Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.     

KSHV strategies for host dsDNA sensing machinery

The innate immune system utilizes pattern recognition receptors cyclic GMP-AMP synthase(cGAS)to sense cytosolic double-stranded(ds) DNA and initiate type 1 interferon signaling and autophagy pathway, which collaborate to limit pathogen infections as well as alarm the adaptive immune response. The genomes of herpesviruses are large dsDNA, which represent a major class of pathogen signatures recognized by cellular DNA sensor cGAS. However, to successfully establish the persistent infection, herpesviruses have evolved their viral genes to modulate different aspects of host immune signaling. This review summarizes the evasion strategies of host cGAS DNA sensing pathway by Kaposi's Sarcoma-associated Herpesvirus(KSHV) and their contributions to KSHV life cycles.     

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.     

Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.     

Regulation of cGAS-STING signalling in cancer: Approach for combination therapy

Innate immunity plays an important role not only during infection but also homeostatic role during stress conditions. Activation of the immune system including innate immune response plays a critical role in the initiation and progression of tumorigenesis. The innate immune sensor recognizes pathogen-associated molecular patterns (PAMPs) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (cGAS-STING) and induces type-1 immune response during viral and bacterial infection. cGAS-STING is regulated differently in conditions like cellular senescence and DNA damage in normal and tumor cells and is implicated in the progression of tumors from different origins. cGAS binds to cytoplasmic dsDNA and synthesize cyclic GMP-AMP (2’3’-cGAMP), which selectively activates STING and downstream IFN and NF-κB activation. We here reviewed the cGAS-STING signalling pathway and its cross-talk with other pathways to modulate tumorigenesis. Further, the review also focused on emerging studies that targeted the cGAS-STING pathway for developing targeted therapeutics and combinatorial regimens for cancer of different origins.     

USP15 promotes cGAS activation through deubiquitylation and liquid condensation

Double-stranded DNA (dsDNA) is recognized as a danger signal by cyclic GMP-AMP synthase (cGAS), which triggers innate immune responses. cGAS activity must be properly regulated to maintain immune homeostasis. However, the mechanism by which cGAS activation is controlled remains to be better understood. In this study, we identified USP15 as a cGAS-interacting partner. USP15 promoted DNA-induced cGAS activation and downstream innate immune responses through a positive feedback mechanism. Specifically, USP15 deubiquitylated cGAS and promoted its activation. In the absence of DNA, USP15 drove cGAS dimerization and liquid condensation through the USP15 intrinsic disordered region (IDR), which prepared cGAS for a rapid response to DNA. Upon DNA stimulation, USP15 was induced to express and boost cGAS activation, functioning as an efficient amplifier in innate immune signal transduction. In summary, the positive role played by USP15-mediated cGAS activation may be a novel regulatory mechanism in the fine-tuning of innate immunity.     

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch

Kaposi’s sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi’s sarcoma (KS). When KSHV infects host’s cells, one of the virus’s proteins, latency-associated nuclear antigen 1 (LANA), binds to the host’s nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA–nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.     

Structural basis for nucleosome-mediated inhibition of cGAS activity

Activation of cyclic GMP-AMP synthase (cGAS) through sensing cytosolic double stranded DNA (dsDNA) plays a pivotal role in innate immunity against exogenous infection as well as cellular regulation under stress. Aberrant activation of cGAS induced by self-DNA is related to autoimmune diseases. cGAS accumulates at chromosomes during mitosis or spontaneously in the nucleus. Binding of cGAS to the nucleosome competitively attenuates the dsDNA-mediated cGAS activation, but the molecular mechanism of the attenuation is still poorly understood. Here, we report two cryo-electron microscopy structures of cGAS–nucleosome complexes. The structures reveal that cGAS interacts with the nucleosome as a monomer, forming 1:1 and 2:2 complexes, respectively. cGAS contacts the nucleosomal acidic patch formed by the H2A–H2B heterodimer through the dsDNA-binding site B in both complexes, and could interact with the DNA from the other symmetrically placed nucleosome via the dsDNA-binding site C in the 2:2 complex. The bound nucleosome inhibits the activation of cGAS through blocking the interaction of cGAS with ligand dsDNA and disrupting cGAS dimerization. R236A or R255A mutation of cGAS impairs the binding between cGAS and the nucleosome, and largely relieves the nucleosome-mediated inhibition of cGAS activity. Our study provides structural insights into the inhibition of cGAS activity by the nucleosome, and advances the understanding of the mechanism by which hosts avoid the autoimmune attack caused by cGAS.Subject terms: Cryoelectron microscopy, Pattern recognition receptors     

Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS

The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.     

Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.     

Nuclear cGAS: sequestration and beyond

The cyclic GMP-AMP(cGAMP)synthase(cGAS)has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway.In the past several years,a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes,DNA replication forks,the double-stranded breaks,and centromeres,suggesting that cGAS may have other functions in addition to its role in DNA sensing.However,while the innate immune function of cGAS is well established,the non-canonical nuclear function of cGAS remains poorly understood.Here,we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function,beyond its well-established role in dsDNA sensing and innate immune response.     

cGAS guards against chromosome endto-end fusions during mitosis and facilitates replicative senescence

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.     

NBS1-CtIP–mediated DNA end resection suppresses cGAS binding to micronuclei

Cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor—in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor—functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head–associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1’s interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.     

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.