Targeting of the cGAS-STING system by DNA viruses

Journal Pre-proofs Commentary Targeting of the cGAS-STING system by DNA viruses Thomas Phelan, Mark A Little, Gareth Brady PII: DOI: Reference:

S0006-2952(20)30041-1 https://doi.org/10.1016/j.bcp.2020.113831 BCP 113831

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Biochemical Pharmacology

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5 December 2019 24 January 2020

Please cite this article as: T. Phelan, M.A. Little, G. Brady, Targeting of the cGAS-STING system by DNA viruses, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.2020.113831

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Targeting of the cGAS-STING system by DNA viruses Thomas Phelan1, Mark A Little2 and Gareth Brady1 Affiliations 1. Trinity Health Kidney Centre, Trinity Translational Medicine Institute, Trinity College Dublin, St. James' Hospital Campus, Dublin, Ireland. Electronic address: [email protected] & [email protected]. Tel: 00353 860556265

2. Trinity Health Kidney Centre, Trinity Translational Medicine Institute, Trinity College Dublin, St. James' Hospital Campus, Dublin, Ireland; Irish Centre for Vascular Biology, Trinity College Dublin, Dublin, Ireland. Electronic address: [email protected].

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Abstract Innate sensing of viruses by cytosolic nucleic acid sensors is a key feature of anti-viral immunity against these pathogens. The DNA sensing pathway through the sensor cyclic GMP–AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) has emerged in recent years as a key, front-line means of driving interferons and pro-inflammatory cytokines in response to DNA virus infection in vertebrates. Unsurprisingly, many DNA viruses have evolved effective inhibitors of this signalling system which target at a wide variety of points from sensing all the way down to the activation of Interferon Regulatory Factor (IRF)-family and Nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB)-family transcription factors which drive a program of pro-inflammatory and anti-viral gene expression. Here we review DNA viruses that have been shown to inhibit this pathway and the inhibitors they have evolved to do it.

Key Words cGAS; STING; DNA virus; NF-κB; IRF

Abbreviations cGAMP, 2’3 cyclic GMP-AMP; cGAS, cyclic GMP–AMP synthase; dsDNA, doublestranded DNA; ER, endoplasmic reticulum; ERGIC, ER-golgi intermediate compartment; GTPase-activating protein SH3 domain–binding protein 1; HCMV, human cytomegalovirus; HSV, herpes simplex virus; IFI16, interferon-γ-inducible factor 16; IFNs, interferons; NAP1, NF-κB-activating kinase-associated protein 1; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; IKK, IκB kinase; IRF, interferon regulatory factor; KSHV, Kaposi’s sarcoma-associated herpesvirus; MCMV, murine cytomegalovirus; MCV, molluscum contagiosum; MDV, Marek’s disease virus; mTORC, mammalian target of rapamycin complex; MVA, modified vaccinia virus Ankara; NEMO, NF-kappa-B essential modulator; PRRs, pathogen recognition receptors; STIM1, stromal interaction molecule 1; STING, stimulator of interferon genes; TANK, TRAF family member-associated NF-kappa-B activator; TBK1, tank-binding kinase 1; TLRs, toll-like receptors; TRAF, TNF receptor associated factor; TRAP, translocon-associated protein; TRIM32, tripartite motif 32; TRIM56, tripartite motif 56; VACV, vaccinia virus; vIRF1, viral interferon regulatory factor 1.

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Contents 1. Introduction.....................................................................................................................4 2. The cGAS-STING Pathway ..........................................................................................6 3. Evasion of DNA sensing by DNA Viruses .............................................................11 4. Herpesviridae................................................................................................................11 5.

Poxviridae ....................................................................................................................18

6.

Viral-based Therapeutics ..........................................................................................24

7.

Concluding Remarks..................................................................................................26

Conflict of interests............................................................................................................26 References............................................................................................................................27

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1. Introduction Viruses exist as obligate intracellular parasites in every known ecosystem and are the most abundant biological entities on Earth. Therefore, it is unsurprising that there is a huge variety of genome structures and sizes, replication mechanisms, and virion structures in the virus world (Krupovič and Bamford, 2010, Koonin and Dolja, 2014). As a result of high levels of variability in the viruses to which they are exposed, their hosts have evolved diverse and effective ways to detect, restrict and eliminate them. The primary, innate, front-line mechanism for detecting viral infection is the vast array of pattern recognition receptors (PRRs) displayed in the cytosol and endosomal compartments of cells. These PRRs include Tolllike receptors (TLR) 3, 7, 8, and 9, the RIG-I-like receptors MDA5 and RIG-I, and cytosolic DNA sensors such as the AIM2 inflammasome and cyclic GMPAMP synthase (cGAS). PRRs can detect a variety of viral ligands including single-stranded (ss) and double-stranded (ds) DNA and RNA, the uncapped 5’ triphosphate of viral RNA and unmethylated CpG viral DNA. These signatures of viral infection function as pathogen associated molecular patterns (PAMPs) due to biochemical distinctions or cellular localisation, enabling the innate immune system to effectively distinguish between self and non-self (Christensen and Paludan, 2017). Activation of PRR pathways during viral infection, while distinct at the point of sensing, overlap considerably in their use of downstream signalling components and culminate in the activation of common transcription factors sets which drive interferon and proinflammatory cytokine production with diverse effects on a 4

wide variety of cell types. Type I interferons (IFNs) are induced by the combined action of IRF-family and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factors (Kim and Maniatis, 1997). Type I IFNs, such as IFN-α and IFN-β, are predominantly induced during the antiviral response and exert their influence by binding to interferon-α/β receptors which activate Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signalling pathway. This results in the upregulation of hundreds of Interferon regulated genes (ISGs) that restrict viral replication, promote growth arrest, and induce apoptosis (Lee and Ashkar, 2018, Kotredes and Gamero, 2013). Type I IFNs exert an interference state on stimulated cells or trigger antiviral behaviour in innate and adaptive immune cells. For example, type I IFNs induce monocytes to produce IL-18, which in turn activates natural killer cells resulting in the production of IFN-γ, a type II IFN. This is a key cytokine in the antiviral response required for macrophage activation and links innate and adaptive immune responses (Lee et al., 2017). NF-κB activation is also induced in response to viral infection, leading to the production of a wide variety of proinflammatory cytokines and chemokines including IL-1β, TNF-α, and IL-6. Proinflammatory cytokines have important central roles in orchestrating inflammation through induction of pro-inflammatory genes, recruitment and activation of various immune cells along with the upregulation of diverse cell surface molecules (Abe and Barber, 2014, Liles and Van Voorhis, 1995). Therefore, both NF-κB- and IRF-activating pathways collaborate in complex ways, with both required for an optimal response to invading viral pathogens (Rubio et al., 2013).

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As a result of the constant selective pressure imposed by host immunity, viruses have evolved diverse toolboxes of immunomodulators and strategies to inhibit or evade the host immune response. A key facet of the innate response to viruses is the cyclic GMP–AMP synthase (cGAS) pathway. This system also plays key roles in the pathology of chronic inflammatory and autoinflammatory conditions and is a therapeutic target of great interest (Lama et al., 2019, Yan, 2017). cGAS is the most recently discovered cytosolic DNA sensor, and, as such, there are relatively few reviews focusing on the evasion of this pathway by DNA viruses. In this review, we describe the strategies that DNA viruses have evolved to suppress and exploit the activation of the cGAS signalling system, thus limiting or subverting interferon and proinflammatory cytokine production and preventing clearance. Furthermore, understanding how DNA viruses target and modulate this pathway at key nodes of activation offers the possibility to highlight both novel facets of signalling and, potentially, new points for therapeutic intervention.

