammasome formation upon transfected DNA was NLRP3 independent (Muruve et al., 2008), it is likely that VZV activates this inflammasome in a DNA-independent fashion. HSV-1 was also shown to transiently activate the NLRP3 inflammasome ( Johnson et al., 2013). Thus, although AIM2 is an important DNA sensor and mediator of the inflammasome response to some DNA viruses, this PRR is probably not involved in recognition of alphaherpesviruses.

2.5. IFI16 Similar to AIM2, IFI16 (and its mouse orthologue p204) belongs to the PYHIN family. IFI16 is also a receptor for intracellular DNA and mediates cytokine induction and inflammasome activation in response to a variety of dsDNA species (Fig. 2). Unterholzner and colleagues identified IFI16 as an intracellular DNA sensor by affinity purification from human monocyte cell extracts using vaccinia virus-derived DNA as bait (Unterholzner et al., 2010). IFI16 binds dsDNA directly, independent of sequence, and this binding is mediated by its two HIN domains, as shown by pull-down assays with tagged and untagged versions of IFI16, by electrophoretic mobility shift assay, by FRET, and by in vitro binding assays, including experiments using purified HIN domains, and by mutations studies with alanine substitution in the HIN domains (Conrady et al., 2012; Dawson & Trapani, 1995b; Morrone et al., 2014; Unterholzner et al., 2010). The crystal structures of the HIN domains of IFI16 and AIM2 in complex with DNA have been solved and reveal binding to the DNA backbone as the basis for the sequence-independent affinity to DNA ( Jin et al., 2012). Alanine substitution assay showed that the DNA-binding capacity of the HIN domains is essential for the DNA-induced immune response in IFI16-transfected HEK293 (human embryonic kidney) cells ( Jin et al., 2012). DNA binding by IFI16 is cooperative and length dependent, with dsDNA fragments


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shorter than 70 bp showing declining affinity (Morrone et al., 2014; Unterholzner et al., 2010). This can be explained by the fact that IFI16 oligomerizes on dsDNA to form filaments, through a process mediated by homotypic interaction between the IFI16 pyrin domains (Morrone et al., 2014). IFI16 (or p204) expression has been demonstrated in many primary and immortalized cell types including myeloid cells, fibroblasts, epithelial cells, and lymphoid cells (Caposio et al., 2007; Conrady et al., 2012; Cristea et al., 2010; Dawson & Trapani, 1995a, 1995b; Duan et al., 2011; Gariano et al., 2012; Hertel et al., 1999; Soby, Laursen, Ostergaard, & Melchjorsen, 2012; Triantafilou et al., 2014; Unterholzner et al., 2010; Veeranki, Duan, Panchanathan, Liu, & Choubey, 2011). In most cell types, e.g., fibroblasts, IFI16 is predominantly localized to the nucleus, but in some cell types, especially macrophage-like cells, a pool of the cellular IFI16 localizes to the cytoplasm ( Johnson et al., 2013; Orzalli, DeLuca, & Knipe, 2012; Unterholzner et al., 2010; Veeranki & Choubey, 2012; Veeranki et al., 2011). In cells commonly used for transfection experiments, such as HeLa and HEK293T, ectopic expression of IFI16 leads to localization of the protein to the nucleus (B€ urckst€ ummer et al., 2009; Hornung et al., 2009). The subcellular localization of IFI16 is regulated by a nonlinear nuclear localization signal (NLS), which can be acetylated by the acetyltransferase p300 in lymphocytes and macrophages, promoting cytosolic localization by inhibiting nuclear import (Li, Diner, Chen, & Cristea, 2012). IFI16-mediated sensing of HSV-1 DNA has been shown to take place independently of nuclear entry of viral DNA in THP-1 cells and monocyte-derived macrophages, in which IFI16 was found to colocalize with HSV-1 DNA in the cytosol (Horan et al., 2013; Unterholzner et al., 2010). However, DNA sensing by IFI16 can also take place in the nucleus. One study showed that in HFFs (human foreskin fibroblasts), recognition of the HSV-1 genome in the nucleus induced translocation of IFI16 to the cytosol where it mediated inflammasome activation ( Johnson et al., 2013). Another study, also using HFFs infected with HSV-1, showed that the nuclear localization of IFI16 did not change upon HSV-1 infection to mediate IFNβ expression via the STING pathway (Orzalli et al., 2012). The NLS of IFI16 was shown to be essential for IFNβ induction upon HSV-1 infection in IFI16-transfected U2OS cells (a human epithelial cell line) and partial relocalization of IFI16 to the cytosol upon infection was observed (Li et al., 2012).

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IFI16/p204 mediates induction of type I IFN and other inflammatory cytokines, such as CXCL10, IL-6, and TNFα, upon HSV-1 infection of human and murine cells (Conrady et al., 2012; Horan et al., 2013; Orzalli et al., 2012; Soby et al., 2012; Unterholzner et al., 2010; Zhu et al., 2014). An important role of IFI16/p204 in the innate immune response has also been proposed by in vivo RNA interference of p204 in mouse corneal epithelium and vaginal mucosa, which led to reduced IFNα induction, and increased viral titers in the cornea and the vaginal lumen after infection with HSV-1 and HSV-2, respectively (Conrady et al., 2012). IFI16-triggered cytokine expression is mediated via the STING/TBK1/ IRF3 pathway and the NF-κB pathway (Fig. 2) (Li, Chen, & Cristea, 2013; Unterholzner et al., 2010). IFI16 RNA interference prevented the nuclear translocation of IRF3 and NF-κB upon HSV-1 infection in murine macrophages cells (Unterholzner et al., 2010). Loss of IFI16 also prevented IRF3, but not IRF7 translocation in THCE (telomerase-immortalized human corneal epithelial) cells in response to HSV-1 (Conrady et al., 2012). The presence of STING was essential for IRF3 signaling in HFFs infected with HSV-1 (Orzalli et al., 2012). The strong maturation of DCs induced by DNA stimulation was partially dependent on IFI16 and STING (KisToth et al., 2011). Coimmunoprecipitations show that IFI16 and STING interact upon DNA stimulation (Unterholzner et al., 2010) and IFI16 and STING colocalize in the cytoplasm in human monocyte-derived macrophages after HSV-1 infection (Horan et al., 2013). However, it remains to be determined if this is a direct interaction and how exactly IFI16mediated STING activation takes place. In addition to its role in type I IFN induction, IFI16 has also been implicated as an activator of the inflammasome (Fig. 2); however, there are also studies that contradict this. Two initial studies that identified the role of AIM2 in inflammasome activation also analyzed IFI16 and failed to show association of IFI16 with the inflammasome adaptor ASC and IFI16mediated inflammasome activation (B€ urckst€ ummer et al., 2009; Hornung et al., 2009). Also, in THP-1 cells endogenous IFI16 did not associate with ASC (Hornung et al., 2009). However, IFI16 has been reported to recognize the HSV-1 genome in the nucleus after HSV-1 infection of HFFs and to subsequently mediate inflammasome assembly ( Johnson et al., 2013). In lytic and latent infections with gammaherpesviruses, IFI16 was also the inducer of inflammasome activation (Ansari et al., 2013; Kerur et al., 2011; Singh et al., 2013). IFI16, in addition to mediating innate immune signaling upon HSV infection, also acts as a restriction factor for


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immediate-early gene expression of HSV-1 by promoting the epigenetic silencing of the viral genome (Orzalli, Conwell, Berrios, DeCaprio, & Knipe, 2013). In conclusion, IFI16 is important for the early host response to HSV infection in many cell types. IFI16 has a DNA-binding domain and can recognize HSV-1 DNA both in the nucleus and in the cytoplasm to mediate type I IFN and inflammatory cytokine responses via the STING and inflammasome pathways. Several interesting questions remain open. It will be important to understand how IFI16 differentiates foreign from host DNA when sensing in the nucleus, despite its sequence-independent DNAbinding domain. Also, the exact mechanisms determining IFI16 localization in different cell types and upon various stimulation conditions await clarification. It needs to be determined which factors influence the type of inflammasome (AIM2 vs. IFI16) formed upon detection of DNA from different intracellular pathogens, but also from closely related viruses (betaherpesviruses vs. alpha- and gammaherpesviruses) and how the cellular context determines the exact role of IFI16 in inflammasome formation. Data on IFI16 sensing of VZV are lacking so far.

