Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway

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.

Figure 1. Identification of a cGAMP synthase (cGAS)

(A) Multiple sequence and structure alignment of putative nucleotidyltransferase (NTase) domain of mouse cGAS, human cGAS and human OAS1 using the PROMALS3D program. Conserved active site residues of 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 alpha helices (H) and beta strands (E). (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. The error bars in this and all other q-RT-PCR assays represent standard errors of the mean (n=3). (D) Immunoblotting of endogenous human proteins in HEK293T and THP1 cells with the indicated antibodies.

Figure 2. cGAS activates IRF3 and induces IFNβ

(A) Expression plasmids (100 and 500 ng) encoding Flag-tagged mouse cGAS (m-cGAS), its active site mutants G198A/S199A (designated as GS>AA), and MAVS were transfected into HEK293T cells or the same cell line stably expressing STING (HEK293T-STING). 24 hr after transfection, IFNβ RNA was measured by q-RT-PCR. (B) Similar to (A), except that cell lysates were analyzed for IRF3 dimerization by native gel electrophoresis (top). The expression levels of the transfected genes were monitored by immunoblotting with a Flag antibody (bottom). h-cGAS: human cGAS; ED>AA: E211A/D213A in mouse cGAS. (C) Expression vectors for the indicated proteins were transfected into HEK293T-STING cells, followed by measurement of IFNβ by q-RT-PCR. (D) Cell lysates shown in (C) were immunoblotted with antibodies against 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 using a Flag antibody. 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.

Figure 3. cGAS is essential for IRF3 activation and IFNβ 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 indicated times, followed by measurement of IFNβ RNA by q-RT-PCR. See fig. S4B for 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 indicated proteins. 24 hr after transfection, IFNβ RNA was measured by q-RT-PCR. see fig. S4C for RNAi efficiency. (C) cGAMP (100 nM) was delivered to digitoninpermeabilized L929/shRNA cells as indicated. IFNβ RNA was measured by q-RT-PCR at indicated times following cGAMP delivery. (D and E) L929-shRNA cells as indicated were infected with HSV1 (ΔICP34.5) or Sendai virus (SeV) for indicated times followed by measurement of IRF3 dimerization. (F) L929/shRNA cells were transfected with HT-DNA or infected with HSV1 for 6 hr, 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 HPLC using a C18 column and the abundance of cGAMP was quantitated by mass spectrometry using SRM.

Figure 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 NTP as indicated. (D) Similar to (B), except that WT and mutant cGAS proteins were expressed and purified from E. coli and assayed for their activities at 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).

Figure 5. cGAS is a DNA binding protein

(A) 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 followed by measurement of IFNβ RNA by q-RT-PCR.

Figure 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 100,000 × g) 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 tranfection, cells were fixed, stained with the Flag antibody or DAPI and imaged by confocal fluorescence microscopy. Inset: magnification of the area outlined in the merged images. These images are representative of at least 10 cells at each time point (representing > 50% of the cells under examination).