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Science 339 (6121): 786-791

Copyright © 2013 by the American Association for the Advancement of Science

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

Lijun Sun1,2,*, Jiaxi Wu1,*, Fenghe Du1,2, Xiang Chen1,2, and Zhijian J. Chen1,2,{dagger}

1 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
2 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.


Figure 1
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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.

 

Figure 2
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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.

 

Figure 3
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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 ({Delta}ICP34.5) (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.

 

Figure 4
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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).

 

Figure 5
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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.

 

Figure 6
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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).

 


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