2.6. cGAS cGAS, a member of the nucleotidyltransferase family, is a recently discovered cytosolic DNA-sensing enzyme that catalyzes the cyclization reaction of ATP and GTP to form cGAMP, which acts as a secondary messenger to activate STING (Fig. 2; Sun et al., 2013; Wu et al., 2013). cGAMP as a secondary messenger activating STING was identified by mass spectrometry of components of heat-inactivated cytosolic extracts from cells transfected with various dsDNA that could mediate STING activation in permeabilized RAW264.7 and THP-1 cells (Wu et al., 2013). Subsequently, cGAMP was found to be synthesized by cGAS by biochemical fractionation and mass spectrometry of the cGAMP-producing fraction (Sun et al., 2013). Purified tagged cGAS could synthesize cGAMP from ATP and GTP in vitro in the presence of various dsDNA, but not without dsDNA stimulation (Sun et al., 2013). So far, cGAS expression has been confirmed in most cell types analyzed, but expression is low in some cell types, including MEFs (Sun et al., 2013). cGAS localizes to the cytosol as shown by subcellular fractionation of THP-1 cells and by confocal microscopy of L929 cells expressing epitopetagged cGAS, with low amounts also present in the nucleus in both cell types (Sun et al., 2013).

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cGAS directly binds dsDNA independent of sequence. Its N-terminal domain is required for DNA binding. Epitope-tagged cGAS, but not N-terminal truncation mutants, associated with biotinylated dsDNA in pull-down assays (Sun et al., 2013). cGAS colocalizes with transfected dsDNA in L929 cells (Sun et al., 2013). The crystal structures of human, murine, and porcine cGAS in combination with GTP and ATP have been solved (Civril et al., 2013; Gao et al., 2013; Kranzusch, Lee, Berger, & Doudna, 2013; Li, Shu, et al., 2013; Zhang et al., 2014). The DNA-binding domain consists of a zinc ribbon/thumb domain (Civril et al., 2013; Kranzusch et al., 2013). DNA binding is based on interactions with the DNA backbone across the minor groove, explaining the sequence independence (Civril et al., 2013). Although initial studies found no evidence that cGAS oligomerizes (Gao et al., 2013), later studies proposed that cGAS forms a 2:2 complex with DNA and that this dimerization is required for activation (Li, Shu, et al., 2013; Zhang et al., 2014). DNA binding to cGAS induces a conformational change, leading to the formation of a nucleotide binding pocket and reorganization of the catalytic site (Civril et al., 2013; Gao et al., 2013; Zhang et al., 2014). STING is essential for the cGAS-activated pathway (Fig. 2). DNA stimulation of L929 cells in the absence of STING did not lead to IRF3 activation (Wu et al., 2013). Overexpression of cGAS in HEK293T cells activated IRF3 and induced IFNβ expression in a manner dependent on STING (Sun et al., 2013). In contrast to other DNA sensors, the mechanism of STING activation by cGAS is now described in detail, with cGAMP binding to dimeric STING inducing a conformational change in the C-terminal domain in STING allowing for activation of TBK1 and downstream signaling to type I IFN expression (Wu et al., 2013; Zhang, Shi, et al., 2013). Interestingly, cGAMP can be transferred from infected cells to neighboring cells via GAP junctions to spread STING activation and IFNβ induction in response to a dsDNA virus (vaccinia virus) as shown in MEFs and transfected HEK293T cells (Ablasser et al., 2013). cGAS activity is regulated by the autophagy protein Beclin-1, which directly interacts with cGAS to stop production of cGAMP. This interaction also relieves Beclin-1 inhibition to activate autophagy of cytosolic DNA, restricting immune signaling after HSV-1 infection (Liang et al., 2014). Cytosolic DNA sensing by cGAS has a clear role in the innate immune response to HSV-1. Infection of THP-1 and L929 cells leads to production of cGAMP and IRF3 activation (Wu et al., 2013). RNA interference of cGAS prevents cGAMP synthesis, IRF3 activation, and the IFNβ response


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to HSV-1 infection in L929 cells and IRF3 activation in THP-1 cells (Sun et al., 2013). Cells from cGAS-deficient mice did not produce type I IFN, CXCL10, and IL-6 and failed to activate IRF3 in response to HSV-1 (Li, Wu, et al., 2013). The importance of cGAS was confirmed in vivo. In the absence of cGAS, IFNα and IFNβ serum levels were reduced in response to intravenous injection of HSV-1, the virus could spread efficiently to the brain, and the cGAS-deficient mice succumbed to the infection much earlier than wild-type mice (Li, Wu, et al., 2013). Data on the involvement of cGAS in immunity to HSV-2 and VZV are lacking so far.

2.7. RNA Pol III and RIG-I Soon after RIG-I was discovered as a cytosolic RNA sensor mediating antiviral innate signaling (Yoneyama et al., 2004), it was reported that RIG-I and its adaptor protein MAVS were also essential for expression of IFNβ in response to AT-rich dsDNA, and to HSV-1 infection in a human hepatoma cell line (Cheng, Zhong, Chung, & Chisari, 2007). Inhibition of the RIG-IMAVS pathway prevented poly(dA:dT)-induced activation of an IFNβ promoter reporter system in HEK293T cells (Ablasser et al., 2009; Chiu, MacMillan, & Chen, 2009). This indicated a role for RIG-I in cytosolic DNA sensing, but RIG-I was not found to bind to dsDNA directly (Cheng et al., 2007). This was followed by the demonstration that RNA extracted from poly(dA:dT)-transfected cells induced IFNβ in human peripheral blood mononuclear cells, indicating that RIG-I activation occurred via an RNA intermediate (Ablasser et al., 2009). The RNA intermediate was identified to be double stranded, to contain a 50 -triphosphate group, and to be synthesized by RNA Pol III (Fig. 2; Ablasser et al., 2009; Chiu et al., 2009). RNA Pol III DNA binding is likely to be dependent on the nucleotide composition of the DNA since GC-rich DNA could not be transcribed (Chiu et al., 2009). At the present stage, the physiological importance on the RNA Pol III pathway in recognition of HSV-1 remains unresolved, since the published data are somewhat contradictory. MEFs from RIG-I-deficient mice were impaired in their IFNβ, but not CCL5 and IL-6 response to HSV-1 DNA (Choi et al., 2009). Deactivation of RNA Pol III activity by a chemical inhibitor prevented IFNβ induction in response to HSV-1 in RAW264.7 cells (Chiu et al., 2009). However, another study found that HEK293T cells, which contain a functional RNA Pol III/RIG-I system, did not respond to HSV60mer DNA and that inhibition of RNA Pol III

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had no effect on HSV-derived DNA- and HSV-1 infection-induced IFNβ expression in RAW264.7 cells, nor on HSV-1-induced IFNβ and TNFα expression in human macrophages, although RNA Pol III inhibition did have an effect on the response to poly(dA:dT) (Melchjorsen et al., 2010; Unterholzner et al., 2010). There are no data yet on HSV-2, VZV, and the RNA Pol III pathway. Further research on the role of RNA Pol III in the antiviral defense in different cell types and especially in vivo is needed.

3. ACCESSIBILITY OF VIRAL DNA TO DNA SENSORS As discussed above, HSV-1 DNA is sensed in endosomes by TLR9, in the cytosol by AIM2, IFI16, cGAS, and RNA Pol III, and in the nucleus by IFI16 (and maybe also by RNA Pol III). The mechanisms through which the viral genome in the capsid is made accessible to the DNA sensors in these subcellular compartments are not fully understood. Since pDCs, in which TLR9 is preferentially expressed, are capable of phagocytosis (Tel et al., 2010), it is likely that virions are delivered to the endolysosomal pathway via this mechanism. Depending on the cell type, alphaherpesviruses enter cells via endocytosis or direct fusion with the plasma membrane (Fig. 1B; Arvin & Gilden, 2013; Roizman et al., 2013). The endocytic entry pathway followed by degradation of the virion would allow for exposure of the viral genome to TLR9 signaling in nonphagocytic cells (Fig. 1B). It is also possible that viral components including the genome are delivered to endosomes via autophagy (Fig. 1B), as autophagy is needed for sensing of ssRNA virus replication intermediates by TLR7 in endolysosomes and as pDCs lacking the autophagy protein ATG5 were severely impaired in their IFNα response to HSV-1 infection (Lee, Lund, Ramanathan, Mizushima, & Iwasaki, 2007). In macrophages, which are nonpermissive for productive HSV-1 replication, the HSV-1 capsid is ubiquitinated and degraded by the proteasome in the cytosol, exposing viral DNA to cytoplasmic sensors (Fig. 1B). However, this does not seem to occur in HFFs and U2OS cells, which are permissive for HSV-1 infection and in which the viral DNA efficiently reaches the nucleus (Horan et al., 2013). Proteasomal capsid degradation may thus be an important mechanism to protect certain cell types from viral infection (Paludan et al., 2011). At present, it is not known which cellular sensors detect the viral capsid, and which E3 ubiquitin ligases are involved; nor is it known whether herpesviruses seek to evade capsid targeting in the cytosol.


