Research Article

Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway

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Science  15 Feb 2013:
Vol. 339, Issue 6121, pp. 786-791
DOI: 10.1126/science.1232458

Abstract

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate–adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

DNA was known to stimulate immune responses long before it was shown to be a genetic material, but the mechanism by which DNA functions as an immune stimulant remains poorly understood (1). Although DNA can stimulate the production of type I interferons in dendritic cells through binding to Toll-like receptor 9 (TLR9) in the endosome, it is still unclear how DNA in the cytosol induces interferon. In particular, the sensor that detects cytosolic DNA in the interferon pathway remains elusive (2). Although several proteins—including DAI, RNA polymerase III, IFI16, DDX41, and several other DNA helicases—have been suggested to function as the potential DNA sensors that induce interferon, no consensus has emerged (3).

Purification and identification of cyclic GMP-AMP synthase (cGAS). We showed that delivery of DNA to mammalian cells or cytosolic extracts triggered the production of cyclic GMP-AMP (cGAMP), which bound to and activated STING, leading to the activation of the transcription factor IRF3 and induction of interferon-β (IFN-β) (4). To identify the cGAMP synthase (cGAS), we fractionated cytosolic extracts (S100) from the murine fibrosarcoma cell line L929, which contains the cGAMP-synthesizing activity. This activity was assayed by incubating the column fractions with adenosine triphosphate and guanosine triphosphate (ATP and GTP) in the presence of herring testis DNA (HT-DNA). After digestion of the DNA with Benzonase (Novagen) and heating at 95°C to denature proteins, the heat-resistant supernatants that contained cGAMP were incubated with perfringolysin O (PFO)–permeabilized Raw264.7 cells (transformed mouse macrophages). cGAMP-induced IRF3 dimerization in these cells was analyzed by native gel electrophoresis (4). Using this assay, we carried out three independent routes of purification, each consisting of four steps of chromatography but differing in the columns or the order of the columns used (fig. S1A). In particular, the third route included an affinity purification step using a biotinylated DNA oligo [a 45–base pair DNA known as immune stimulatory DNA (ISD)]. We estimated that we achieved a range of 8000- to 15,000-fold purification and 2 to 5% recovery of the activity from these routes of fractionation. However, in the last step of each of these purification routes, silver staining of the fractions did not reveal clear protein bands that copurified with the cGAS activity, which suggests that the abundance of the putative cGAS protein might be very low in L929 cytosolic extracts.

We developed a quantitative mass spectrometry strategy to identify a list of proteins that copurified with the cGAS activity at the last step of each purification route. We reasoned that the putative cGAS protein must copurify with its activity in all three purification routes, whereas most "contaminating" proteins would not. Thus, from the last step of each purification route, we chose fractions that contained most of the cGAS activity (peak fractions) and adjacent fractions that contained very weak or no activity (fig. S1B). The proteins in each fraction were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and identified by nano–liquid chromatography–mass spectrometry (nano-LC-MS). The data were analyzed by label-free quantification using the MaxQuant software (5); the proteins that copurified with the cGAS activity are shown in table S1 and illustrated in a Venn diagram (fig. S1C). Remarkably, although many proteins copurified with the cGAS activity in one or two purification routes, only three proteins copurified in all three routes. All three were putative uncharacterized proteins: E330016A19 (NCBI accession number NP_775562), Arf-GAP with dual PH domain–containing protein 2 (NP_742145), and signal recognition particle 9-kD protein (NP_036188). Among these, more than 24 unique peptides were identified in E330016A19, representing 41% coverage in this protein of 507 amino acids (fig. S2A).