2. The cGAS-STING Pathway Molecular phylogenetic evidence suggests that the cGAS-STING pathway evolved around 600 million years ago in a common ancestor of humans and cnidarians, though its antiviral function appears to be a more recent development (Margolis et al., 2017). cGAS was first identified in 2013 by Sun et al. and is now recognised as a crucial element in the host DNA immune response pathway (Sun et al., 2013). cGAS acts in conjunction with the downstream effector, stimulator of interferon genes (STING) (Ishikawa and Barber, 2008), in response to dsDNA to induce the production of interferons and

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proinflammatory cytokines via activation of IRF and NF-κB family transcription factors (Abe and Barber, 2014). A conformational change is triggered upon direct binding of cGAS to dsDNA, inducing catalytic activity of the enzyme which converts adenosine triphosphate (ATP) and guanosine triphosphate (GTP) into a second cyclic messenger called 2’3 cyclic GMP-AMP (cGAMP). This binds to STING with a high affinity compared to cGAMP molecules with other combinations of phosphodiester linkages (Zhang et al., 2013, Wu et al., 2013). The extent of cGAS activation is influenced by several factors. The length of the DNA bound to cGAS has a significant impact on the enzymatic activity with short dsDNA (<20bp) inducing activation less efficiently (Li et al., 2013b). Large cGAS complex formation is enhanced by GTPase-activating protein SH3 domain–binding protein 1 (G3BP1). G3BP1 enhances cGAS oligomerisation, promoting its DNA binding ability, and is crucial for mediating DNA sensing independent of DNA length (Liu et al., 2019b). The E3-ligase, tripartite motif 56 (TRIM56), has been shown to induce monoubiquitination of cGAS at Lys335 in the amino-terminal regulatory domain. TRIM56 monoubiquitination of cGAS has been shown to increase DNA binding and promotes cGAS dimerization (Seo et al., 2018). While TRIM56 was previously believed to also polyubiquitinate STING in response to dsDNA detection by cGAS (Tsuchida et al., 2010), more recent research disputed this finding using two-step co-immunoprecipitation. This method involves two rounds of antibody purification prior to immunoblotting and is designed to minimise coprecipitating contaminants. Sciuto et al (2019) provides a detailed explanation and protocol for this type of immunoprecipitation. Using a two-step co-immunoprecipitation method, it was demonstrated that TRIM56 did not 7

directly interact with STING and is likely ubiquitinating another element copurifying with this adapter. These researchers observed a similar effect for tripartite motif 32 (TRIM32), previously thought to drive direct activation of STING by polyubiquitination (Wang et al., 2014, Zhang et al., 2012). Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor protein involved in a wide range of protein-protein interactions and also displays E3 ligase ability. TRAF6 has also been shown to polyubiquitinate cGAS and positively regulate its activation and downstream induction of IFNs (Chen and Chen, 2019). However, the mechanistic requirement of this polyubiquitination remains to be fully elucidated. STING resides at the endoplasmic reticulum (ER) in an inactive state through interactions with the Ca2+ sensor, stromal interaction molecule 1 (STIM1). This interaction is disrupted in response to a conformational change in STING induced by cGAMP binding, resulting in STING trafficking through the ER Golgi intermediate compartment (ERGIC) (Srikanth et al., 2019, Motwani et al., 2019). Recently, Cryo-electron microscopy revealed the mechanism by which cGAMP activates STING. Binding of cGAMP to the ligand binding domain of STING leads to a 180° conformational change relative to the transmembrane domain, enabling STING oligomerisation and translocation (Shang et al., 2019). This translocation of STING enables the C-terminal tail to insert into a groove in tankbinding kinase 1 (TBK1) located between the scaffold and dimerization domain and the kinase domain of adjacent TBK1 subunits. STING oligomerisation is essential for activation of TBK1 as it enables trans-autophosphorylation of adjacent TBK1 molecules, a process that would not be otherwise feasible due to geometric molecular constraints (Larabi et al., 2013, Zhang et al., 2019). 8

Phosphorylation of the C-terminal tail of STING by TBK1 then provides a docking site for IRF3 where it becomes phosphorylated by TBK1 (Zhao et al., 2016). Activated IRF3 dimers then translocate into the nucleus upon activation where they bind target gene promoters and activate transcription of genes including type I interferons (Figure 1) (Honda et al., 2006). While robust IRF activation by the cGAS-STING signalling pathway is wellcharacterised, NF-κB is also activated by the system and plays an important role in regulating gene expression in the DNA sensing response (Ishikawa and Barber, 2008). However, the molecular events leading up the activation of NFκB in this pathway remain poorly understood. A number of proteins are believed to be involved in linking the two activation events including the E3 ligases TRIM32, TRIM56, TRAF6, the kinases TBK1, IKKα and IKKβ, and the ubiquitin binding protein NF-kappa-B essential modulator (NEMO) (Fang et al., 2017, Abe and Barber, 2014, Dunphy et al., 2018). The involvement of TRAF familygenerated polyubiquitin chains in NF-κB signalling has been well- documented, particularly in the TLR pathways (Dhillon et al., 2019). Ubiquitin chains generated by TRAF6 bind to and activate NEMO, an essential component of the IKK complex, leading to IKKα and IKKβ phosphorylation and subsequent phosphorylation and degradation of IκB. Degradation of this inhibitory protein enables NF-κB to translocate into the nucleus and regulate transcription of proinflammatory cytokines and IFNs through cooperation with IRF3 (Dhillon et al., 2019, Kim and Maniatis, 1997). Studies also demonstrate a role for TRAF6 in the activation of NF-κB via the cGAS-STING pathway in response to dsDNA, whereby overexpression of TRAF6 in HEK293T cells results in enhanced NFκB promoter activity, likely through the ubiquitination of STING (Abe and Barber, 9

2014). Similarly, TRAF3 overexpression enhances non-canonical activation of NF-κB via the NF-κB inducing kinase NIK-IKKα axis (Abe and Barber, 2014). Furthermore, an alternative pathway to STING-induced NF-κB activation has been revealed in keratinocytes in response to dsDNA damage, independent of cGAS (Dunphy et al., 2018). Detection of dsDNA breaks in the nucleus leads to translocation of the DNA binding protein, interferon-γ-inducible factor 16 (IFI16), and the tumour suppressor p53, from the nucleus where it forms a complex with STING. IFI16 was shown to be required for the recruitment of TRAF6 to STING which catalyses the formation of k63-linked polyubiquitin chains resulting the activation of the IKK complex and subsequent NF-κB activation (Dunphy et al., 2018). However, it is uncertain if this pathway plays any role during virus infection. While TRAF-family proteins are required in most known cases of NF-κB activation, recent research by Fang et al. describes a mechanism by which the cGAS-STING system utilises TRIM32/56 generated polyubiquitin chains to activate the IKK complex via NEMO (Fang et al., 2017). However, lower levels of NF-κB activation were reported in comparison to TRAF6-mediated signalling, and IKKβ but not IKKα phosphorylation was required for this pathway. Furthermore, TBK1 was shown to be required to fully activate the IKK complex and the NEMO-IKK axis was also required for optimal activation of IRF3. However, the involvement of TBK1 in STING-mediated NF-κB activation was disputed in more recent research investigating an inhibitor of STING-mediated IRF signalling produced by murine cytomegalovirus, where

STING

translocation was not required for NF-κB activation (Stempel et al., 2019). Furthermore, this process was crucial for the STING-TBK1 interaction, and 10

overexpression of STING in TBK1 siRNA knockdown HEK293T cells resulted in similar levels of NF-κB activation in both wild type and TBK1 deficient cells. While the exact mechanisms surrounding STING-mediated NF-κB activation remain unclear, the mechanism of STING-mediated IRF activation seems clear and the robust activation of both by the cGAS-STING system appears to be essential to efficiently eliminate invading pathogens displaying dsDNA as a PAMP.

3. Evasion of DNA sensing by DNA Viruses As previously described, the cGAS-STING system is activated by DNA from both endogenous and exogenous sources during infection. After long periods of co-evolution of viruses with their vertebrate hosts, many DNA viruses have evolved to subvert this system in order evade innate anti-viral immunity. Numerous viral evasion mechanisms and immunomodulators have been identified in DNA viruses which target activation of the cGAS-STING pathway at a variety of levels.

4. Herpesviridae Herpesviruses are a family of enveloped dsDNA viruses that include herpes simplex virus (HSV), and varicella zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus (HHV), and Epstein Barr Virus (EBV). Virtually all humans become latently infected with one or more of these viruses during their lifetime (Sehrawat et al., 2018). Herpesviruses are divided into three subfamilies, Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae, based on their target cells and the site of latency (Cruz-Muñoz and Fuentes-Pananá, 11

2018). Herpesviruses have two distinct lifecycles: latent and lytic. During latent infection, a subset of latent genes are expressed enabling the virus to remain within the host after initial infection with intermittent reactivation of the virus. During lytic infection, a different repertoire of viral genes are expressed in a linear, time-dependant manner. These are grouped into immediate early genes (encoding regulatory proteins), early genes (encoding enzymes required for DNA replication), and late genes (encoding structural proteins), culminating in the release of virions (Speck and Ganem, 2010, Li et al., 2016). It is important to note that these viruses replicate in the nuclei of infected cells and a viral nucleocapsid protects the dsDNA genome from detection by cytosolic DNA sensors upon entry into the cell. However, it has been shown that, in HSV-1 infected macrophages, the viral capsid becomes polyubiquitinated in the cytosol, resulting in its degradation by the proteasome, thus releasing the viral DNA for detection (Horan et al., 2013). Herpesviruses have been shown to target the cGAS-STING signalling system at a variety of levels (Figure 2). Kaposi’s sarcoma-associated herpesvirus (KSHV) contains a tegument protein known as ORF52, a late gene produced during the lytic replication cycle, which has been shown to inhibit the activity of cGAS (Li et al., 2016). ORF52 is only found in the cytosol and binds to and sequesters DNA. This was reported to be insufficient to inhibit type I IFN activation on its own as cGAS binds to DNA with a higher affinity. ORF52 was also reported to directly interact with the catalytic domain of cGAS, resulting in reduced enzymatic activity and inhibition of downstream IRF3 activation. However, ORF52 deficient viruses only exhibited a marginal increase in the level of IRF3 activation (Wu et al., 2015) likely due to the vast array of other inhibitors expressed by the KSHV genome that target 12