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In cells permissive for infection, the viral capsid is delivered to nuclear pores, where HSV DNA is transported into the nucleus (Roizman et al., 2013). In the nucleus, the viral DNA is not protected by the capsid and hence potentially exposed to nuclear PRRs (Fig. 1B). In this context, the question is not so much how the exposure occurs, but rather how nuclear DNA sensors can distinguish between self and foreign DNA. The observed recognition of viral DNA in the nucleus by IFI16 challenges the concept of the nucleus as an “immunoprivileged” subcellular compartment and the notion that intracellular DNA sensor reacts to DNA purely based on their aberrant localization in the cytoplasm. This question is especially intriguing, as IFI16 recognizes DNA sequence independently by interactions with the backbone ( Jin et al., 2012). It is possible that additional cofactors guide IFI16 specifically to viral DNA. This may involve components of the DNA damage response, some of which have been shown to have role in DNA sensing (Ferguson, Mansur, Peters, Ren, & Smith, 2012; Kondo et al., 2013; Zhang et al., 2011) and which are known to associate with alphaherpesviral DNA based on their ability to recognize dsDNA breaks (Weitzman, Lilley, & Chaurushiya, 2010). Interestingly, DNA damage can induce expression of IFN and inflammatory genes (Brzostek-Racine, Gordon, Van Scoy, & Reich, 2011). Additionally, histones may protect the host genome from recognition by PRRs. This may contribute to the fact that during latent infection, when viral DNA is circularized and associated with histones, it does not induce a strong innate immune response anymore.

4. EVASION OF DNA-INDUCED SIGNALING Evasion of the innate and adaptive immune system is very important for the ability of alphaherpesviruses to establish and maintain infection. Therefore, these viruses have evolved mechanisms to target all steps of the immune response. As discussed above, multiple PRRs recognize herpesviruses and converge at common signaling pathways, especially IRF3 and NF-κB signaling. So in addition to inhibiting certain PRRs directly, alphaherpesviruses target the downstream signaling events such as IRF3 and NF-κB activation (Fig. 3). STING as a crucial mediator of intracellular nucleic acid-induced signaling has been shown to be targeted by various RNA viruses, but not yet by DNA viruses (Ran et al., 2014). Since the intracellular DNA sensors have been discovered only in recent years, it can be expected that more viral immune evasion molecules inhibiting these specifically will be discovered soon. As the host–pathogen interaction very early in

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Figure 3 Evasion of innate DNA sensing by alphaherpesviruses. HSV ICP0 inhibits IFI16, NF-κB, and IRF3 signaling in the cytosol and nucleus. ICP0 also promotes cytoplasmic translocation of USP7, which inhibits NEMO when at this location. BoHV-1 ICP0 targets IRF3 for degradation. HSV ICP34.5 interferes with the action of TBK1 and the IKK complex and binds to Beclin-1 to inhibit autophagy, which may prevent delivery of PAMPs to the endosome for TLR9 sensing. HSV US3 blocks nuclear translocation of IRF3 and NF-κB and inhibits signaling from MyD88 to the IKK complex. HSV ICP27 binds to IκB, preventing its degradation. HSV VP16 interacts with a coactivator of IRF3 (CBP) and with NF-κB to prevent gene expression. HSV US11 interacts with RIG-I to prevent activation of MAVS. HSV UL36 impairs MAVS signaling. VZV IE62 blocks IRF3 activation by TBK1. VZV ORF47 kinase prevents nuclear translocation of IRF3, like HSV US3. Like ICP0, VZV ORF61 targets IRF3 for degradation.

infection determines the outcome of infection, many innate immune evasion molecules are tegument or immediate-early proteins. Herpesvirus proteins are often multifunctional and many viral inhibitors of innate signaling interfere through several mechanisms. More research needs to be done to elucidate in detail the mechanism of action of many of the immune evasion molecules. The HSV ICP0 is an immediate-early protein with multiple functions, including E3 ubiquitin ligase activity via its RING finger domain. ICP0 directly targets IFI16 for proteasomal degradation, and this interferes with IRF3-induced IFNβ expression and inflammasome-mediated secretion of mature IL-1β ( Johnson et al., 2013; Orzalli et al., 2012). ICP0 also


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sequesters activated IRF3 and its coactivators in the nucleus and enhances IRF3 degradation to prevent IFNβ gene induction (Melroe, Silva, Schaffer, & Knipe, 2007; Orzalli et al., 2012). Another study confirmed that ICP0 inhibits IRF3 signaling, but in this study cytosolic localization of ICP0 was essential and IRF3 was not degraded (Paladino, Collins, & Mossman, 2010). The bovine herpesvirus 1 (BoHV-1) ICP0 has been reported to target IRF3 for proteasomal degradation (Saira, Zhou, & Jones, 2007). USP7 is a host protein, which, when in the cytoplasm, downregulates NF-κB signaling by deubiquitinating TRAF6 and NEMO. This function is promoted by ICP0, which binds to nuclear USP7 and transfers it to the cytoplasm (Daubeuf et al., 2009). ICP0 also directly interacts with the NF-κB subunits p65 and p50 to prevent nuclear translocation and to induce degradation, respectively (Zhang, Wang, Wang, & Zheng, 2013). The HSV neurovirulence factor ICP34.5 interferes with the STINGTBK1-IRF3 signaling axis by binding to TBK1 to prevent complex formation with IRF3, thus inhibiting IRF3 activation. This requires the N-terminal part of the protein for this action (Ma et al., 2012; Verpooten, Ma, Hou, Yan, & He, 2009). ICP34.5 also binds to IKKα/β and protein phosphatase 1 to induce dephosphorylation of IKKα/β, hindering it from phosphorylating IκB, thus preventing activation of NF-κB, and ultimately impairing DC maturation, although this was studied in the context of TLR4 signaling ( Jin, Yan, Ma, Cao, & He, 2011). Interestingly, ICP34.5 also binds to Beclin-1 to inhibit autophagy, which may prevent delivery of PAMPs to the endosome for TLR9 sensing (Orvedahl et al., 2007). The HSV pUS3 kinase was also shown to inhibit IRF3 and NF-κB signaling (Peri et al., 2008; Sen, Liu, Roller, & Knipe, 2013; Wang, Ni, Wang, & Zheng, 2014; Wang, Wang, Lin, & Zheng, 2013). pUS3 interacts directly with IRF3 and NF-κB (p65 subunit) and mediates their hyperphosphorylation. This blocks dimerization in case of IRF3 and in both cases prevents nuclear translocation and gene induction (Wang et al., 2014; Wang, Wang, Lin, et al., 2013). pUS3 also reduces the polyubiquitination of TRAF6, which is needed for activation of the IKK complex and thus for NF-κB signaling (Sen et al., 2013). This study focused on TLR2 signaling, and it will be interesting to learn whether pUS3 also inhibits TLR9 signaling. The HSV immediate-early ICP27 protein is also involved in the suppression of IRF3 and NF-κB-mediated innate signaling, as an ICP27 deletion mutant of HSV-1 elicited higher cytokine responses than a wild-type virus