Bioinformatic analysis drew our attention to E330016A19, which exhibited structural and sequence homology to the catalytic domain of human oligoadenylate synthase (OAS1) (Fig. 1A). In particular, E330016A19 contains a conserved Gly[Gly/Ser]x9–13[Glu/Asp]h[Glu/Asp]h motif, where x9–13 indicates 9 to 13 flanking residues consisting of any amino acid and h indicates a hydrophobic amino acid. This motif is found in the nucleotidyltransferase (NTase) family (6). Besides OAS1, this family includes adenylate cyclase, polyadenylate polymerase, and DNA polymerases. The C terminus of E330016A19 contained a Mab21 (male abnormal 21) domain, which was first identified in the Caenorhabditis elegans protein Mab21 (7). Sequence alignment revealed that the C-terminal NTase and Mab21 domains are highly conserved from zebrafish to human (fig. S2, B and C), whereas the N-terminal sequences are much less conserved (8). The human homolog of E330016A19, C6orf150 (also known as MB21D1), was recently identified as one of several positive hits in a screen for interferon-stimulated genes (ISGs) whose overexpression inhibited viral replication (9). For clarity, and on the basis of evidence presented below, we propose to name the mouse protein E330016A19 as m-cGAS and the human homolog C6orf150 as h-cGAS.

Fig. 1

Identification of a cGAMP synthase (cGAS). (A) Multiple sequence and structure alignment of putative NTase domain of mouse cGAS, human cGAS, and human OAS1 using the PROMALS3D program. Conserved active-site residues of the NTase superfamily are highlighted in black, identical amino acids in red, and conserved amino acids in yellow. Predicted secondary structure is indicated above the alignment as α helices (H) and β strands (E). Abbreviations for amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (B and C) Quantitative RT-PCR analyses of cGAS RNA levels in different murine (B) and human (C) cell lines. MEF(imt), immortalized MEF; Raw, Raw264.7; SDM, spleen-derived macrophage; BMDM, bone marrow–derived macrophage. Here and in all other qRT-PCR assays, error bars denote SEM (n = 3). (D) Immunoblotting of endogenous human proteins in HEK293T and THP1 cells with the indicated antibodies.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the expression of m-cGAS was low in immortalized mouse embryo fibroblasts (MEFs) but high in L929 cells, Raw264.7 cells, and bone marrow–derived macrophages (Fig. 1B). Similarly, the expression of h-cGAS RNA was very low in human embryonic kidney (HEK) 293T cells but high in the human monocytic cell line THP1 (Fig. 1C). Immunoblotting further confirmed that h-cGAS protein was expressed in THP1 cells but not HEK293T cells (Fig. 1D; no mouse cGAS antibody is available yet). Thus, the expression levels of m-cGAS and h-cGAS in different cell lines correlated with the ability of these cells to produce cGAMP and induce IFN-β in response to cytosolic DNA (4, 10).

Catalysis by cGAS triggers type I interferon production. Overexpression of m-cGAS in HEK293T, which lacks STING expression (Fig. 1D), did not induce IFN-β, whereas stable expression of STING in HEK293T cells rendered these cells highly competent in IFN-β induction by m-cGAS (Fig. 2A). Point mutations of the putative catalytic residues Gly198 and Ser199 to alanine abolished the ability of m-cGAS to induce IFN-β. These mutations, as well as mutations of the other putative catalytic residues Glu211 and Asp213 to alanine, also abrogated the ability of m-cGAS to induce IRF3 dimerization in HEK293T-STING cells (Fig. 2B).

Fig. 2

cGAS activates IRF3 and induces IFN-β. (A) Expression plasmids (100 and 500 ng) encoding Flag-tagged mouse cGAS (m-cGAS), its active-site mutants G198A and S199A (designated as GS>AA), and MAVS were transfected into HEK293T cells or the same cell line stably expressing STING (HEK293T-STING). IFN-β RNA was measured by qRT-PCR 24 hours after transfection. (B) Similar to (A), except that cell lysates were analyzed for IRF3 dimerization by native gel electrophoresis (top). Expression levels of the transfected genes were monitored by immunoblotting with antibody to Flag (bottom). h-cGAS, human cGAS; ED>AA, E211A and D213A in mouse cGAS. (C) Expression vectors for the indicated proteins were transfected into HEK293T-STING cells, followed by measurement of IFN-β by qRT-PCR. (D) Cell lysates shown in (C) were immunoblotted with antibodies to Flag and IRF3 after SDS-PAGE and native PAGE, respectively (top two panels). Aliquots of the cell extracts were assayed for the presence of cGAMP activity, which was measured by detecting IRF3 dimerization after delivery into permeabilized Raw264.7 cells (bottom). (E) Human and mouse cGAS were expressed in HEK293T cells and affinity-purified with antibody to Flag. The proteins were incubated with ATP and GTP in the presence or absence of HT-DNA, and the synthesis of cGAMP was assessed by its ability to induce IRF3 dimerization in Raw264.7 cells.