IRF activation at many different levels (Gao et al., 1997, Hwang et al., 2009, Wu et al., 2015). Another KSHV protein, viral interferon regulatory factor 1 (vIRF1), is also a potent inhibitor of type I IFN and cGAS-STING signalling, and has no analogues in other human herpesviruses (Ma et al., 2015). vIRF1 can inhibit cGAS-STING signalling at multiple points throughout the pathway. Some mechanisms of inhibition by this multifunctional inhibitor target far downstream where pathways from other PRRs intersect and thus is not specific for cGAS-STING signalling. An example of this is the ability of vIRF1 to prevent IRF3 recruitment of the p300/CBP transcriptional cofactor or preventing p300 histone acetyltransferase activity leading to an altered chromatin structure and subsequent inefficient transcription (Li et al., 2000). However, vIRF1 was also found to directly prevent the interaction of STING and TBK1 thus inhibiting downstream phosphorylation of IRF3 by TBK1 (Ma et al., 2015). Cytomegalovirus (CMV) has developed effective strategies for evading the host immune system. However, CMV evasion mechanisms of innate immunity remain poorly understood at present. Murine cytomegalovirus (MCMV) is a wellestablished model for CMV infection (Lisnić et al., 2015). Recently, the first MCMV protein to subvert STING-mediated signalling was described (Stempel et al., 2019). The MCMV M152 protein was shown to have distinct effects on STING-associated IRF and NF-κB activation. M152 specifically inhibits cGASSTING induced IFNβ levels and promoter activity but has negligible effects on NF-κB signalling. Further investigation revealed that M152 was able to prevent the trafficking of STING from the ER to the ERGIC, therefore inhibiting the interaction between STING and TBK1, preventing the downstream type I IFN 13

response. Interestingly, using an active STING mutant that restricts STING translocation to the ER, NF-κB activation levels remained high with relatively low levels of inhibition compared to the IRF-activated reporter, suggesting that STING activates NF-κB while resident in the ER. This also suggests that TBK1 is not crucial to the activation of NF-κB as previous reports suggested, as STING trafficking is a prerequisite to TBK1 activation (Liu et al., 2015, Fang et al., 2017, Stempel et al., 2019). Interestingly, NF-κB activation was beneficial for MCMV as it promoted viral replication in this study. This result was attributed to corresponding sites on the HCMV major immediately early promoter (MIEP) and NF-κB, which was previously described to be essential to initiate the viral transcriptional program (Stempel et al., 2019, Caposio et al., 2007). Human cytomegalovirus (HCMV) infection can have major negative effects on reproduction in humans causing congenital conditions that lead to severe disability along with severe complications in immunocompromised individuals (Britt, 2017). Infection of primary human fibroblasts induces both strong Type I IFN and proinflammatory cytokine production, but HCMV produces a number of proteins that have been recently shown to block elements of the cGAS-STING pathway. One such protein is pp65 (pUL83), a tegument protein, which was originally shown to inhibit IRF3 activation (Abate et al., 2004). The exact mechanism of this inhibition remained elusive up until recently as it would be nearly a decade until the identification of cGAS. Reports from a study using human foreskin fibroblasts, infected with a pp65 deficient strain of HCMV, revealed that type I IFN induction was increased compared to wild type infection (Biolatti et al., 2018). This study revealed that pp65 directly bound via its Nterminal domain to cGAS and prevented the enzymatic production of cGAMP, 14

thus dampening downstream signalling. pp65 also mediates its inhibitory effects on the cGAS-STING pathway through an indirect mechanism. IFI16 has been shown to act on STING synergistically with cGAMP in keratinocytes to promote STING phosphorylation and translocation (Almine et al., 2017). pp65 has been shown to inhibit IFI16 during detection of nuclear HCMV DNA by preventing oligomerization of the IFI16 pyrin domain (Li et al., 2013a). As a result, downstream activation of IRF and NF-κB were significantly impeded. This interaction is also required to activate viral gene expression as pp65 recruits IFI16 to the major immediate early promoter. Another cGAS-STING antagonist produced by HCMV is the tegument protein UL42 which acts on both cGAS and STING to inhibit type I IFN production (Fu et al., 2019). Overexpression of UL42 was shown to inhibit cGAMP production and activation of downstream effector genes. This effect was attributed to the ability of UL42 to prevent the interaction of cGAS with DNA and cGAS oligomerization. Furthermore, UL42 was also shown to act on STING through interaction with autophagy associated proteins p62 and LC3B. LC3B can localize to autophagic membranes, and p62 (also known as sequestosome 1) can bind to LC3B and deliver cargo for degradation (Jiang and Mizushima, 2015). The translocon-associated protein (TRAP) complex is an important accessory translocon component that stimulates transocation of proteins through the ER membrane (Fons et al., 2003). p62 delivers the TRAPβ subunit of the complex to autophagosomes in conjunction with LC3B whereby it subsequently undergoes degradation by lysosomal enzymes leading to impaired trafficking of STING (Fu et al., 2019). Similarly, the HCMV protein UL82 also disrupts STING trafficking from the ER. iRhom2 is an ER-associated 15

protein that bridges the interaction between STING and TRAPβ and also maintains STING stability by promoting deubiquitinase recruitment (Luo et al., 2016). UL82 interacts with both STING and iRhom2 to prevent association with TRAPβ, thereby inhibiting STING trafficking and downstream IRF signalling (Fu et al., 2017b). Like UL42, another HCMV protein known as UL31 blocks cGAS enzymatic activity. It achieves this through direct interaction with cGAS and disassociation of DNA from the enzyme. Subsequently, cGAMP production is hindered and downstream IRF and NF-κB activation is thus reduced. While UL31 can directly bind DNA, it does so to a much lower extent than the cGAS-DNA interaction, indicating that this effect is not a result of competitive DNA binding but due to the direct interaction with cGAS (Huang et al., 2018b). Marek’s Disease Virus (MDV) is highly contagious in poultry and causes T cell lymphomas with high fatality. As such, while also being an economically important due to its effect on poultry, this virus also serves as a model organism for virus-induced T cell lymphomas (Biggs and Nair, 2012). The chicken interferon pathway is poorly characterised compared to mammalian counterparts. However, studies have shown that IRF7 and NF-κB are activated in response to DNA sensing by the cGAS-STING system. IRF7 is then phosphorylated by TBK1, dimerizes, and translocates into the nucleus where it binds to the IFNβ promoter (Santhakumar et al., 2017). MDV protein RLORF4 was previously shown to be important for viral pathogenicity as its deletion disrupts MDV replication (Jarosinski et al., 2005). However, these researchers were unable to uncover the exact function of this protein. A more refined role for this protein was recently described by Liu et al. by demonstrating its potent 16

inhibitory effects on both NF-κB and IFN-β, but not IRF7 (Liu et al., 2019a). These effects were attributed to the ability of RLORF4 to bind to the NF-κB subunits, p60 and p50, and suppress nuclear translocation. Since p60 and p50 translocation is a prerequisite for IFN-β induction, the antiviral response is also dampened (Liu et al., 2019a, Kim and Maniatis, 1997). MDV protein VP23 has been reported to interact and inhibit downstream signalling of the cGAS-STING system in chick embryo fibroblasts. VP23 prevents nuclear translocation of IRF7 by inhibiting its phosphorylation and dimerization mediated by TBK1. This is a direct result of VP23’s ability to bind to the domain on IRF7 that is involved in the interaction with TBK1 thus disrupting their association. Furthermore, the effects of VP23 were shown to be IRF-specific as NF-κB reporter activity remained unaffected. Interestingly, the interaction between VP23 and IRF7 appears to be indispensable for MDV replication as a VP23 deletion mutant virus could not be rescued (Gao et al., 2019). The C-terminus was not required to mediate its effects of IFN signalling, and a previous study (Kim et al., 2011) looking at HSV-1 VP23 showed the importance of this ortholog for closing the capsid shell into an icosahedral structure during virus assembly (Gao et al., 2019). This would suggest that VP23 has additional roles beyond immunomodulation. HSV-1 is the causative agent behind herpes labialis (cold sores). It induces an acute lytic infection and thereafter resides in a latent state in neurons for the duration of the host’s life with intermittent reactivation (Su et al., 2016). The HSV-1 protein VP22 is a tegument protein encoded by UL49 gene and is involved in both viral replication and host immune evasion. VP22 is involved in viral replication and pathogenesis by performing a number of different functions such as microtubule 17

