Messenger cGAMP Research Articles

Abstract A116: Lipid control of DNA-stimulated innate immunity

Abstract Type I interferon (IFN) plays essential roles in both spontaneous and iatrogenic tumor immunogenicity. Tumor-derived DNA is recognized by 2’,3’-cyclic GMP-AMP (cGAMP) synthase (cGAS) that is important in tumor immunogenicity. Upon DNA binding, cGAS produces the endogenous second messenger cGAMP that binds to and activates stimulator of IFN genes (STING), a signaling adaptor. cGAMP binding triggers STING trafficking from the endoplasmic reticulum (ER) to perinuclear compartments, with simultaneous activation of serine/threonine-protein kinase TBK1 that in turn phosphorylates the transcription factor IRF3, leading to upregulation of type I interferons. However, where and how TBK1 is activated by STING upon cGAMP stimulation is unclear. Our study focuses on the regulation of cGAMP-stimulated STING trafficking and activation by lipid messengers with essential roles in subcellular protein/membrane trafficking and signaling. Through in vitro signaling reconstitution, we identified a cellular lipid as an essential factor of STING signaling. We found both STING and TBK1 were lipid effectors. Lipid binding not only promotes STING trafficking but also stimulates TBK1 activation. These results reveal a new component of the STING-TBK1 complex that controls cytosolic DNA-stimulated innate immune signaling. Citation Format: Xiaojun Tan, Conggang Zhang, Zhijian J. Chen. Lipid control of DNA-stimulated innate immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A116.

ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

Cyclic GMP-AMP synthase (cGAS) senses double-strand (ds) DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the adaptor MITA/STING to initiate innate antiviral response. How cGAS activity is regulated remains enigmatic. Here, we identify ZCCHC3, a CCHC-type zinc-finger protein, as a positive regulator of cytosolic dsDNA- and DNA virus-triggered signaling. We show that ZCCHC3-deficiency inhibits dsDNA- and DNA virus-triggered induction of downstream effector genes, and that ZCCHC3-deficient mice are more susceptible to lethal herpes simplex virus type 1 or vaccinia virus infection. ZCCHC3 directly binds to dsDNA, enhances the binding of cGAS to dsDNA, and is important for cGAS activation following viral infection. Our results suggest that ZCCHC3 is a co-sensor for recognition of dsDNA by cGAS, which is important for efficient innate immune response to cytosolic dsDNA and DNA virus.

Manganese Increases the Sensitivity of the cGAS-STING Pathway for Double-Stranded DNA and Is Required for the Host Defense against DNA Viruses

Manganese (Mn) is essential for many physiological processes, but its functions in innate immunity remain undefined. Here, we found that Mn2+ was required for the host defense against DNA viruses by increasing the sensitivity of the DNA sensor cGAS and its downstream adaptor protein STING. Mn2+ was released from membrane-enclosed organelles upon viral infection and accumulated in the cytosol where it bound directly to cGAS. Mn2+ enhanced the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity, enabling cGAS to produce secondary messenger cGAMP inthe presence of low concentrations of dsDNA that would otherwise be non-stimulatory. Mn2+ alsoenhanced STING activity by augmenting cGAMP-STING binding affinity. Mn-deficient mice showed diminished cytokine production and were more vulnerable to DNA viruses, and Mn-deficient STING-deficient mice showed no increased susceptibility. These findings indicate that Mn is critically involved and required for the host defense against DNA viruses.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing

Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity.

TREX1 is a checkpoint for innate immune sensing of DNA damage that fosters cancer immune resistance.