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in macrophages (Melchjorsen, Siren, Julkunen, Paludan, & Matikainen, 2006). ICP27 binds to the NF-κB inhibitor IκB, prevents its phosphorylation and ubiquitination, and stabilizes it in this way to prevent NF-κB signaling (Kim et al., 2008). It is currently unknown how ICP27-mediated inhibition of the IRF3-pathway occurs and what the physiological importance of this is. Although the HSV tegument protein VP16 was first reported not to have a direct role in evasion of cytokine expression, but rather to support the expression of evasion molecules, e.g., ICP27 (Mogensen, Melchjorsen, Malmgaard, Casola, & Paludan, 2004), a recent study shows that VP16 blocks both IRF3 and NF-κB signaling (Xing et al., 2013). VP16 was found not to block IRF3 activation directly, but rather to interact with the coactivator CBP (CREB-binding protein) to prevent gene expression. The HSV tegument protein pUS11 inhibits RNA virus-induced IFNβ signaling by interacting with RIG-I and MDA5 to prevent their association with the adaptor MAVS (Xing, Wang, Lin, Mossman, & Zheng, 2012). The tegument protein pUL36 impairs RIG-I-mediated IRF3 activation and IFNβ induction by deubiquitinating TRAF3, which is needed in ubiquitinated form for the recruitment of TBK1 (Wang, Wang, Li, & Zheng, 2013). Since the cytosolic DNA sensor RNA Pol III signals via RIG-I, it is likely that DNA-induced signaling is also inhibited via these mechanism. Although less well studied, VZV also encodes for proteins evading early innate signaling. The immediate-early protein IE62 blocks IRF3 phosphorylation by TBK1, which prevents its activation, although IE62 could not be shown to interact with IRF3 or TBK1 and the IRF3–TBK1 complex remained intact (Sen et al., 2010). Interestingly, the immediate-early ORF47 (open reading frame 47) kinase directly hyperphosphorylates IRF3, which prevents dimerization and thus activation (Zhu et al., 2011). The immediate-early protein ORF61, which contains an E3 ubiquitin ligase RING finger domain homologous to the ICP0 RING finger domain, targets activated IRF3 for proteasomal degradation (Vandevenne et al., 2011).

5. RELEVANCE FOR VACCINE DESIGN Finding efficient prophylactic and therapeutic vaccines against HSV-1 and HSV-2 has been an important but so far unachieved goal of vaccine research for many decades, while safe and useful vaccines against VZV are


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already in use (Coleman & Shukla, 2013; Gershon, 2013). The experimental HSV vaccines tried so far have not provided long-term protection and sterilizing immunity to prevent asymptomatic shedding of the virus (Coleman & Shukla, 2013). It is thought that optimization of DNA vaccines, on which much research effort has been focused in the past 20 years, could lead to progress toward the goal of achieving efficient and lasting humoral and cellular immune responses (Coban, Kobiyama, Jounai, Tozuka, & Ishii, 2013). Especially induction of a strong CD8+ T cell response is lacking in conventional vaccination (Rappuoli, 2007). A plasmid DNA vaccine against HSV-2 is currently in a phase I/II clinical trial (Awasthi & Friedman, 2014), and DNA vaccines are already in use against other viruses in veterinary medicine (Coban et al., 2013). However, the main issue with current DNA vaccines is low immunogenicity (Coban et al., 2013). The recent research on intracellular DNA sensing has revealed that the administered vaccine plasmid fulfills two functions, mediating expression of the antigen for (cross-)presentation by DCs and for activation of T and B cells and binding to cytosolic DNA sensors, thus inducing the cytokine response discussed above, resulting in an adjuvant effect of the vaccine plasmid (Coban et al., 2013). Components of the nucleic acid-sensing system, such as STING and TBK1, are important for successful generation of an efficient adaptive immune response after DNA vaccination (Ishii et al., 2008; Ishikawa et al., 2009). Several attempts have been made to increase this adjuvant effect based on the new knowledge about the DNA recognition system. For example, immunization of mice with plasmids encoding a model antigen and the first proposed DNA sensor DAI resulted in improved proliferation and activation (IFNγ production) of antigen-specific CD8+ T cells and the induction of memory. The Th1 response was also improved (Lladser et al., 2010). CDNs have been suggested as novel adjuvants, which mediate their function by binding to STING and inducing cytokine expression (Dubensky, Kanne, & Leong, 2013). In this context, the recent discovery that cGAS produces cGAMP is of great relevance. Mice immunized intramuscularly with a model antigen (purified OVA protein) in combination with cGAMP as an adjuvant displayed strong, STING-dependent antibody production that was not present in the OVA-only control and CD4+ and CD8+ T cells from these mice produced more cytokines (IFNγ and IL-2) (Li, Wu, et al., 2013). Thus, the new advances in the field of intracellular DNA sensing have already contributed to vaccine development and have potential to be applied in vaccines in the future, especially against intracellular pathogen, including

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alphaherpesviruses. However, further research on the exact mechanisms of action of DNA vaccines, especially in the context of DNA sensing, is needed to develop methods to accurately manipulate the resulting adaptive immune response.

6. CONCLUSIONS AND FUTURE PERSPECTIVE Great progress has been made in the past decade on the innate immune recognition of alphaherpesviruses. We have learned that viral nucleic acids, in particular DNA, are potent stimulators of the first line of defense against these viruses. This has broadened our perception of PAMPs and revealed of a number of new principles of the innate system. The identification of cGAS represents the single most important advance in this field, since the first discovery of innate DNA sensing. For the research that has formed the basis for the current knowledge, HSV-1 has often served as the model pathogen, which is why there is abundant data on DNA sensing of this virus in particular. The knowledge on innate immune sensing of HSV-1 DNA is of importance for vaccine design and development of immune modulation therapy, which in combination with antiviral therapy could lead to improved clinical efficacy of treatment of, e.g., herpes simplex encephalitis. Finally, with the great body of knowledge on immune activation and evasion by HSV1, experimental evidence regarding other alphaherpesviruses, including HSV-2, VZV, and alphaherpesviruses of veterinary relevance, is bound to follow soon. However, before this research can find medical application in humans, numerous questions remain to be investigated. A challenge for future research will be to find experimental procedures to differentiate between “true” DNA sensors primarily responsible for initiation of the immune response to DNA, and the numerous DNA-binding proteins also proposed to be bone fide DNA sensors, but more likely involved in the signaling network downstream in the pathways. Refinement of reconstitution systems will be useful for answering this question. Another central question is by what mechanisms STING can be activated and STING’s exact role within the innate signaling network. This is especially interesting as STING also seems to be involved in immune signaling following virus–cell membrane fusion (Holm et al., 2012) and ER stress (Petrasek et al., 2013), thus suggesting the existence of different types of STING activation. It is already clear that the signaling network employed by PPRs is much more complex and intertwined than previously thought and includes many possibilities for


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redundancy, which complicates research. Unraveling the interplay between the many PRRs and signaling adaptors will be crucial. Moreover, the discovery of nuclear DNA sensors such as IFI16 challenges the concept of an “immunoprivileged nucleus” and necessitates research on how host and pathogen DNA are distinguished in this compartment. It will be interesting to investigate how innate DNA recognition and its evasion by viral proteins are involved in the establishment and maintenance of alphaherpesviral latency. Importantly, the relevance of the novel DNA sensors needs to be confirmed in humans. The detailed study of primary immunodeficiencies which render humans more susceptible to diseases caused by alphaherpesvirus infections, as is known for TLR3 deficiency (Zhang et al., 2007), will play a key role in achieving this goal.