reorganization to enhance intracellular transport (Elliott and O'Hare, 1998) and accumulation of virion host shutoff protein which degrades host mRNAs (Taddeo et al., 2007). Amongst its many other functions, VP22 also targets cGAS and inhibits its enzymatic activity through direct interaction with the enzyme thereby inhibiting downstream IFNβ production (Huang et al., 2018a). The HSV-1 protein UL46 also appears to have multiple roles during infection and is a highly abundant tegument protein. Interestingly, UL46 appears to bind to STING and TBK1 on separate domains resulting in disruption of their functions (Deschamps and Kalamvoki, 2017). However, the precise mechanism behind this was not elucidated. Both STING and TBK1 were able to interact with each other in the presence of UL46 suggesting that this effect was not caused by blockage of STING-TBK1 binding sites. This work also revealed that expression of UL46 in cells leads to deficiency in STING and IFI16 transcripts, but this was not observed in cells during infection with wild type HSV-1. A recent study has shown that UL46 exerts its inhibitory effects by preventing TBK1mediated signalling. As previously mentioned, adjacent TBK1 molecules are required to enable trans-autophosphorylation and activation due to geometrical constraints (Zhang et al., 2019). UL46 was shown to disrupt the dimerization of TBK1 molecules, leading to a reduction in the phosphorylation and translocation of IRF3 (You et al., 2019).

5. Poxviridae Poxviruses are among the most feared viruses in human history. This is due to the devastation caused by variola virus, the causative agent of smallpox, resulting in the deaths of nearly half a billion people over the course of the 20th 18

century alone (Thèves et al., 2014). Poxviruses are large viruses that contain a complex dsDNA genome, protected by enveloped capsid, and can only replicate within the cytoplasm of cells. They are divided into two subfamilies: Chordopoxvirinae and Entomopoxvirinae, which infect vertebrates and invertebrates, respectively. Many chordopoxviruses have the ability to infect a wide range of hosts while others can only infect one host species (Oliveira et al., 2017). Approximately 50 genes have analogs conserved across all poxvirus species that have been sequenced, many of which are centrally located on the genome, with another 40 conserved across chordopoxviruses (Lefkowitz et al., 2006). In contrast, many of the genes that are involved in host-specificity and modulation of the host antiviral response are located at the terminal ends of the genome (Brady and Bowie, 2014). The reason for this disparity in the localisation of genes is due to the fact that the terminal regions of the genome have a higher capacity for recombination with exogenous sequences than the internal regions. This is likely due to fact that replication is initiated at the termini, and because repeat sequences in the termini encourage homologous recombination (Yao et al., 1997). While a long history of catastrophic epidemics involving poxviruses serve as a reminder to be vigilant of their potential danger, their study has been essential for the advancement of vaccines, virotherapy, and cell biology. This has enabled the development of new immunization strategies, treatments for various forms of cancer and elucidating evasion mechanisms in intracellular signalling pathways (García-Arriaza et al., 2014, Chan and McFadden, 2014, Brady and Bowie, 2014). Poxviruses have also been shown to target the cGAS-STING signalling system at a variety of levels (Figure 3).

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Modified vaccinia virus Ankara (MVA) is an attenuated form of vaccinia virus that was developed to increase safety of the smallpox vaccine. Serial passaging of MVA in chick embryo fibroblasts resulted in deletions and mutations of various genes involved virulence and evasion, leading to a virus with replication restricted to avian cells (Altenburg et al., 2014). As a result of the deletion of many immunomodulators, it is no surprise that MVA induces cGAS-STING mediated type I IFN signalling in conventional dendritic cells. The vaccinia virus (VACV) protein N1 is an immunomodulatory protein that is altered by a 4 amino acid frameshift in MVA, resulting in loss of the protein function (Dai et al., 2014, Meisinger-Henschel et al., 2007). N1 has been previously shown to inhibit TLR mediated NF-κB signalling by targeting the IKK complex and IRF3 signalling by targeting TBK1 (DiPerna et al., 2004). A decade later, cGAS was shown to be the main cytosolic sensor of MVA as cGAS-deficient murine dendritic cells were unable to mount an effective response to infection compared to wild-type cells (Dai et al., 2014). Although the exact mechanism of inhibition remains to be elucidated, in light of earlier studies, the effect is likely due to the interaction between N1 and TBK1 along with its ability to inhibit at the level of the IKK complex. Further downstream, the related VACV protein N2, inhibits signalling at the level of nuclear IRF3. Inhibition of upstream signalling is not observed in response to the presence of N2 but nuclear translocation of IRF3 is still able to occur. However, reduced expression of the IRF3 promoter dependent chemokine CXCL10, indicates that this effect is due to N2-mediated inhibition of IRF3 (Ferguson et al., 2013). The VACV protein C6 is yet another immunomodulator of downstream targets of the cGAS-STING system and specifically inhibits type I IFN production, while 20

having no effect on NF-κB activation. C6 was shown to interact with TBK1 and IκB kinase-ε (IKKε), both of which are upstream IRF3/7 and required to phosphorylate and activate these IRFs. C6 associates with the adaptor proteins NF-Kappa-B-Activating Kinase-Associated Protein 1 (NAP1), TRAF family member-associated NF-kappa-B activator (TANK) and TBK1 binding protein (or SINTBAD) which are constitutively associated with TBK1 and IKKε and appears to disrupt their function (Unterholzner et al., 2011). The TBK1 and IKKε level of signalling is a key point of convergence for a number of PRRs such as TLRs, RLRs and cytosolic DNA sensors, so it is unsurprising that inhibition of this region has a profound effect on many innate virus sensing pathways (Verhelst et al., 2013). The poxvirus protein F17 is conserved across poxvirus species and is involved in virion formation (Wickramasekera and Traktman, 2010). Until recently, F17 has had no known function outside of its role as a structural protein, but it has now been reported to indirectly induce cGAS degradation by manipulating the mammalian Target of Rapamycin complexes (mTORC)1/2 (Meade et al., 2018). mTORC1 and mTORC2 are distinguished by the presence of their N-terminal regulators known as Regulatory associated protein of mTOR (Raptor) and Rapamycin-insensitive companion of mTOR (Rictor), respectively. These two regulatory proteins bind their target proteins antagonistically in relation to one another. mTORC1 activity occurs in response to mitogenic signals such as those induced by the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) axis. PI3K is an intracellular lipid kinase and AKT is a serine/threonine kinase and these exert their effect by phosphorylating downstream targets such as mTORC1. This influences metabolic processes and increases protein 21

synthesis. mTORC2 acts in a similar manner as it is responsive to mitogenic stimuli such as growth factors but differs in its regulatory function as it controls cytoskeletal formation and cell survival. Activity of mTORC1 leads to mTORC2 inhibition through another kinase p70S6K, meaning activities of both are finely balanced (Meade et al., 2018, Jhanwar-Uniyal et al., 2019). VACV activates mTOR signalling during the early stages of infection allowing the virus to benefit from metabolic signalling and establish infection. F17 was able to interact with both raptor and rictor, leading to hyperactivation of mTOR and enhanced phosphorylation of p70S6K and 4E-BP1, both of which are downstream targets of mTORC1. This dysregulated signalling of mTOR by F17 drove cGAS degradation leading to inhibition of downstream signalling. Furthermore, STING and mTOR cooperate to resolve stressed ER membranes as a result of STING activation which is required for optimal signalling in this system. These ER membranes are sequestered and degraded by autophagy induced by the inhibition of mTORC1 (Moretti et al., 2017). Therefore, the persistent activation of mTORC1 by F17 likely leads to a build-up of ER stress thus dampening the IFN response (Meade et al., 2018). Following its production by cGAS, cGAMP potently induces innate immune responses by activating STING, making it a key element of this DNA sensing pathway (Zhang et al., 2013). Hence, one would assume that a strong selective pressure exists for viruses to target cGAMP directly. Until recently, the only known enzyme with the ability to degrade cGAMP is the phosphodiesterase ecto-nucleotide pyrophosphatase/phosphodiesterase ENPP1, an extracellular protein which is not found in the cytosol (Li et al., 2014). However, a recent study revealed that poxviruses can also target cGAMP using poxvirus immune 22