The magnitude of IFN-β induction by c-GAS was comparable to that induced by MAVS (an adaptor protein that functions downstream of the RNA sensor RIG-I) and was higher than that induced by other putative DNA sensors, including DAI, IFI16, and DDX41, by several orders of magnitude (Fig. 2C). To determine whether overexpression of cGAS and other putative DNA sensors led to the production of cGAMP in cells, we incubated supernatants from heat-treated cell extracts with PFO-permeabilized Raw264.7 cells, followed by measurement of IRF3 dimerization (Fig. 2D, bottom). Among all the proteins expressed in HEK293T-STING cells, only cGAS was capable of producing the cGAMP activity in the cells.

To test whether cGAS could synthesize cGAMP in vitro, we purified wild-type and mutant Flag-cGAS proteins from transfected HEK293T cells. Wild-type m-cGAS and h-cGAS, but not the catalytically inactive mutants of cGAS, were able to produce the cGAMP activity, which stimulated IRF3 dimerization in permeabilized Raw264.7 cells (fig. S3A). We found that the in vitro activities of both m-cGAS and h-cGAS were dependent on the presence of HT-DNA (Fig. 2E). To test whether DNA enhances IFN-β induction by cGAS in cells, we transfected different amounts of cGAS expression plasmid, with or without HT-DNA, into HEK293T-STING cells (fig. S3B). HT-DNA markedly enhanced IFN-β induction by low (10 and 50 ng) but not high (200 ng) doses of cGAS plasmid. It is possible that the transfected cGAS plasmid DNA activated the cGAS protein in the cells, resulting in IFN-β induction. In contrast to cGAS, IFI16 and DDX41 did not induce IFN-β even when HT-DNA was cotransfected.

cGAS is required for IFN-β induction by DNA transfection and DNA virus infection. We used two different pairs of small interfering RNA (siRNA) to knock down m-cGAS in L929 cells, and found that both siRNA oligos strongly inhibited IFN-β induction by HT-DNA; moreover, the degree of inhibition correlated with the efficiency of knocking down m-cGAS RNA (fig. S4A). We also established two L929 cell lines stably expressing short hairpin RNA (shRNA) sequences that targeted distinct regions of m-cGAS (fig. S4B). The ability of these cells to induce IFN-β in response to HT-DNA was severely compromised relative to another cell line expressing a control shRNA against green fluorescent protein (shGFP; Fig. 3A).

Fig. 3

cGAS is essential for IRF3 activation and IFN-β induction by DNA transfection and DNA virus infection. (A) L929 cell lines stably expressing shRNA targeting GFP (control) or two different regions of m-cGAS were transfected with HT-DNA for the indicated times, followed by measurement of IFN-β RNA by qRT-PCR. See fig. S4B for RNA interference (RNAi) efficiency. (B) L929 cells stably expressing shRNA against GFP, cGAS, or STING were transfected with pcDNA3 (vector) or the same vector driving the expression of the indicated proteins. IFN-β RNA was measured by qRT-PCR 24 hours after transfection; see fig. S4C for RNAi efficiency. (C) cGAMP (100 nM) was delivered to digitonin-permeabilized L929 cells stably expressing shRNA against GFP, cGAS, or STING. IFN-β RNA was measured by qRT-PCR at the indicated times after cGAMP delivery. (D and E) L929 cells stably expressing shRNA against GFP or cGAS were infected with HSV1 (ΔICP34.5) (D) or Sendai virus (SeV) (E) for the indicated times, followed by measurement of IRF3 dimerization. (F) L929 cells stably expressing shRNA against GFP or cGAS were transfected with HT-DNA or infected with HSV1 for 6 hours, followed by measurement of IRF3 dimerization (top). Extracts from these cells were used to prepare heat-resistant supernatants, which were delivered to permeabilized Raw264.7 cells to stimulate IRF3 dimerization (bottom). (G) The heat-resistant supernatants in (F) were fractionated by high-performance liquid chromatography using a C18 column; the abundance of cGAMP was quantitated by mass spectrometry using SRM.