REFERENCES Abe, T., & Barber, G. N. (2014). Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-kappaB activation through TBK1. Journal of Virology, 88(10), 5328–5341. Ablasser, A., Bauernfeind, F., Hartmann, G., Latz, E., Fitzgerald, K. A., & Hornung, V. (2009). RIG-I-dependent sensing of poly (dA: dT) through the induction of an RNA polymerase III–transcribed RNA intermediate. Nature Immunology, 10(10), 1065–1072. Ablasser, A., Schmid-Burgk, J. L., Hemmerling, I., Horvath, G. L., Schmidt, T., Latz, E., et al. (2013). Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature, 503(7477), 530–534. Ahmad-Nejad, P., Ha¨cker, H., Rutz, M., Bauer, S., Vabulas, R. M., & Wagner, H. (2002). Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. European Journal of IMMUNOLOGY, 32(7), 1958–1968. Ank, N., Iversen, M. B., Bartholdy, C., Staeheli, P., Hartmann, R., Jensen, U. B., et al. (2008). An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity. Journal of Immunology (Baltimore Md.:1950), 180(4), 2474–2485. Ansari, M. A., Singh, V. V., Dutta, S., Veettil, M. V., Dutta, D., Chikoti, L., et al. (2013). Constitutive interferon-inducible protein 16-inflammasome activation during EpsteinBarr virus latency I, II, and III in B and epithelial cells. Journal of Virology, 87(15), 8606–8623. Arvin, A., & Gilden, D. (2013). Varicella-zoster virus. In D. Knipe & P. Howley (Eds.), Fields virology (6th ed.). Philadelphia: Lippincott Williams & Wilkins (Chapter 63). Atianand, M. K., & Fitzgerald, K. A. (2013). Molecular basis of DNA recognition in the immune system. Journal of Immunology (Baltimore Md.: 1950), 190(5), 1911–1918. Awasthi, S., & Friedman, H. M. (2014). Status of prophylactic and therapeutic genital herpes vaccines. Current Opinion in Virology, 6, 6–12. Boivin, N., Menasria, R., Piret, J., & Boivin, G. (2012). Modulation of TLR9 response in a mouse model of herpes simplex virus encephalitis. Antiviral Research, 96(3), 414–421. Bourke, E., Bosisio, D., Golay, J., Polentarutti, N., & Mantovani, A. (2003). The toll-like receptor repertoire of human B lymphocytes: Inducible and selective expression of TLR9 and TLR10 in normal and transformed cells. Blood, 102(3), 956–963. Brzostek-Racine, S., Gordon, C., Van Scoy, S., & Reich, N. C. (2011). The DNA damage response induces IFN. Journal of Immunology (Baltimore Md.: 1950), 187(10), 5336–5345.

92


B€ urckst€ ummer, T., Baumann, C., Bl€ uml, S., Dixit, E., D€ urnberger, G., Jahn, H., et al. (2009). An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nature Immunology, 10(3), 266–272. Burdette, D. L., & Vance, R. E. (2013). STING and the innate immune response to nucleic acids in the cytosol. Nature Immunology, 14(1), 19–26. Caposio, P., Gugliesi, F., Zannetti, C., Sponza, S., Mondini, M., Medico, E., et al. (2007). A novel role of the interferon-inducible protein IFI16 as inducer of proinflammatory molecules in endothelial cells. The Journal of Biological Chemistry, 282(46), 33515–33529. Cheng, G., Zhong, J., Chung, J., & Chisari, F. V. (2007). Double-stranded DNA and double-stranded RNA induce a common antiviral signaling pathway in human cells. Proceedings of the National Academy of Sciences of the United States of America, 104(21), 9035–9040. Chiu, Y., MacMillan, J. B., & Chen, Z. J. (2009). RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell, 138(3), 576–591. Choi, M. K., Wang, Z., Ban, T., Yanai, H., Lu, Y., Koshiba, R., et al. (2009). A selective contribution of the RIG-I-like receptor pathway to type I interferon responses activated by cytosolic DNA. Proceedings of the National Academy of Sciences of the United States of America, 106(42), 17870–17875. Civril, F., Deimling, T., de Oliveira Mann, C. C., Ablasser, A., Moldt, M., Witte, G., et al. (2013). Structural mechanism of cytosolic DNA sensing by cGAS. Nature, 498(7454), 332–337. Coban, C., Kobiyama, K., Jounai, N., Tozuka, M., & Ishii, K. J. (2013). DNA vaccines: a simple DNA sensing matter? Human Vaccines & Immunotherapeutics, 9, 2216–2221. Coleman, J. L., & Shukla, D. (2013). Recent advances in vaccine development for herpes simplex virus types I and II. Human Vaccines & Immunotherapeutics, 9, 729–735. Conrady, C. D., Zheng, M., Fitzgerald, K. A., Liu, C., & Carr, D. J. (2012). Resistance to HSV-1 infection in the epithelium resides with the novel innate sensor, IFI-16. Mucosal Immunology, 5(2), 173–183. Cradock-Watson, J., Ridehalgh, M. K., & Bourne, M. (1979). Specific immunoglobulin responses after varicella and herpes zoster. Journal of Hygiene, 82(02), 319–336. Cristea, I. M., Moorman, N. J., Terhune, S. S., Cuevas, C. D., O’Keefe, E. S., Rout, M. P., et al. (2010). Human cytomegalovirus pUL83 stimulates activity of the viral immediateearly promoter through its interaction with the cellular IFI16 protein. Journal of Virology, 84(15), 7803–7814. Daubeuf, S., Singh, D., Tan, Y., Liu, H., Federoff, H. J., Bowers, W. J., et al. (2009). HSV ICP0 recruits USP7 to modulate TLR-mediated innate response. Blood, 113(14), 3264–3275. Dawson, M. J., & Trapani, J. A. (1995a). IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines. Journal of Cellular Biochemistry, 57(1), 39–51. Dawson, M. J., & Trapani, J. A. (1995b). The interferon inducible autoantigen, Ifi 16: Localization to the nucleolus and identification of a DNA-binding domain. Biochemical and Biophysical Research Communications, 214(1), 152–162. Duan, X., Ponomareva, L., Veeranki, S., Panchanathan, R., Dickerson, E., & Choubey, D. (2011). Differential roles for the interferon-inducible IFI16 and AIM2 innate immune sensors for cytosolic DNA in cellular senescence of human fibroblasts. Molecular Cancer Research: MCR, 9(5), 589–602. Dubensky, T. W., Kanne, D. B., & Leong, M. L. (2013). Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants. Therapeutic Advances in Vaccines, 1, 131–143. http://dx.doi.org/10.1177/2051013613501988.


93

Egan, K. P., Wu, S., Wigdahl, B., & Jennings, S. R. (2013). Immunological control of herpes simplex virus infections. Journal of Neurovirology, 19(4), 328–345. El Falaky, I. H., Vestergaard, B. F., & Hornsleth, A. (1977). IgG, IgA and IgM antibody responses in patients with acute genital and non-genital herpetic lesions determined by the indirect fluorescent antibody method. Scandinavian Journal of Infectious Diseases, 9(2), 79–84. Ferguson, B. J., Mansur, D. S., Peters, N. E., Ren, H., & Smith, G. L. (2012). DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. eLife, 1, e00047. Fernandes-Alnemri, T., Yu, J., Datta, P., Wu, J., & Alnemri, E. S. (2009). AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature, 458(7237), 509–513. Furr, S. R., Chauhan, V. S., Moerdyk-Schauwecker, M. J., & Marriott, I. (2011). A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells. Journal of Neuroinflammation, 8(99). Gao, P., Ascano, M., Wu, Y., Barchet, W., Gaffney, B. L., Zillinger, T., et al. (2013). Cyclic [G (20 , 50 ) pA (30 , 50 ) p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell, 153(5), 1094–1107. Gariano, G. R., Dell’Oste, V., Bronzini, M., Gatti, D., Luganini, A., De Andrea, M., et al. (2012). The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication. PLoS Pathogens, 8(1), e1002498. Gershon, A. A. (2013). Varicella zoster vaccines and their implications for development of HSV vaccines. Virology, 435(1), 29–36. Goubau, D., Deddouche, S., & Reis e Sousa, C. (2013). Cytosolic sensing of viruses. Immunity, 38(5), 855–869. Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S., & Wagner, H. (2000). Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. The Journal of Experimental Medicine, 192(4), 595–600. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., et al. (2000). A Tolllike receptor recognizes bacterial DNA. Nature, 408(6813), 740–745. Hertel, L., De Andrea, M., Azzimonti, B., Rolle, A., Gariglio, M., & Landolfo, S. (1999). The interferon-inducible 204 gene, a member of the Ifi 200 family, is not involved in the antiviral state induction by IFN-α, but is required by the mouse cytomegalovirus for its replication. Virology, 262(1), 1–8. Hochrein, H., Schlatter, B., O’Keeffe, M., Wagner, C., Schmitz, F., Schiemann, M., et al. (2004). Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways. Proceedings of the National Academy of Sciences of the United States of America, 101(31), 11416–11421. Holm, C. K., Jensen, S. B., Jakobsen, M. R., Cheshenko, N., Horan, K. A., Moeller, H. B., et al. (2012). Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING. Nature Immunology, 13(8), 737–743. Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., et al. (2005). IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature, 434(7034), 772–777. Horan, K. A., Hansen, K., Jakobsen, M. R., Holm, C. K., Søby, S., Unterholzner, L., et al. (2013). Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors. The Journal of Immunology, 190(5), 2311–2319. Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D. R., et al. (2009). AIM2 recognizes cytosolic dsDNA and forms a caspase1-activating inflammasome with ASC. Nature, 458(7237), 514–518.