nucleases (poxins) (Eaglesham et al., 2019). In this work, an array of mammalian viruses was screened for nuclease activity against cGAMP resulting in the identification of the B2R gene encoding a family of poxins. VACV B2R gene encodes poxin that destabilises cGAMP through hydrolysis of the canonical 3′–5′ bond. While many poxvirus studies aiming to identify host immune-evasion mechanisms focus on proteins found in VACV, relatively few studies have looked in detail at a human-adapted poxvirus. Molluscum Contagiosum virus (MCV) is a large doubled-stranded DNA virus that is specifically adapted to long-term human infection. MCV is common throughout the developed world and is the primary poxvirus causing human disease (Chen et al., 2013). By investigating the mechanisms MCV has evolved to subvert human immunity, this virus may offer insights into human pathways not offered by non-human adapted family members of poxviruses. However, progress in this area is hampered by the lack of an adequate laboratory cell culture system that enables MCV replication which partially explains the paucity of research in this area (Chen et al., 2013). Despite this, MCV has been shown to produce immunomodulatory proteins that interact with various elements of the canonical NF-κB signalling pathway. Some examples include 2 viral FLICE-like proteins (vFLIPS): MC159 and MC160. These can block NF-κB signalling by binding to NEMO and preventing IKKβ phosphorylation and by inducing IKKα degradation, respectively, and block TNFα receptor-induced apoptosis (Shisler, 2014). The MCV protein MC132 recruits the p65 subunit of NF-κB to an elongin B/C Cullin 5 E3 ligase complex where it ubiquitinates it, tagging it for degradation by the proteasome (Brady et 23

al., 2015). MC005 is another MCV protein that inhibits NF-κB activation further upstream. This mediates its inhibitory effects by binding to the NEMO subunit of the IKK complex and preventing conformational change required for the exposure for phosphorylation sites on IKKβ (Brady et al., 2017). Due to their inhibition at common activation nodes downstream of many PRRs, it is unsurprising that both MC132 and MC005 have been shown to inhibit NF-κB activation by cGAS. As the majority of the MCV genome remains to be investigated it is currently unclear whether this virus possesses inhibitors acting directly at the level of cGAS or STING.

6. Viral-based Therapeutics Inflammation lies at the centre of human disease and is triggered via intracellular signalling pathways. This leads to a wide variety of autoimmune and autoinflammatory conditions, such as systemic lupus erythematosus, during which aberrant activation of the cGAS-STING pathway plays a significant pathological role (Mustelin et al., 2019). Innate immune signalling underlies and controls inflammation and, as such, understanding and manipulating the key elements of these pathways is crucial for the development of new antiinflammatory therapies. Since viruses have specifically evolved to subvert the host immune system, allowing viral growth and replication, investigation of their mechanisms of immunomodulation offer and excellent tool for the identification of novel therapeutic targets. As viruses produce a vast array of inhibitory proteins to block inflammation and antiviral responses these may prove to be useful tools on which to base novel therapeutics to ameliorate human disease driven by aberrant innate responses.

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Protein-based therapeutics represent a major leap in medicine with over 100 proteins currently approved for clinical use. Recombinant proteins such as insulin-like growth factor (Fintini et al., 2009) and antibodies such as rituximab (Maloney, 2012) are among many examples of highly effective protein therapeutics. However, the majority of these therapeutics exert their effects on extracellular targets emphasising the challenges of designing effective protein therapeutics with targets not exposed to the extracellular environment. The cell membrane presents as a major obstacle for therapies aiming to target intracellular mechanisms. As a result, there has been a recent surge in studies focusing on alternative drug delivery systems (Chatin et al., 2015, Ye et al., 2016, Patra et al., 2018). Cell-penetrating peptides (CPPs) are short peptides containing a motif such as poly-arginine that enables either passive or active transport across biological membranes. Currently there are upwards of 25 CPPconjugated drugs in clinical trials that have the potential to treat a variety of diseases including those involving inflammation (Habault and Poyet, 2019, Fu et al., 2017a). There has been some success in the intracellular delivery of a DAN virus-derived peptides using CPPs, one example being viral inhibitor peptide of TLR4 (VIPER), an 11-amino acid peptide derived from the VACV protein A46, which potently inhibits LPS-activated NF-κB with systemic efficacy in murine LPS-induced sepsis models (Lysakova-Devine et al., 2010). VIPER inhibits NF-κB by masking binding sites on MyD88 adaptor-like (MAL) and TRIFrelated adaptor molecule (TRAM), thus preventing downstream signalling. This highlights the potential therapeutic value of reducing the anti-inflammatory activity of viral immunomodulators with intracellular targets to CPP-tagged peptides, particularly those derived from viruses adapted to human infection.

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Through further peptide screening of emerging viral immunomodulators targeting key pathways in human disease (like the cGAS-STING system), more effective therapeutics for treating human disease may be derived by allowing virus evolution to select concise points of inhibition.

7. Concluding Remarks While the innate immune system of hosts has evolved many ways to detect and eliminate invading pathogens, such as the employment of receptors that recognise conserved pathogenic elements, viruses have evolved in step to counteract this system. We have described the current understanding of many strategies employed by herpesviruses and poxviruses to subvert and inhibit DNA sensing and signalling through the cGAS-STING system. The importance of targeting cGAS-STING signalling is emphasised in many cases by the number of nonredundant proteins employed by the same virus that inhibit this system at multiple sites. Much can be learned about our own cellular defence mechanisms by studying host-pathogen interactions. The immunomodulatory proteins produced by viruses can highlight key elements of the pathways involved in inflammation and thus reveal highly sought-after therapeutic targets. Furthermore, by exploiting the inhibitory properties of these viral proteins, more effective treatments may become available to maintain chronic inflammatory conditions.

Conflict of interests There is no conflict of interest to declare.

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References ABATE, D. A., WATANABE, S. & MOCARSKI, E. S. 2004. Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response. Journal of virology, 78, 10995-11006. ABE, T. & BARBER, G. N. 2014. Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-kappaB activation through TBK1. J Virol, 88, 5328-41. ALMINE, J. F., O’HARE, C. A. J., DUNPHY, G., HAGA, I. R., NAIK, R. J., ATRIH, A., CONNOLLY, D. J., TAYLOR, J., KELSALL, I. R., BOWIE, A. G., BEARD, P. M. & UNTERHOLZNER, L. 2017. IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes. Nature Communications, 8, 14392. ALTENBURG, A. F., KREIJTZ, J. H. C. M., DE VRIES, R. D., SONG, F., FUX, R., RIMMELZWAAN, G. F., SUTTER, G. & VOLZ, A. 2014. Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases. Viruses, 6, 2735-2761. BIGGS, P. M. & NAIR, V. 2012. The long view: 40 years of Marek's disease research and Avian Pathology. Avian Pathology, 41, 3-9. BIOLATTI, M., DELL'OSTE, V., PAUTASSO, S., GUGLIESI, F., VON EINEM, J., KRAPP, C., JAKOBSEN, M. R., BORGOGNA, C., GARIGLIO, M., DE ANDREA, M. & LANDOLFO, S. 2018. Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING. Journal of virology, 92, e0177417. BRADY, G. & BOWIE, A. G. 2014. Innate immune activation of NFκB and its antagonism by poxviruses. Cytokine & Growth Factor Reviews, 25, 611-620. BRADY, G., HAAS, D. A., FARRELL, P. J., PICHLMAIR, A. & BOWIE, A. G. 2015. Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-B Activation by Targeting p65 for Degradation. Journal of virology, 89, 8406-8415. BRADY, G., HAAS, D. A., FARRELL, P. J., PICHLMAIR, A. & BOWIE, A. G. 2017. Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation. Journal of virology, 91. BRITT, W. J. 2017. Congenital Human Cytomegalovirus Infection and the Enigma of Maternal Immunity. Journal of virology, 91, e02392-16. CAPOSIO, P., LUGANINI, A., HAHN, G., LANDOLFO, S. & GRIBAUDO, G. 2007. Activation of the virusinduced IKK/NF-κB signalling axis is critical for the replication of human cytomegalovirus in quiescent cells. Cellular Microbiology, 9, 2040-2054. CHAN, W. M. & MCFADDEN, G. 2014. Oncolytic Poxviruses. Annual review of virology, 1, 119-141. CHATIN, B., MÉVEL, M., DEVALLIÈRE, J., DALLET, L., HAUDEBOURG, T., PEUZIAT, P., COLOMBANI, T., BERCHEL, M., LAMBERT, O., EDELMAN, A. & PITARD, B. 2015. Liposome-based Formulation for Intracellular Delivery of Functional Proteins. Molecular Therapy - Nucleic Acids, 4, e244. CHEN, X., ANSTEY, A. V. & BUGERT, J. J. 2013. Molluscum contagiosum virus infection. Lancet Infect Dis, 13, 877-88. CHEN, X. & CHEN, Y. 2019. Ubiquitination of cGAS by TRAF6 regulates anti-DNA viral innate immune responses. Biochemical and Biophysical Research Communications, 514, 659-664. CHRISTENSEN, M. H. & PALUDAN, S. R. 2017. Viral evasion of DNA-stimulated innate immune responses. Cellular & molecular immunology, 14, 4-13. CRUZ-MUÑOZ, M. E. & FUENTES-PANANÁ, E. M. 2018. Beta and Gamma Human Herpesviruses: Agonistic and Antagonistic Interactions with the Host Immune System. Frontiers in microbiology, 8, 2521-2521. DAI, P., WANG, W., CAO, H., AVOGADRI, F., DAI, L., DREXLER, I., JOYCE, J. A., LI, X.-D., CHEN, Z., MERGHOUB, T., SHUMAN, S. & DENG, L. 2014. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway. PLoS pathogens, 10, e1003989-e1003989.