Expression of cGAS in the L929–sh-cGAS cells restored IFN-β induction (Fig. 3B). Expression of STING or MAVS in these cells (Fig. 3B) or delivery of cGAMP to these cells (Fig. 3C) also induced IFN-β. In contrast, expression of cGAS or delivery of cGAMP failed to induce IFN-β in L929-shSTING cells, whereas expression of STING or MAVS restored IFN-β induction in these cells (Fig. 3, B and C). Quantitative RT-PCR analyses confirmed the specificity and efficiency of knocking down cGAS and STING in the L929 cell lines stably expressing the corresponding shRNAs (fig. S4C). These results indicate that cGAS functions upstream of STING and is required for IFN-β induction by cytosolic DNA.

Herpes simplex virus 1 (HSV-1) is a DNA virus known to induce interferons through the activation of STING and IRF3 (3). L929 cells expressing shRNA against m-cGAS, but not against GFP, were severely compromised in IRF3 dimerization in response to HSV-1 infection (Fig. 3D). In contrast, knockdown of cGAS did not affect IRF3 activation by Sendai virus, an RNA virus (Fig. 3E). To determine whether cGAS is required for the generation of cGAMP in cells, we transfected HT-DNA into L929-shGFP and L929–sh-cGAS cells or infected these cells with HSV-1, then prepared heat-resistant fractions that contained cGAMP, which was subsequently delivered to permeabilized Raw264.7 cells to measure IRF3 activation. Knockdown of cGAS largely abolished the cGAMP activity generated by DNA transfection or HSV-1 infection (Fig. 3F, bottom). Quantitative mass spectrometry using selective reaction monitoring (SRM) showed that the abundance of cGAMP induced by DNA transfection or HSV-1 infection was markedly reduced in L929 cells depleted of cGAS (Fig. 3G). Taken together, these results demonstrate that cGAS is essential for producing cGAMP and activating IRF3 in response to DNA transfection or HSV-1 infection.

To determine whether cGAS is important in the DNA sensing pathway in human cells, we established a THP1 cell line stably expressing a shRNA targeting h-cGAS (fig. S4D). The knockdown of h-cGAS strongly inhibited IFN-β induction by HT-DNA transfection or infection by vaccinia virus, another DNA virus, but not by Sendai virus (fig. S4D). The knockdown of h-cGAS also inhibited IRF3 dimerization induced by HSV-1 infection in THP1 cells (fig. S4E). This result was further confirmed in another THP1 cell line expressing a shRNA targeting a different region of h-cGAS (fig. S4F). The strong and specific effects of multiple cGAS shRNA sequences in inhibiting DNA-induced IRF3 activation and IFN-β induction in both mouse and human cell lines demonstrate a key role of cGAS in the STING-dependent DNA sensing pathway.

Recombinant cGAS protein catalyzes cGAMP synthesis from ATP and GTP in a DNA-dependent manner. To test whether cGAS is sufficient to catalyze cGAMP synthesis, we expressed Flag-tagged h-cGAS in HEK293T cells and purified it to apparent homogeneity (Fig. 4A). In the presence of HT-DNA, purified c-GAS protein catalyzed the production of cGAMP activity, which stimulated IRF3 dimerization in permeabilized Raw264.7 cells (Fig. 4B). Deoxyribonuclease I (DNase I) treatment abolished this activity. The cGAS activity was also stimulated by other DNAs, including poly(deoxyadenosine-deoxythymidine), poly(deoxyguanosine-deoxycytidine), and ISD, but not by the RNA poly(inosine-cytidine). The synthesis of cGAMP by cGAS required both ATP and GTP but did not require cytidine triphosphate (CTP) or uridine triphosphate (UTP) (Fig. 4C). These results indicate that the cyclase activity of purified cGAS protein was stimulated by DNA but not by RNA.