94


Ishii, K. J., Coban, C., Kato, H., Takahashi, K., Torii, Y., Takeshita, F., et al. (2005). A Tolllike receptor–independent antiviral response induced by double-stranded B-form DNA. Nature Immunology, 7(1), 40–48. Ishii, K. J., Kawagoe, T., Koyama, S., Matsui, K., Kumar, H., Kawai, T., et al. (2008). TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature, 451(7179), 725–729. Ishikawa, H., Ma, Z., & Barber, G. N. (2009). STING regulates intracellular DNAmediated, type I interferon-dependent innate immunity. Nature, 461(7265), 788–792. Jin, T., Perry, A., Jiang, J., Smith, P., Curry, J. A., Unterholzner, L., et al. (2012). Structures of the HIN domain: DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity, 36(4), 561–571. Jin, H., Yan, Z., Ma, Y., Cao, Y., & He, B. (2011). A herpesvirus virulence factor inhibits dendritic cell maturation through protein phosphatase 1 and Ikappa B kinase. Journal of Virology, 85(7), 3397–3407. Johnson, K. E., Chikoti, L., & Chandran, B. (2013). Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and NLRP3 inflammasomes. Journal of Virology, 87(9), 5005–5018. Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R. W., Kastelein, R. A., Bazan, F., et al. (2001). Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. The Journal of Experimental Medicine, 194(6), 863–869. Kaiser, W. J., Upton, J. W., & Mocarski, E. S. (2008). Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNAdependent activator of IFN regulatory factors. Journal of Immunology (Baltimore Md.: 1950), 181(9), 6427–6434. Kawai, T., & Akira, S. (2011). Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity, 34(5), 637–650. Kerur, N., Veettil, M. V., Sharma-Walia, N., Bottero, V., Sadagopan, S., Otageri, P., et al. (2011). IFI16 acts as a nuclear pathogen sensor to induce the inflammasome in response to Kaposi Sarcoma-associated herpesvirus infection. Cell Host & Microbe, 9(5), 363–375. Kim, K., Khayrutdinov, B. I., Lee, C. K., Cheong, H. K., Kang, S. W., Park, H., et al. (2011). Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs. Proceedings of the National Academy of Sciences of the United States of America, 108(17), 6921–6926. Kim, J. C., Lee, S. Y., Kim, S. Y., Kim, J. K., Kim, H. J., Lee, H. M., et al. (2008). HSV-1 ICP27 suppresses NF-κB activity by stabilizing IκBα. FEBS Letters, 582(16), 2371–2376. Kis-Toth, K., Szanto, A., Thai, T. H., & Tsokos, G. C. (2011). Cytosolic DNA-activated human dendritic cells are potent activators of the adaptive immune response. Journal of Immunology (Baltimore Md.: 1950), 187(3), 1222–1234. Kondo, T., Kobayashi, J., Saitoh, T., Maruyama, K., Ishii, K. J., Barber, G. N., et al. (2013). DNA damage sensor MRE11 recognizes cytosolic double-stranded DNA and induces type I interferon by regulating STING trafficking. Proceedings of the National Academy of Sciences of the United States of America, 110(8), 2969–2974. Kranzusch, P. J., Lee, A. S., Berger, J. M., & Doudna, J. A. (2013). Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Reports, 3(5), 1362–1368. Krug, A., Luker, G. D., Barchet, W., Leib, D. A., Akira, S., & Colonna, M. (2004). Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. Blood, 103(4), 1433–1437. Ku, C. L., von Bernuth, H., Picard, C., Zhang, S. Y., Chang, H. H., Yang, K., et al. (2007). Selective predisposition to bacterial infections in IRAK-4-deficient children: IRAK-4dependent TLRs are otherwise redundant in protective immunity. The Journal of Experimental Medicine, 204(10), 2407–2422.


95

Latz, E., Verma, A., Visintin, A., Gong, M., Sirois, C. M., Klein, D. C., et al. (2007). Ligandinduced conformational changes allosterically activate Toll-like receptor 9. Nature Immunology, 8(7), 772–779. Lee, H. K., Lund, J. M., Ramanathan, B., Mizushima, N., & Iwasaki, A. (2007). Autophagydependent viral recognition by plasmacytoid dendritic cells. Science (New York, N.Y.), 315(5817), 1398–1401. Li, T., Chen, J., & Cristea, I. M. (2013). Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion. Cell Host & Microbe, 14(5), 591–599. Li, T., Diner, B. A., Chen, J., & Cristea, I. M. (2012). Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proceedings of the National Academy of Sciences of the United States of America, 109(26), 10558–10563. Li, X., Shu, C., Yi, G., Chaton, C. T., Shelton, C. L., Diao, J., et al. (2013). Cyclic GMPAMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity, 39(6), 1019–1031. Li, X. D., Wu, J., Gao, D., Wang, H., Sun, L., & Chen, Z. J. (2013). Pivotal roles of cGAScGAMP signaling in antiviral defense and immune adjuvant effects. Science (New York, N.Y.), 341(6152), 1390–1394. Liang, Q., Seo, G. J., Choi, Y. J., Kwak, M., Ge, J., Rodgers, M. A., et al. (2014). Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses. Cell Host & Microbe, 15(2), 228–238. Lima, G. K., Zolini, G. P., Mansur, D. S., Freire Lima, B. H., Wischhoff, U., Astigarraga, R. G., et al. (2010). Toll-like receptor (TLR) 2 and TLR9 expressed in trigeminal ganglia are critical to viral control during herpes simplex virus 1 infection. The American Journal of Pathology, 177(5), 2433–2445. Lippmann, J., Rothenburg, S., Deigendesch, N., Eitel, J., Meixenberger, K., Van Laak, V., et al. (2008). IFNβ responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI). Cellular Microbiology, 10(12), 2579–2588. Lladser, A., Mougiakakos, D., Tufvesson, H., Ligtenberg, M. A., Quest, A. F., Kiessling, R., et al. (2010). DAI (DLM-1/ZBP1) as a genetic adjuvant for DNA vaccines that promotes effective antitumor CTL immunity. Molecular Therapy, 19(3), 594–601. Lund, J. M., Linehan, M. M., Iijima, N., & Iwasaki, A. (2006). Cutting edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ. Journal of Immunology (Baltimore Md.: 1950), 177(11), 7510–7514. Lund, J., Sato, A., Akira, S., Medzhitov, R., & Iwasaki, A. (2003). Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. The Journal of Experimental Medicine, 198(3), 513–520. Ma, Y., Jin, H., Valyi-Nagy, T., Cao, Y., Yan, Z., & He, B. (2012). Inhibition of TANK binding kinase 1 by herpes simplex virus 1 facilitates productive infection. Journal of Virology, 86(4), 2188–2196. Malmgaard, L., Melchjorsen, J., Bowie, A. G., Mogensen, S. C., & Paludan, S. R. (2004). Viral activation of macrophages through TLR-dependent and -independent pathways. Journal of Immunology (Baltimore Md.: 1950), 173(11), 6890–6898. Megjugorac, N. J., Young, H. A., Amrute, S. B., Olshalsky, S. L., & Fitzgerald-Bocarsly, P. (2004). Virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of T and NK cells. Journal of Leukocyte Biology, 75(3), 504–514. Melchjorsen, J., Rintahaka, J., Soby, S., Horan, K. A., Poltajainen, A., Ostergaard, L., et al. (2010). Early innate recognition of herpes simplex virus in human primary macrophages is mediated via the MDA5/MAVS-dependent and MDA5/MAVS/RNA polymerase III-independent pathways. Journal of Virology, 84(21), 11350–11358. Melchjorsen, J., Siren, J., Julkunen, I., Paludan, S. R., & Matikainen, S. (2006). Induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages

96


and dendritic cells is dependent on virus replication and is counteracted by ICP27 targeting NF-kappaB and IRF-3. The Journal of General Virology, 87(Pt 5), 1099–1108. Melroe, G. T., Silva, L., Schaffer, P. A., & Knipe, D. M. (2007). Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: Potential role in blocking IFN-β induction. Virology, 360(2), 305–321. Mogensen, T. H. (2009). Pathogen recognition and inflammatory signaling in innate immune defenses. Clinical Microbiology Reviews, 22(2), 240–273 (Table of Contents). Mogensen, T. H., Melchjorsen, J., Malmgaard, L., Casola, A., & Paludan, S. R. (2004). Suppression of proinflammatory cytokine expression by herpes simplex virus type 1. Journal of Virology, 78(11), 5883–5890. Morrone, S. R., Wang, T., Constantoulakis, L. M., Hooy, R. M., Delannoy, M. J., & Sohn, J. (2014). Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy. Proceedings of the National Academy of Sciences of the United States of America, 111(1), E62–E71. Muruve, D. A., Pe´trilli, V., Zaiss, A. K., White, L. R., Clark, S. A., Ross, P. J., et al. (2008). The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response. Nature, 452(7183), 103–107. Nie, Y., & Wang, Y. (2013). Innate immune responses to DNA viruses. Protein & Cell, 4(1), 1–7. Nour, A. M., Reichelt, M., Ku, C. C., Ho, M. Y., Heineman, T. C., & Arvin, A. M. (2011). Varicella-zoster virus infection triggers formation of an interleukin-1beta (IL-1beta)processing inflammasome complex. The Journal of Biological Chemistry, 286(20), 17921–17933. Orvedahl, A., Alexander, D., Tallo´czy, Z., Sun, Q., Wei, Y., Zhang, W., et al. (2007). HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host & Microbe, 1(1), 23–35. Orzalli, M. H., Conwell, S. E., Berrios, C., DeCaprio, J. A., & Knipe, D. M. (2013). Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA. Proceedings of the National Academy of Sciences of the United States of America, 110(47), E4492–E4501. Orzalli, M. H., DeLuca, N. A., & Knipe, D. M. (2012). Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein. Proceedings of the National Academy of Sciences of the United States of America, 109(44), E3008–E3017. Osterrieder, N., Kamil, J. P., Schumacher, D., Tischer, B. K., & Trapp, S. (2006). Marek’s disease virus: From miasma to model. Nature Reviews Microbiology, 4(4), 283–294. Paladino, P., Collins, S. E., & Mossman, K. L. (2010). Cellular localization of the herpes simplex virus ICP0 protein dictates its ability to block IRF3-mediated innate immune responses. PLoS One, 5(4), e10428. Paludan, S. R., Bowie, A. G., Horan, K. A., & Fitzgerald, K. A. (2011). Recognition of herpesviruses by the innate immune system. Nature Reviews Immunology, 11(2), 143–154. Pellett, P., & Roizman, B. (2013). Herpesviridae. In D. Knipe & P. Howley (Eds.), Fields virology (6th ed.). Philadelphia: Lippincott Williams & Wilkins (Chapter 59). Peri, P., Mattila, R. K., Kantola, H., Broberg, E., Karttunen, H. S., Waris, M., et al. (2008). Herpes simplex virus type 1 Us3 gene deletion influences toll-like receptor responses in cultured monocytic cells. Virology Journal, 5, 140. Petrasek, J., Iracheta-Vellve, A., Csak, T., Satishchandran, A., Kodys, K., Kurt-Jones, E. A., et al. (2013). STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease. Proceedings of the National Academy of Sciences of the United States of America, 110(41), 16544–16549. Pham, T. H., Kwon, K. M., Kim, Y. E., Kim, K. K., & Ahn, J. H. (2013). DNA sensingindependent inhibition of herpes simplex virus 1 replication by DAI/ZBP1. Journal of Virology, 87(6), 3076–3086.


97

Pomeranz, L. E., Reynolds, A. E., & Hengartner, C. J. (2005). Molecular biology of pseudorabies virus: Impact on neurovirology and veterinary medicine. Microbiology and Molecular Biology Reviews: MMBR, 69(3), 462–500. Ran, Y., Shu, H., & Wang, Y. (2014). MITA/STING: A central and multifaceted mediator in innate immune response. Cytokine & Growth Factor Reviews. http://dx.doi.org/ 10.1016/j.cytogfr.2014.05.003 [Epub ahead of print]. Rappuoli, R. (2007). Bridging the knowledge gaps in vaccine design. Nature Biotechnology, 25(12), 1361–1366. Rasmussen, S. B., Jensen, S. B., Nielsen, C., Quartin, E., Kato, H., Chen, Z. J., et al. (2009). Herpes simplex virus infection is sensed by both Toll-like receptors and retinoic acidinducible gene- like receptors, which synergize to induce type I interferon production. The Journal of General Virology, 90(Pt. 1), 74–78. Rasmussen, S. B., Sorensen, L. N., Malmgaard, L., Ank, N., Baines, J. D., Chen, Z. J., et al. (2007). Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems. Journal of Virology, 81(24), 13315–13324. Rathinam, V. A., Jiang, Z., Waggoner, S. N., Sharma, S., Cole, L. E., Waggoner, L., et al. (2010). The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nature Immunology, 11(5), 395–402. Roberts, T. L., Idris, A., Dunn, J. A., Kelly, G. M., Burnton, C. M., Hodgson, S., et al. (2009). HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science (New York, N.Y.), 323(5917), 1057–1060. Roizman, B., Knipe, D., & Whitley, R. (2013). Herpes simplex virus. In D. Knipe & P. Howley (Eds.), Fields virology (6th ed.). Philadelphia: Lippincott Williams & Wilkins (Chapter 60). Sagulenko, V., Thygesen, S., Sester, D., Idris, A., Cridland, J., Vajjhala, P., et al. (2013). AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death & Differentiation, 20(9), 1149–1160. Saira, K., Zhou, Y., & Jones, C. (2007). The infected cell protein 0 encoded by bovine herpesvirus 1 (bICP0) induces degradation of interferon response factor 3 and, consequently, inhibits beta interferon promoter activity. Journal of Virology, 81(7), 3077–3086. Sarangi, P. P., Kim, B., Kurt-Jones, E., & Rouse, B. T. (2007). Innate recognition network driving herpes simplex virus-induced corneal immunopathology: Role of the toll pathway in early inflammatory events in stromal keratitis. Journal of Virology, 81(20), 11128–11138. Sato, A., Linehan, M. M., & Iwasaki, A. (2006). Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells. Proceedings of the National Academy of Sciences of the United States of America, 103(46), 17343–17348. Schnare, M., Takeda, K., Akira, S., & Medzhitov, R. (2000). Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Current Biology, 10(18), 1139–1142. Sen, J., Liu, X., Roller, R., & Knipe, D. M. (2013). Herpes simplex virus US3 tegument protein inhibits Toll-like receptor 2 signaling at or before TRAF6 ubiquitination. Virology, 439(2), 65–73. Sen, N., Sommer, M., Che, X., White, K., Ruyechan, W. T., & Arvin, A. M. (2010). Varicella-zoster virus immediate-early protein 62 blocks interferon regulatory factor 3 (IRF3) phosphorylation at key serine residues: A novel mechanism of IRF3 inhibition among herpesviruses. Journal of Virology, 84(18), 9240–9253. Singh, V. V., Kerur, N., Bottero, V., Dutta, S., Chakraborty, S., Ansari, M. A., et al. (2013). Kaposi’s sarcoma-associated herpesvirus latency in endothelial and B cells activates