27

DESCHAMPS, T. & KALAMVOKI, M. 2017. Evasion of the STING DNA-Sensing Pathway by VP11/12 of Herpes Simplex Virus 1. Journal of virology, 91, e00535-17. DHILLON, B., ALEITHAN, F., ABDUL-SATER, Z. & ABDUL-SATER, A. A. 2019. The Evolving Role of TRAFs in Mediating Inflammatory Responses. Frontiers in Immunology, 10, 104. DIPERNA, G., STACK, J., BOWIE, A. G., BOYD, A., KOTWAL, G., ZHANG, Z., ARVIKAR, S., LATZ, E., FITZGERALD, K. A. & MARSHALL, W. L. 2004. Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors. J Biol Chem, 279, 36570-8. DUNPHY, G., FLANNERY, S. M., ALMINE, J. F., CONNOLLY, D. J., PAULUS, C., JØNSSON, K. L., JAKOBSEN, M. R., NEVELS, M. M., BOWIE, A. G. & UNTERHOLZNER, L. 2018. Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage. Molecular cell, 71, 745-760.e5. EAGLESHAM, J. B., PAN, Y., KUPPER, T. S. & KRANZUSCH, P. J. 2019. Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling. Nature, 566, 259-263. ELLIOTT, G. & O'HARE, P. 1998. Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules. Journal of virology, 72, 6448-6455. FANG, R., WANG, C., JIANG, Q., LV, M., GAO, P., YU, X., MU, P., ZHANG, R., BI, S., FENG, J. M. & JIANG, Z. 2017. NEMO-IKKbeta Are Essential for IRF3 and NF-kappaB Activation in the cGAS-STING Pathway. J Immunol, 199, 3222-3233. FERGUSON, B. J., BENFIELD, C. T. O., REN, H., LEE, V. H., FRAZER, G. L., STRNADOVA, P., SUMNER, R. P. & SMITH, G. L. 2013. Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence. The Journal of general virology, 94, 2070-2081. FINTINI, D., BRUFANI, C. & CAPPA, M. 2009. Profile of mecasermin for the long-term treatment of growth failure in children and adolescents with severe primary IGF-1 deficiency. Therapeutics and clinical risk management, 5, 553-559. FONS, R. D., BOGERT, B. A. & HEGDE, R. S. 2003. Substrate-specific function of the transloconassociated protein complex during translocation across the ER membrane. The Journal of cell biology, 160, 529-539. FU, L.-S., WU, Y.-R., FANG, S.-L., TSAI, J.-J., LIN, H.-K., CHEN, Y.-J., CHEN, T.-Y. & CHANG, M. D.-T. 2017a. Cell Penetrating Peptide Derived from Human Eosinophil Cationic Protein Decreases Airway Allergic Inflammation. Scientific Reports, 7, 12352. FU, Y.-Z., GUO, Y., ZOU, H.-M., SU, S., WANG, S.-Y., YANG, Q., LUO, M.-H. & WANG, Y.-Y. 2019. Human cytomegalovirus protein UL42 antagonizes cGAS/MITA-mediated innate antiviral response. PLOS Pathogens, 15, e1007691. FU, Y. Z., SU, S., GAO, Y. Q., WANG, P. P., HUANG, Z. F., HU, M. M., LUO, W. W., LI, S., LUO, M. H., WANG, Y. Y. & SHU, H. B. 2017b. Human Cytomegalovirus Tegument Protein UL82 Inhibits STING-Mediated Signaling to Evade Antiviral Immunity. Cell Host Microbe, 21, 231-243. GAO, L., LI, K., ZHANG, Y., LIU, Y., LIU, C., ZHANG, Y., GAO, Y., QI, X., CUI, H., WANG, Y. & WANG, X. 2019. Inhibition of DNA-Sensing Pathway by Marek's Disease Virus VP23 Protein through Suppression of Interferon Regulatory Factor 7 Activation. Journal of Virology, 93, e01934-18. GAO, S. J., BOSHOFF, C., JAYACHANDRA, S., WEISS, R. A., CHANG, Y. & MOORE, P. S. 1997. KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene, 15, 1979-85. GARCÍA-ARRIAZA, J., CEPEDA, V., HALLENGÄRD, D., SORZANO, C. Ó. S., KÜMMERER, B. M., LILJESTRÖM, P. & ESTEBAN, M. 2014. A Novel Poxvirus-Based Vaccine, MVA-CHIKV, Is Highly Immunogenic and Protects Mice against Chikungunya Infection. Journal of Virology, 88, 3527. HABAULT, J. & POYET, J.-L. 2019. Recent Advances in Cell Penetrating Peptide-Based Anticancer Therapies. Molecules (Basel, Switzerland), 24, 927. HONDA, K., TAKAOKA, A. & TANIGUCHI, T. 2006. Type I interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors. Immunity, 25, 349-60. 28

HORAN, K. A., HANSEN, K., JAKOBSEN, M. R., HOLM, C. K., SØBY, S., UNTERHOLZNER, L., THOMPSON, M., WEST, J. A., IVERSEN, M. B., RASMUSSEN, S. B., ELLERMANN-ERIKSEN, S., KURT-JONES, E., LANDOLFO, S., DAMANIA, B., MELCHJORSEN, J., BOWIE, A. G., FITZGERALD, K. A. & PALUDAN, S. R. 2013. Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors. Journal of immunology (Baltimore, Md. : 1950), 190, 2311-2319. HUANG, J., YOU, H., SU, C., LI, Y., CHEN, S. & ZHENG, C. 2018a. Herpes Simplex Virus 1 Tegument Protein VP22 Abrogates cGAS/STING-Mediated Antiviral Innate Immunity. Journal of Virology, 92, e00841-18. HUANG, Z. F., ZOU, H. M., LIAO, B. W., ZHANG, H. Y., YANG, Y., FU, Y. Z., WANG, S. Y., LUO, M. H. & WANG, Y. Y. 2018b. Human Cytomegalovirus Protein UL31 Inhibits DNA Sensing of cGAS to Mediate Immune Evasion. Cell Host Microbe, 24, 69-80.e4. HWANG, S., KIM, K. S., FLANO, E., WU, T.-T., TONG, L. M., PARK, A. N., SONG, M. J., SANCHEZ, D. J., O'CONNELL, R. M., CHENG, G. & SUN, R. 2009. Conserved herpesviral kinase promotes viral persistence by inhibiting the IRF-3-mediated type I interferon response. Cell host & microbe, 5, 166-178. ISHIKAWA, H. & BARBER, G. N. 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature, 455, 674-678. JAROSINSKI, K. W., OSTERRIEDER, N., NAIR, V. K. & SCHAT, K. A. 2005. Attenuation of Marek's disease virus by deletion of open reading frame RLORF4 but not RLORF5a. Journal of virology, 79, 11647-11659. JHANWAR-UNIYAL, M., WAINWRIGHT, J. V., MOHAN, A. L., TOBIAS, M. E., MURALI, R., GANDHI, C. D. & SCHMIDT, M. H. 2019. Diverse signaling mechanisms of mTOR complexes: mTORC1 and mTORC2 in forming a formidable relationship. Advances in Biological Regulation, 72, 51-62. JIANG, P. & MIZUSHIMA, N. 2015. LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods, 75, 13-18. KIM, H. S., HUANG, E., DESAI, J., SOLE, M., PRYCE, E. N., OKOYE, M. E., PERSON, S. & DESAI, P. J. 2011. A domain in the herpes simplex virus 1 triplex protein VP23 is essential for closure of capsid shells into icosahedral structures. Journal of virology, 85, 12698-12707. KIM, T. K. & MANIATIS, T. 1997. The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome. Mol Cell, 1, 119-29. KOONIN, E. V. & DOLJA, V. V. 2014. Virus world as an evolutionary network of viruses and capsidless selfish elements. Microbiology and molecular biology reviews : MMBR, 78, 278-303. KOTREDES, K. P. & GAMERO, A. M. 2013. Interferons as inducers of apoptosis in malignant cells. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 33, 162-170. KRUPOVIČ, M. & BAMFORD, D. H. 2010. Order to the viral universe. Journal of virology, 84, 1247612479. LAMA, L., ADURA, C., XIE, W., TOMITA, D., KAMEI, T., KURYAVYI, V., GOGAKOS, T., STEINBERG, J. I., MILLER, M., RAMOS-ESPIRITU, L., ASANO, Y., HASHIZUME, S., AIDA, J., IMAEDA, T., OKAMOTO, R., JENNINGS, A. J., MICHINO, M., KUROITA, T., STAMFORD, A., GAO, P., MEINKE, P., GLICKMAN, J. F., PATEL, D. J. & TUSCHL, T. 2019. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nature communications, 10, 2261-2261. LARABI, A., DEVOS, JULIETTE M., NG, S.-L., NANAO, MAX H., ROUND, A., MANIATIS, T. & PANNE, D. 2013. Crystal Structure and Mechanism of Activation of TANK-Binding Kinase 1. Cell Reports, 3, 734-746. LEE, A. J. & ASHKAR, A. A. 2018. The Dual Nature of Type I and Type II Interferons. Frontiers in immunology, 9, 2061-2061. LEE, A. J., CHEN, B., CHEW, M. V., BARRA, N. G., SHENOUDA, M. M., NHAM, T., VAN ROOIJEN, N., JORDANA, M., MOSSMAN, K. L., SCHREIBER, R. D., MACK, M. & ASHKAR, A. A. 2017. 29

Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection. The Journal of Experimental Medicine, 214, 1153. LEFKOWITZ, E. J., WANG, C. & UPTON, C. 2006. Poxviruses: past, present and future. Virus Research, 117, 105-118. LI, L., YIN, Q., KUSS, P., MALIGA, Z., MILLÁN, J. L., WU, H. & MITCHISON, T. J. 2014. Hydrolysis of 2'3'cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nature chemical biology, 10, 10431048. LI, M., DAMANIA, B., ALVAREZ, X., OGRYZKO, V., OZATO, K. & JUNG, J. U. 2000. Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor. Molecular and cellular biology, 20, 8254-8263. LI, T., CHEN, J. & CRISTEA, I. M. 2013a. Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion. Cell host & microbe, 14, 591-599. LI, W., AVEY, D., FU, B., WU, J.-J., MA, S., LIU, X. & ZHU, F. 2016. Kaposi's Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny Viruses. Journal of virology, 90, 53295342. LI, X., SHU, C., YI, G., CHATON, C. T., SHELTON, C. L., DIAO, J., ZUO, X., KAO, C. C., HERR, A. B. & LI, P. 2013b. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity, 39, 1019-1031. LILES, W. C. & VAN VOORHIS, W. C. 1995. Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response. J Infect Dis, 172, 1573-80. LISNIĆ, B., LISNIĆ, V. J. & JONJIĆ, S. 2015. NK cell interplay with cytomegaloviruses. Current Opinion in Virology, 15, 9-18. LIU, S., CAI, X., WU, J., CONG, Q., CHEN, X., LI, T., DU, F., REN, J., WU, Y.-T., GRISHIN, N. V. & CHEN, Z. J. 2015. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science, 347, aaa2630. LIU, Y., GAO, L., XU, Z., LUO, D., ZHANG, Y., GAO, Y., LIU, C., QI, X., CUI, H., LI, K. & WANG, X. 2019a. Marek's Disease Virus RLORF4 Inhibits Type I Interferon Production by Antagonizing NFkappaB Activation. J Virol, 93. LIU, Z.-S., CAI, H., XUE, W., WANG, M., XIA, T., LI, W.-J., XING, J.-Q., ZHAO, M., HUANG, Y.-J., CHEN, S., WU, S.-M., WANG, X., LIU, X., PANG, X., ZHANG, Z.-Y., LI, T., DAI, J., DONG, F., XIA, Q., LI, A.-L., ZHOU, T., LIU, Z.-G., ZHANG, X.-M. & LI, T. 2019b. G3BP1 promotes DNA binding and activation of cGAS. Nature Immunology, 20, 18-28. LUO, W.-W., LI, S., LI, C., LIAN, H., YANG, Q., ZHONG, B. & SHU, H.-B. 2016. iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING. Nature Immunology, 17, 1057. LYSAKOVA-DEVINE, T., KEOGH, B., HARRINGTON, B., NAGPAL, K., HALLE, A., GOLENBOCK, D. T., MONIE, T. & BOWIE, A. G. 2010. Viral Inhibitory Peptide of TLR4, a Peptide Derived from Vaccinia Protein A46, Specifically Inhibits TLR4 by Directly Targeting MyD88 Adaptor-Like and TRIF-Related Adaptor Molecule. The Journal of Immunology, 185, 4261. MA, Z., JACOBS, S. R., WEST, J. A., STOPFORD, C., ZHANG, Z., DAVIS, Z., BARBER, G. N., GLAUNSINGER, B. A., DITTMER, D. P. & DAMANIA, B. 2015. Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses. Proceedings of the National Academy of Sciences of the United States of America, 112, E4306-E4315. MALONEY, D. G. 2012. Anti-CD20 antibody therapy for B-cell lymphomas. N Engl J Med, 366, 200816. MARGOLIS, S. R., WILSON, S. C. & VANCE, R. E. 2017. Evolutionary Origins of cGAS-STING Signaling. Trends in Immunology, 38, 733-743. MEADE, N., FUREY, C., LI, H., VERMA, R., CHAI, Q., ROLLINS, M. G., DIGIUSEPPE, S., NAGHAVI, M. H. & WALSH, D. 2018. Poxviruses Evade Cytosolic Sensing through Disruption of an mTORC1mTORC2 Regulatory Circuit. Cell, 174, 1143-1157.e17. 30

MEISINGER-HENSCHEL, C., SCHMIDT, M., LUKASSEN, S., LINKE, B., KRAUSE, L., KONIETZNY, S., GOESMANN, A., HOWLEY, P., CHAPLIN, P., SUTER, M. & HAUSMANN, J. 2007. Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J Gen Virol, 88, 3249-59. MORETTI, J., ROY, S., BOZEC, D., MARTINEZ, J., CHAPMAN, J. R., UEBERHEIDE, B., LAMMING, D. W., CHEN, Z. J., HORNG, T., YERETSSIAN, G., GREEN, D. R. & BLANDER, J. M. 2017. STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum. Cell, 171, 809-823.e13. MOTWANI, M., PESIRIDIS, S. & FITZGERALD, K. A. 2019. DNA sensing by the cGAS–STING pathway in health and disease. Nature Reviews Genetics, 20, 657-674. MUSTELIN, T., LOOD, C. & GILTIAY, N. V. 2019. Sources of Pathogenic Nucleic Acids in Systemic Lupus Erythematosus. Frontiers in immunology, 10, 1028-1028. OLIVEIRA, G. P., RODRIGUES, R. A. L., LIMA, M. T., DRUMOND, B. P. & ABRAHÃO, J. S. 2017. Poxvirus Host Range Genes and Virus-Host Spectrum: A Critical Review. Viruses, 9, 331. PATRA, J. K., DAS, G., FRACETO, L. F., CAMPOS, E. V. R., RODRIGUEZ-TORRES, M. D. P., ACOSTATORRES, L. S., DIAZ-TORRES, L. A., GRILLO, R., SWAMY, M. K., SHARMA, S., HABTEMARIAM, S. & SHIN, H.-S. 2018. Nano based drug delivery systems: recent developments and future prospects. Journal of Nanobiotechnology, 16, 71. RUBIO, D., XU, R. H., REMAKUS, S., KROUSE, T. E., TRUCKENMILLER, M. E., THAPA, R. J., BALACHANDRAN, S., ALCAMI, A., NORBURY, C. C. & SIGAL, L. J. 2013. Crosstalk between the type 1 interferon and nuclear factor kappa B pathways confers resistance to a lethal virus infection. Cell Host Microbe, 13, 701-10. SANTHAKUMAR, D., RUBBENSTROTH, D., MARTINEZ-SOBRIDO, L. & MUNIR, M. 2017. Avian Interferons and Their Antiviral Effectors. Frontiers in immunology, 8, 49-49. SCIUTO, M. R., COPPOLA, V., IANNOLO, G., DE MARIA, R. & HAAS, T. L. 2019. Two-Step CoImmunoprecipitation (TIP). Current Protocols in Molecular Biology, 125, e80. SEHRAWAT, S., KUMAR, D. & ROUSE, B. T. 2018. Herpesviruses: Harmonious Pathogens but Relevant Cofactors in Other Diseases? Frontiers in Cellular and Infection Microbiology, 8, 177. SEO, G. J., KIM, C., SHIN, W.-J., SKLAN, E. H., EOH, H. & JUNG, J. U. 2018. TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing. Nature Communications, 9, 613. SHANG, G., ZHANG, C., CHEN, Z. J., BAI, X.-C. & ZHANG, X. 2019. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP–AMP. Nature, 567, 389-393. SHISLER, J. L. 2014. Viral and Cellular FLICE-Inhibitory Proteins: a Comparison of Their Roles in Regulating Intrinsic Immune Responses. Journal of Virology, 88, 6539. SPECK, S. H. & GANEM, D. 2010. Viral latency and its regulation: lessons from the gammaherpesviruses. Cell host & microbe, 8, 100-115. SRIKANTH, S., WOO, J. S., WU, B., EL-SHERBINY, Y. M., LEUNG, J., CHUPRADIT, K., RICE, L., SEO, G. J., CALMETTES, G., RAMAKRISHNA, C., CANTIN, E., AN, D. S., SUN, R., WU, T.-T., JUNG, J. U., SAVIC, S. & GWACK, Y. 2019. The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum. Nature Immunology, 20, 152-162. STEMPEL, M., CHAN, B., JURANIĆ LISNIĆ, V., KRMPOTIĆ, A., HARTUNG, J., PALUDAN, S. R., FÜLLBRUNN, N., LEMMERMANN, N. A. & BRINKMANN, M. M. 2019. The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection. The EMBO journal, 38, e100983. SU, C., ZHAN, G. & ZHENG, C. 2016. Evasion of host antiviral innate immunity by HSV-1, an update. Virology journal, 13, 38-38. 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, 786-791. TADDEO, B., SCIORTINO, M. T., ZHANG, W. & ROIZMAN, B. 2007. Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for 31