Genomic instability is a hallmark of neoplastic transformation that leads to the accumulation of mutations, and generates a state of replicative stress in neoplastic cells associated with dysregulated DNA damage repair (DDR) responses. The importance of increasing mutations in driving cancer progression is well established, whereas relatively little attention has been devoted to the DNA displaced to the cytosol of cancer cells, a byproduct of genomic instability and of the ensuing DDR response. The presence of DNA in the cytosol promotes the activation of viral defense pathways in all cells, leading to activation of innate and adaptive immune responses. In fact, the improper accumulation of cytosolic DNA in normal cells is known to drive severe autoimmune pathology. Thus, cancer cells must evade cytoplasmic DNA detection pathways to avoid immune-mediated destruction. The main sensor for cytoplasmic DNA is the cyclic GMP-AMP synthase, cGAS. Upon activation by cytosolic DNA, cGAS catalyzes the formation of the second messenger cGAMP, which activates STING (stimulator of IFN genes), leading to the production of type I interferon (IFN-I). IFN-I is a critical effector of cell-mediated antiviral and antitumor immunity, and its production by cancer cells can be subverted by several mechanisms. However, the key upstream regulator of cytosolic DNA-mediated immune stimulation is the DNA exonuclease 3'-repair exonuclease 1 (TREX1). Here, we will discuss evidence in support of a role of TREX1 as an immune checkpoint that, when up-regulated, hinders the development of antitumor immune responses.

Palmitic acid dysregulates the Hippo–YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS–STING–IRF3 signaling mechanism

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

Intracellular DNA sensing pathway of cGAS-cGAMP is decreased in human newborns and young children

Newborns are highly susceptible to DNA virus infections, which may result from the characteristics of neonatal innate immune systems. Here we analyzed for the first time the development of innate immune sensing and signaling of intracellular DNA virus infection in human newborns and young children. Both mRNA and protein expression of cGAS, an intracellular DNA sensor, were shown to be significantly reduced in neonatal peripheral blood mononuclear cells (PBMCs). In addition, cGAS expression in neonatal PBMCs could be induced upon herpes simplex virus type 1 (HSV-1) or interferon-α (IFNα) stimulation. Furthermore, production of the second messenger cGAMP and activation of the transcriptional factor IRF3 was severely decreased in neonatal cord blood mononuclear cells (CBMCs) or PBMCs compared with adults. In contrast, the downstream signaling STING-TBK1-IRF3 appeared to be functional in neonatal PBMCs, as demonstrated by the fact that IRF3 phosphorylation and IFNβ production in these cells could be activated by cGAMP. Intriguingly, decreased expression of cGAS in neonatal cells can be rescued by DNA demethylation, with concomitant enhancement in IFNβ induction by HSV-1. Thus, cGAS restoration or STING stimulation by small molecules during infancy might improve the age-dependent susceptibility to DNA virus infection.

Inhibition of hepatitis B virus replication by activation of the cGAS-STING pathway.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) senses cytosolic DNA and catalyses synthesis of the second messenger cGAMP, which activates the downstream signalling adaptor protein STING, leading to the expression of type I interferons. Hepatitis B virus (HBV) is a small DNA virus, and the cGAS-STING pathway may inhibit HBV RNA synthesis and viral assembly in cell culture, but the exact roles of the cGAS pathway in the restriction of HBV replication in infection systems remain to be elucidated. In this study, replication of HBV was significantly inhibited both in cell culture and in vivo in a mouse model when the cGAS-STING pathway was activated by dsDNA or cGAMP. In contrast, the presence of enzymatically inactive cGAS mutant did not influence HBV replication. Moreover, knockdown of cGAS in human peripheral blood monocytes led to a higher level of intracellular HBV DNA. Collectively, our data indicate that the cGAS-STING pathway plays a role in the surveillance of HBV infection and may be exploited for development of novel anti-HBV strategies.

Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.

The recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor STING. The cGAS-STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. Thus, the cGAS pathway must be properly regulated. Here we review the recent advances in understanding of the cGAS-STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease.

Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer.

SUMMARYBrain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. Breast and lung cancer cells express protocadherin 7 (PCDH7) to favor the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells employ these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines IFNα and TNFα. As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, which support tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle for the applicability of this therapeutic strategy to treat established brain metastasis.