98


gamma interferon-inducible protein 16-mediated inflammasomes. Journal of Virology, 87(8), 4417–4431. Soby, S., Laursen, R. R., Ostergaard, L., & Melchjorsen, J. (2012). HSV-1-induced chemokine expression via IFI16-dependent and IFI16-independent pathways in human monocyte-derived macrophages. Herpesviridae, 3(1), 6. Sorensen, L. N., Reinert, L. S., Malmgaard, L., Bartholdy, C., Thomsen, A. R., & Paludan, S. R. (2008). TLR2 and TLR9 synergistically control herpes simplex virus infection in the brain. Journal of immunology (Baltimore Md.: 1950), 181(12), 8604–8612. Stetson, D. B., & Medzhitov, R. (2006). Recognition of cytosolic DNA activates an IRF3dependent innate immune response. Immunity, 24(1), 93–103. Strowig, T., Henao-Mejia, J., Elinav, E., & Flavell, R. (2012). Inflammasomes in health and disease. Nature, 481(7381), 278–286. Sun, L., Wu, J., Du, F., Chen, X., & Chen, Z. J. (2013). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science (New York, N.Y.), 339(6121), 786–791. Takaoka, A., Wang, Z., Choi, M. K., Yanai, H., Negishi, H., Ban, T., et al. (2007). DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature, 448(7152), 501–505. Takeda, S., Miyazaki, D., Sasaki, S., Yamamoto, Y., Terasaka, Y., Yakura, K., et al. (2011). Roles played by toll-like receptor-9 in corneal endothelial cells after herpes simplex virus type 1 infection. Investigative Ophthalmology & Visual Science, 52(9), 6729–6736. Takeuchi, O., & Akira, S. (2010). Pattern recognition receptors and inflammation. Cell, 140(6), 805–820. Tanaka, Y., & Chen, Z. J. (2012). STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Science Signaling, 5(214), ra20. Tel, J., Lambeck, A. J., Cruz, L. J., Tacken, P. J., de Vries, I. J., & Figdor, C. G. (2010). Human plasmacytoid dendritic cells phagocytose, process, and present exogenous particulate antigen. Journal of Immunology (Baltimore Md.: 1950), 184(8), 4276–4283. Triantafilou, K., Eryilmazlar, D., & Triantafilou, M. (2014). Herpes simplex virus 2–induced activation in vaginal cells involves Toll-like receptors 2 and 9 and DNA sensors DAI and IFI16. American Journal of Obstetrics and Gynecology, 210(2), 122.e1–122.e10. Unterholzner, L., Keating, S. E., Baran, M., Horan, K. A., Jensen, S. B., Sharma, S., et al. (2010). IFI16 is an innate immune sensor for intracellular DNA. Nature Immunology, 11(11), 997–1004. Vandevenne, P., Lebrun, M., El Mjiyad, N., Ote, I., Di Valentin, E., Habraken, Y., et al. (2011). The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3. PLoS One, 6(2), e16870. Veeranki, S., & Choubey, D. (2012). Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: Regulation of subcellular localization. Molecular Immunology, 49(4), 567–571. Veeranki, S., Duan, X., Panchanathan, R., Liu, H., & Choubey, D. (2011). IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes. PLoS One, 6(10), e27040. Verpooten, D., Ma, Y., Hou, S., Yan, Z., & He, B. (2009). Control of TANK-binding kinase 1-mediated signaling by the gamma(1)34.5 protein of herpes simplex virus 1. The Journal of Biological Chemistry, 284(2), 1097–1105. Wagner, H. (2004). The immunobiology of the TLR9 subfamily. Trends in Immunology, 25(7), 381–386. Wagner, M., Poeck, H., Jahrsdoerfer, B., Rothenfusser, S., Prell, D., Bohle, B., et al. (2004). IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. Journal of Immunology (Baltimore Md.: 1950), 172(2), 954–963.


99

Wang, Z., Choi, M. K., Ban, T., Yanai, H., Negishi, H., Lu, Y., et al. (2008). Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules. Proceedings of the National Academy of Sciences of the United States of America, 105(14), 5477–5482. Wang, K., Ni, L., Wang, S., & Zheng, C. (2014). Herpes simplex virus 1 protein kinase US3 hyperphosphorylates p65/RelA and dampens NF-kappaB activation. Journal of Virology, 88(14), 7941–7951. Wang, S., Wang, K., Li, J., & Zheng, C. (2013). Herpes simplex virus 1 ubiquitin-specific protease UL36 inhibits beta interferon production by deubiquitinating TRAF3. Journal of Virology, 87(21), 11851–11860. Wang, S., Wang, K., Lin, R., & Zheng, C. (2013). Herpes simplex virus 1 serine/threonine kinase US3 hyperphosphorylates IRF3 and inhibits beta interferon production. Journal of Virology, 87(23), 12814–12827. Weitzman, M. D., Lilley, C. E., & Chaurushiya, M. S. (2010). Genomes in conflict: Maintaining genome integrity during virus infection. Annual Review of Microbiology, 64, 61–81. Wu, J., & Chen, Z. J. (2014). Innate immune sensing and signaling of cytosolic nucleic acids. Annual Review of Immunology, 32, 461–488. Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., et al. (2013). Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science (New York, N.Y.), 339(6121), 826–830. Wuest, T., Austin, B. A., Uematsu, S., Thapa, M., Akira, S., & Carr, D. J. (2006). Intact TRL 9 and type I interferon signaling pathways are required to augment HSV-1 induced corneal CXCL9 and CXCL10. Journal of Neuroimmunology, 179(1), 46–52. Xing, J., Ni, L., Wang, S., Wang, K., Lin, R., & Zheng, C. (2013). Herpes simplex virus 1-encoded tegument protein VP16 abrogates the production of beta interferon (IFN) by inhibiting NF-kappaB activation and blocking IFN regulatory factor 3 to recruit its coactivator CBP. Journal of Virology, 87(17), 9788–9801. Xing, J., Wang, S., Lin, R., Mossman, K. L., & Zheng, C. (2012). Herpes simplex virus 1 tegument protein US11 downmodulates the RLR signaling pathway via direct interaction with RIG-I and MDA-5. Journal of Virology, 86(7), 3528–3540. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., et al. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature Immunology, 5(7), 730–737. Yu, H., Huang, H., Kuo, H., Sheen, J., Ou, C., Hsu, T., et al. (2011). IFN-α production by human mononuclear cells infected with varicella-zoster virus through TLR9-dependent and-independent pathways. Cellular & Molecular Immunology, 8(2), 181–188. Zhang, X., Brann, T. W., Zhou, M., Yang, J., Oguariri, R. M., Lidie, K. B., et al. (2011). Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN. Journal of Immunology (Baltimore Md.: 1950), 186(8), 4541–4545. Zhang, S. Y., Jouanguy, E., Ugolini, S., Smahi, A., Elain, G., Romero, P., et al. (2007). TLR3 deficiency in patients with herpes simplex encephalitis. Science (New York, N.Y.), 317(5844), 1522–1527. Zhang, X., Shi, H., Wu, J., Zhang, X., Sun, L., Chen, C., et al. (2013). Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Molecular cell, 51(2), 226–235. Zhang, J., Wang, K., Wang, S., & Zheng, C. (2013). Herpes simplex virus 1 E3 ubiquitin ligase ICP0 protein inhibits tumor necrosis factor alpha-induced NF-kappaB activation by interacting with p65/RelA and p50/NF-kappaB1. Journal of Virology, 87(23), 12935–12948. Zhang, X., Wu, J., Du, F., Xu, H., Sun, L., Chen, Z., et al. (2014). The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Reports, 6(3), 421–430.

100


Zheng, M., Klinman, D. M., Gierynska, M., & Rouse, B. T. (2002). DNA containing CpG motifs induces angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 99(13), 8944–8949. Zhong, B., Yang, Y., Li, S., Wang, Y., Li, Y., Diao, F., et al. (2008). The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity, 29(4), 538–550. Zhu, W., Liu, P., Yu, L., Chen, Q., Liu, Z., Yan, K., et al. (2014). p204-initiated innate antiviral response in mouse Leydig cells. Biology of Reproduction, 91, 8. Zhu, H., Zheng, C., Xing, J., Wang, S., Li, S., Lin, R., et al. (2011). Varicella-zoster virus immediate-early protein ORF61 abrogates the IRF3-mediated innate immune response through degradation of activated IRF3. Journal of Virology, 85(21), 11079–11089. Zolini, G. P., Lima, G. K., Lucinda, N., Silva, M. A., Dias, M. F., Pessoa, N. L., et al. (2014). Defense against HSV-1 in a murine model is mediated by iNOS and orchestrated by the activation of TLR2 and TLR9 in trigeminal ganglia. Journal of Neuroinflammation, 11(1), 1–12.

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