accumulation of mRNA. Proceedings of the National Academy of Sciences of the United States of America, 104, 12163-12168. THÈVES, C., BIAGINI, P. & CRUBÉZY, E. 2014. The rediscovery of smallpox. Clinical Microbiology and Infection, 20, 210-218. TSUCHIDA, T., ZOU, J., SAITOH, T., KUMAR, H., ABE, T., MATSUURA, Y., KAWAI, T. & AKIRA, S. 2010. The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular doublestranded DNA. Immunity, 33, 765-76. UNTERHOLZNER, L., SUMNER, R. P., BARAN, M., REN, H., MANSUR, D. S., BOURKE, N. M., RANDOW, F., SMITH, G. L. & BOWIE, A. G. 2011. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. PLoS pathogens, 7, e1002247-e1002247. VERHELST, K., VERSTREPEN, L., CARPENTIER, I. & BEYAERT, R. 2013. IκB kinase ɛ (IKKɛ): A therapeutic target in inflammation and cancer. Biochemical Pharmacology, 85, 873-880. WANG, Q., LIU, X., CUI, Y., TANG, Y., CHEN, W., LI, S., YU, H., PAN, Y. & WANG, C. 2014. The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING. Immunity, 41, 919-33. WICKRAMASEKERA, N. T. & TRAKTMAN, P. 2010. Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity. Journal of Virology, 84, 6846. WU, J., SUN, L., CHEN, X., DU, F., SHI, H., CHEN, C. & CHEN, Z. J. 2013. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science, 339, 826-30. WU, J.-J., LI, W., SHAO, Y., AVEY, D., FU, B., GILLEN, J., HAND, T., MA, S., LIU, X., MILEY, W., KONRAD, A., NEIPEL, F., STÜRZL, M., WHITBY, D., LI, H. & ZHU, F. 2015. Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein. Cell host & microbe, 18, 333-344. YAN, N. 2017. Immune Diseases Associated with TREX1 and STING Dysfunction. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 37, 198-206. YAO, X. D., MATECIC, M. & ELIAS, P. 1997. Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4. Journal of virology, 71, 6842-6849. YE, J., LIU, E., YU, Z., PEI, X., CHEN, S., ZHANG, P., SHIN, M.-C., GONG, J., HE, H. & YANG, V. C. 2016. CPP-Assisted Intracellular Drug Delivery, What Is Next? International journal of molecular sciences, 17, 1892. YOU, H., ZHENG, S., HUANG, Z., LIN, Y., SHEN, Q. & ZHENG, C. 2019. Herpes Simplex Virus 1 Tegument Protein UL46 Inhibits TANK-Binding Kinase 1-Mediated Signaling. mBio, 10, e00919-19. ZHANG, C., SHANG, G., GUI, X., ZHANG, X., BAI, X.-C. & CHEN, Z. J. 2019. Structural basis of STING binding with and phosphorylation by TBK1. Nature, 567, 394-398. ZHANG, J., HU, M.-M., WANG, Y.-Y. & SHU, H.-B. 2012. TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination. The Journal of biological chemistry, 287, 28646-28655. ZHANG, X., SHI, H., WU, J., ZHANG, X., SUN, L., CHEN, C. & CHEN, Z. J. 2013. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Molecular cell, 51, 226-235. ZHAO, B., SHU, C., GAO, X., SANKARAN, B., DU, F., SHELTON, C. L., HERR, A. B., JI, J.-Y. & LI, P. 2016. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins. Proceedings of the National Academy of Sciences of the United States of America, 113, E3403-E3412.

CRediT author statement 32

The following details author contributions to this manuscript: Thomas Phelan: Writing - Original Draft. Mark Little: Writing - Review & Editing. Gareth Brady: Writing - Original Draft, Supervision.

Figure 1- Sensing of DNA viruses by the cGAS-STING Pathway. When a DNA virus enters a cell, it un-coats, releasing the DNA genome. (1) The cytosolic DNA sensor cGAS binds to viral DNA and becomes monoubiquitinated by TRIM56. G3BP1 also interacts with cGAS to form large cGAS complexes. cGAS catalyses the conversion of ATP and GTP to form 2’3 cGAMP, a cyclic second messenger, which binds into a groove in the ligand binding domain between STING dimers. Ligand binding induces a 180° rotation of the binding domain resulting in STING activation. E3 ligases, such as TRAF6 or TRIM32, polyubiquitinate components in complex with STING. (2) Activated STING disassociates from STIM1 and translocates from the endoplasmic reticulum to the ER-golgi-intermediate compartment where it oligomerizes and interacts with TBK1, enabling phosphorylation of IRF3. Both TBK1 and IKKε phosphorylate IRF3 and these kinases exist in complex with the scaffold proteins NAP1, TANK and SINTBAD. Phosphorylated, dimeric IRF3 migrates into the nucleus where it can activate the transcription of genes like type I interferons. (3) The exact mechanism leading to STING-mediated NF-κB activation remains unclear but may involve ubiquitin chains generated by specific E3 ligases, as is the case with TRAF6 in the TLR/IL-1R pathways. NEMO binds to ubiquitin chains triggering phosphorylation of IKKα and IKKβ. IKKβ phosphorylates the NF-κB inhibitory protein, IκB, leading to its proteasomal degradation. NF-κB then 33

migrates into the nucleus and activates transcription of target genes encoding proinflammatory cytokines. The forms of the individual proteins were adapted from Servier Medical Art (https://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

Figure 2- Inhibition of the cGAS-STING Pathway by Herpesvirus proteins. Herpesvirus proteins inhibit the cGAS-STING system at various points in the pathway to prevent the activation of IRF3/7 and NF-κB. The black arrow indicates IFI16 oligomers migrating out of the nucleus following detection of nuclear viral DNA. Subsequently, IFI16 interacts with STING to initiate downstream signalling. The oligomerization of IFI16 molecules is blocked by pp65 which prevents the interaction between IFI16 pyrin domains. The forms of the

individual

proteins

were

adapted

from

Servier

Medical

Art

(https://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

Figure 3- Inhibition of the cGAS-STING Pathway by Poxvirus proteins. Poxvirus proteins inhibit the cGAS-STING system at various points in the pathway to prevent the activation of IRF3/7 and NF-κB. The MCV proteins MC159/160/132/005, while not directly involved in inhibition of the cGAS-STING signalling, prevent activation of NF-κB downstream of STING and thus have been included. The forms of the individual proteins were adapted from Servier Medical Art (https://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Generic License.

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