Fig. 4

DNA-dependent synthesis of cGAMP by purified cGAS. (A) Silver staining of Flag–h-cGAS expressed and purified from HEK293T cells. (B) Purified Flag–h-cGAS as shown in (A) was incubated with ATP and GTP in the presence of different forms of nucleic acids as indicated. Generation of cGAMP was assessed by its ability to induce IRF3 dimerization in Raw264.7 cells. (C) Similar to (B), except that reactions contained HT-DNA and different combinations of nucleotide triphosphates as indicated. (D) Similar to (B), except that wild-type and mutant cGAS proteins were expressed and purified from E. coli and assayed for their activities at the indicated concentrations. (E) Purified m-cGAS from E. coli was incubated with ATP, GTP, and DNA for 0 or 60 min, and the production of cGAMP was analyzed by IRF3 dimerization assay (top) and mass spectrometry using SRM (bottom).

We also expressed m-cGAS in Escherichia coli as a SUMO (small ubiquitin-related modifier protein) fusion protein. After purification, SUMO–m-cGAS generated the cGAMP activity in a DNA-dependent manner (fig. S5, A and C). However, after the SUMO tag was removed by a SUMO protease, the m-cGAS protein catalyzed cGAMP synthesis in a DNA-independent manner (fig. S5, B and C). The reason for this loss of DNA dependency is unclear but could be due to some conformational changes after SUMO removal. Titration experiments showed that recombinant cGAS protein at less than 1 nM led to detectable IRF3 dimerization, whereas the catalytically inactive mutant of cGAS failed to activate IRF3 even at high concentrations (Fig. 4D). To formally prove that cGAS catalyzes the synthesis of cGAMP, we analyzed the reaction products by nano-LC-MS using SRM. cGAMP was detected in a 60-min reaction containing purified cGAS, ATP, and GTP (Fig. 4E). The identity of cGAMP was further confirmed by ion fragmentation using collision-induced dissociation (CID). The fragmentation pattern of cGAMP synthesized by purified cGAS revealed product ions whose mass/charge ratio (m/z) values matched those of chemically synthesized cGAMP (fig. S5D). Collectively, these results demonstrate that purified cGAS catalyzes the synthesis of cGAMP from ATP and GTP.

cGAS binds to DNA. The stimulation of cGAS activity by DNA suggests that c-GAS is a DNA sensor (Fig. 4B). Indeed, both GST (glutathione S-transferase) fused to the N terminus of m-CGAS (GST–m-cGAS) and GST–h-cGAS, but not GST–RIG-I N terminus [RIG-I(N)], were precipitated by biotinylated ISD (Fig. 5A). In contrast, biotinylated RNA did not bind cGAS (Fig. 5B). Deletion analyses showed that the h-cGAS N-terminal fragment containing residues 1 to 212, but not the C-terminal fragment (residues 213 to 522), bound to ISD (Fig. 5C). A longer C-terminal fragment containing residues 161 to 522 did bind to ISD, which suggests that the sequence 161 to 212 may be important for DNA binding. However, deletion of residues 161 to 212 from h-cGAS did not impair ISD binding, which suggests that cGAS contains another DNA binding domain at the N terminus. Indeed, the N-terminal fragment containing residues 1 to 160 also bound ISD (Fig. 5C). Thus, cGAS may contain two separate DNA binding domains at the N terminus. Our attempts to express the cGAS fragment containing residues 161 to 212 in E. coli or HEK293T cells have not been successful, so at present we do not know whether this sequence alone is sufficient to bind DNA. Nonetheless, it is clear that the N terminus of h-cGAS containing residues 1 to 212 is both necessary and sufficient to bind DNA.

Fig. 5

cGAS is a DNA binding protein. (A) The indicated GST fusion proteins were expressed and purified from E. coli and then incubated with streptavidin beads in the presence of ISD or biotin-ISD. Bound proteins were eluted with SDS sample buffer and detected by immunoblotting with a GST antibody. (B) Flag–h-cGAS was expressed and purified from HEK293T cells and then incubated with streptavidin beads as described in (A), except that a Flag antibody was used in immunoblotting and a biotin-RNA was also tested for binding to cGAS. (C) Flag-tagged full-length or truncated human cGAS proteins were expressed in HEK293T cells and affinity-purified. Their ability to bind biotin-ISD was assayed as described in (B). Right panel: Expression plasmids encoding full-length and deletion mutants of h-cGAS were transfected into HEK293T-STING cells, and IFN-β RNA was then measured by qRT-PCR.

Different deletion mutants of h-cGAS were overexpressed in HEK293T-STING cells to determine their ability to activate IRF3 and induce IFN-β and the cytokine tumor necrosis factor–α (TNF-α) (Fig. 5C and fig. S6A). The protein fragment containing residues 1 to 382, which lacks the C-terminal 140 residues including much of the Mab21 domain, failed to induce IFN-β (Fig. 5C, right) or TNF-α or to activate IRF3 (fig. S6A), which suggests that an intact Mab21 domain is important for cGAS function. As expected, deletion of the N-terminal 212 residues (residues 213 to 522), which include part of the NTase domain, abolished the cGAS activity (Fig. 5C and fig. S6A). An internal deletion of just four amino acids (Lys171, Leu172, Lys173, and Leu174) within the first helix of the NTase fold preceding the catalytic residues also destroyed the cGAS activity (fig. S6A).

Deletion of the N-terminal 160 residues did not affect IRF3 activation or cytokine induction by cGAS (Fig. 5C and fig. S6A). In vitro assay showed that this protein fragment (residues 161 to 522) still activated the IRF3 pathway in a DNA-dependent manner (fig. S6B). Thus, the N-terminal 160 amino acids of h-cGAS, whose primary sequence is not highly conserved evolutionarily, appear to be largely dispensable for DNA binding and catalysis by cGAS. In contrast, the NTase and Mab21 domains are important for cGAS activity.

cGAS is predominantly localized in the cytosol. To determine whether cGAS is a cytosolic DNA sensor, we prepared cytosolic and nuclear extracts from THP1 cells and analyzed the localization of endogenous h-cGAS by immunoblotting. h-cGAS was detected in the cytosolic extracts but was barely detectable in the nuclear extracts (Fig. 6A). The THP1 extracts were further subjected to differential centrifugation to separate subcellular organelles from one another and from the cytosol (Fig. 6B). Similar amounts of h-cGAS were detected in S100 and in the pellet after 100,000g centrifugation, which suggests that this protein is soluble in the cytoplasm but that a substantial fraction of the protein is associated with light vesicles or organelles. The cGAS protein was not detected in the pellet after 5000g centrifugation, which contained mitochondria and endoplasmic reticulum (ER) as evidenced by the presence of VDAC and STING, respectively. cGAS was also not detectable in the pellet after 20,000g centrifugation, which contained predominantly ER and heavy vesicles (Fig. 6B).

Fig. 6

cGAS binds to DNA in the cytoplasm. (A) Nuclear and cytoplasmic fractions were prepared from THP-1 cells and analyzed by immunoblotting with the indicated antibodies. (B) THP-1 cells were homogenized in hypotonic buffer and subjected to differential centrifugation. Pellets at different speeds of centrifugation (e.g., P100: pellets after 100,000g) and S100 were immunoblotted with the indicated antibodies. (C) L929 cells stably expressing Flag-cGAS (green) were transfected with Cy3-ISD (red). At different time points after transfection, cells were fixed, stained with antibody to Flag or with 4′,6-diamidino-2-phenylindole (DAPI), and imaged by confocal fluorescence microscopy. The insets in the merged images are magnifications of the small boxed areas. These images are representative of at least 10 cells at each time point (representing >50% of the cells under examination).

We also examined the localization of cGAS by confocal immunofluorescence microscopy of L929 cells stably expressing Flag–m-cGAS (Fig. 6C). The cGAS protein was distributed throughout the cytoplasm but could also be observed in the nuclear or perinuclear region. After the cells were transfected with Cyanine 3 (Cy3)–labeled ISD for 2 or 4 hours, punctate forms of cGAS were observed, and they overlapped with the DNA fluorescence. Such colocalization and apparent aggregation of cGAS and Cy3-ISD was observed in more than 50% of the cells under observation. These results, together with the biochemical evidence of direct binding of cGAS with DNA, suggest that cGAS binds to DNA in the cytoplasm.

Discussion. We have developed a strategy that combines quantitative mass spectrometry with conventional protein purification to identify biologically active proteins partially purified from crude cell extracts. This strategy may be generally applicable to proteins that are difficult to purify to homogeneity because of very low abundance, labile activity, or scarce starting materials. As a proof of principle, we used this strategy to identify the mouse protein E330016A19 as the enzyme that synthesizes cGAMP. This discovery led to the identification of a large family of cGAS that is conserved from fish to human, formally demonstrating that vertebrate animals contain evolutionarily conserved enzymes that synthesize cyclic dinucleotides, which were previously found only in bacteria, archaea, and protozoa (1113). Vibrio cholerae can synthesize cGAMP through its cyclase DncV (VC0179), which contains an NTase domain but has no obvious primary sequence homology to the mammalian cGAS (12).

Our results not only demonstrate that cGAS is a cytosolic DNA sensor that triggers the type I interferon pathway, but also reveal a mechanism of immune signaling in which cGAS generates the second messenger cGAMP, which binds to and activates STING (4), thereby triggering type I interferon production. It remains to be determined whether STING evolved first to detect cyclic dinucleotides from bacteria, or to detect endogenous cGAMP in the host as a mechanism of responding to cytosolic DNA. Although STING can directly detect certain cyclic dinucleotides produced by some bacteria, the deployment of cGAS as a cytosolic DNA sensor greatly expands the repertoire of microorganisms detected by the host immune system. In principle, all microorganisms that can carry DNA into the host cytoplasm—such as DNA viruses, bacteria, parasites (e.g., malaria), and retroviruses (e.g., HIV)—could potentially trigger the cGAS-STING pathway (14, 15). The enzymatic synthesis of cGAMP by cGAS provides a mechanism of signal amplification for a robust and sensitive immune response. However, the detection of self DNA in the host cytoplasm by cGAS could also lead to autoimmune diseases, such as systemic lupus erythematosus, Sjögren's syndrome, and Aicardi-Goutières syndrome (1618).

Several other DNA sensors, such as DAI, IFI16, and DDX41, have been reported to induce type I interferons (1921). Overexpression of DAI, IFI16, or DDX41 did not lead to the production of cGAMP. We also found that knockdown of DDX41 and p204 (a mouse homolog of IFI16) by siRNA did not inhibit the generation of cGAMP activity in HT-DNA–transfected L929 cells (fig. S7). Nonetheless, it is possible that distinct DNA sensors exist in different cell types. Unlike other putative DNA sensors and most pattern recognition receptors (e.g., TLRs), cGAS is a cyclase that is likely more amenable to inhibition by small-molecule compounds. These inhibitors may be developed into therapeutic agents for the treatment of human autoimmune diseases.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1232458/DC1

Materials and Methods

Figs. S1 to S7

Table S1

References (22, 23)

References and Notes

  1. Acknowledgments: We thank W. Li for helpful discussion on bioinformatics analyses. The GenBank accession numbers for human and mouse cGAS sequences are KC294566 and KC294567, respectively. Supported by NIH grant AI-093967. Z.J.C. is an investigator of Howard Hughes Medical Institute.
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