Uncovering the mechanism of E1A antagonism of the cGAS-STING pathway

Abstract The cGAS-STING pathway is critical for antiviral defense. Cytosolic DNA is detected by cGAS, which synthesizes the second messenger cGAMP that activates the signaling adaptor STING. Like the RNA sensing pathway, the cGAS-STING pathway converges on kinase TBK-1, which phosphorylates IRF-3, allowing it to translocate to the nucleus to induce production of type I interferons. Many viruses encode inhibitors of this pathway to avoid the ensuing antiviral state. Previously, we discovered that oncoproteins E7 and E1A encoded by human papillomavirus and adenovirus, respectively, specifically block the cGAS-STING pathway and that these proteins bind to STING. However, the mechanism underlying the antagonism of the cGAS-STING pathway is unknown. To dissect the mechanism for E1A, we used HEK 293 cells, which express E1A, to assess known events in the pathway: TBK-1 and IRF-3 phosphorylation, IRF-3 dimerization and IRF-3 nuclear translocation. Consistent with a specific block in the DNA-activated interferon response in HEK 293 cells, we found that IRF-3 dimerization and nuclear translocation were prevented in response to DNA, but not to RNA stimulation. Surprisingly, we found that early events were intact in response to DNA, including robust phosphorylation of IRF-3 on serine 386, which is required for dimerization. However, phosphorylation of serine 396, an event with unclear importance for IRF-3 activation, was impaired after DNA stimulation. This reveals that E1A blocks a potentially unappreciated late step in the pathway, preventing complete IRF-3 phosphorylation and subsequent dimerization. Ongoing work will address the temporal and spatial regulation of this novel step in STING-dependent IRF-3 activation and its antagonism by E1A.

How Low Cholesterol Is Good for Anti-viral Immunity

York etal. identify a role for decreased cholesterol biosynthesis in virally infected cells as a critical event in the induction of the anti-viral response. The mechanism involves enhanced signaling by STING in the ER membrane in response to the second messenger cGAMP, promoting increased transcription of type I interferons. The authors suggest that a "lipid code" is being sensed by STING to allow it to signal.

Viruses pack antiviral mediators

Viruses often hijack host proteins for their own use, turning host cells into virion-spewing machines. However, Bridgeman et al. and Gentili et al. now report a sneaky way that the host can fight back (see the Perspective by Schoggins). Host cells that expressed the enzyme cGAS, an innate immune receptor that senses cytoplasmic DNA, packaged the cGAS-generated second messenger cGAMP into virions. Virions could then transfer cGAMP to neighboring cells, triggering an antiviral gene program in these newly infected cells. Such transfer of an antiviral mediator may help to speed up the immune response to put the brakes on viral spread. A. Bridgeman, J. Maelfait, T. Davenne, T. Partridge, Y. Peng, A. Mayer, T. Dong, V. Kaever, P. Borrow, J. Rehwinkel, Viruses transfer the antiviral second messenger cGAMP between cells. Science 349 , 1228–1232 (2015). [Abstract][Full Text] M. Gentili, J. Kowal, M. Tkach, T. Satoh, X. Lahaye, C. Conrad, M. Boyron, B. Lombard, S. Durand, G. Kroemer, D. Loew, M. Dalod, C. Thery, N. Manel, Transmission of innate immune signaling by packaging of cGAMP in viral particles. Science 349 , 1232–1236 (2015).[Abstract][Full Text] J. W. Schoggins, Viruses carry antiviral cargo. Science 349 , 1166–1167 (2015). [Abstract][Full Text]

Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein.

Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS binding and DNA binding. Moreover, ORF52 homologs in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52 null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing.

Viruses transfer the antiviral second messenger cGAMP between cells.

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.

Transmission of innate immune signaling by packaging of cGAMP in viral particles.

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.

Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape.

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular ‘arms races.’ Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2’-5’-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.

Single amino acid change in STING leads to constitutive active signaling.

The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS) activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.

Molecular cloning and functional characterization of porcine cyclic GMP–AMP synthase

Cyclic GMP–AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation.