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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 A IFNβ RNA (fold) Plasmid: B IB: IRF3 IB: Flag cGAS WT MAVS Vector m-cGAS WT m-cGAS GS>AA m-cGAS ED>AA Lane: 1 2 3 4 5 HEK293T-STING cGAS WT h-cGAS WT 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). cGAS GS>AA HEK293T HEK293T-STING (IRF3)2 IRF3 IB:Flag MAVS endogenous IRF3 cGAMP activity assay Vector DAI C IFNβ RNA (fold) - Plasmid: D E IFI16 DDX41 cGAS MAVS Lane: 1 2 3 4 5 6 MW(kDa) 100 75 50 (IRF3)2 IRF3 (IRF3)2 IRF3 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. 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 20 200 2000 20 200 2000 20 200 2000 20 200 2000 20 200 2000 DAI IFI16 DDX41 cGAS MAVS IB: IRF3 RESEARCH ARTICLES Control h-cGAS m-cGAS DNA: - + - + - + Lane: 1 2 3 4 5 6 ng (IRF3)2 IRF3 Fig. 2. cGAS activates IRF3 and induces IFN-b. (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-b 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-b by qRT-PCR. (D) Celllysatesshownin(C)wereimmunoblottedwithantibodiesto 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 wereincubatedwithATPandGTPinthepresenceorabsenceofHT-DNA,andthe synthesis of cGAMP was assessed by its ability to induce IRF3 dimerization in Raw264.7 cells. www.sciencemag.org SCIENCE VOL 339 15 FEBRUARY 2013 787 on February 14, 2013 www.sciencemag.org Downloaded from
RESEARCH ARTICLES 788 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-cGASproteinwasexpressedinTHP1cellsbut 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-b 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-b, whereas stable expression of STING in HEK293T cells rendered these cells highly competent in IFN-b induction by m-cGAS (Fig. 2A). Point mutations of the putative catalytic residues Gly 198 and Ser 199 to alanine abolished the ability of m-cGAS to induce IFN-b. These mutations, as well as mutations of the other putative catalytic residues Glu 211 and Asp 213 to alanine, also abrogated the ability of m-cGAS to induce IRF3 dimerization in HEK293T-STING cells (Fig. 2B). The magnitude of IFN-b 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, includ- 80 60 40 20 IFNβ RNA (fold) 100 0 shGFP sh-cGAS-a sh-cGAS-b 0 1 2 3 4 5 6 7 8 9 Hours post DNA transfection ing 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 FlagcGAS 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-b 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-b 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-b in- A B C D E shGFP sh-cGAS HSV1: 0 2 4 6 9 0 2 4 6 9 hours IB: IRF3 IB: IRF3 shGFP sh-cGAS SeV: 0 4 8 0 4 8 hours (IRF3)2 (IRF3)2 IFNβ RNA (fold) F G shGFP sh-cGAS endogenous IRF3 cGAMP activity assay 50 40 30 20 15 10 5 Mock DNA shGFP sh-cGAS shSTING Fig. 3. cGAS is essential for IRF3 activation and IFN-b 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-b 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-b RNA was measured by qRT-PCR 24 hours after transfection; see fig. S4C for RNAi efficiency. (C)cGAMP(100nM) was delivered to digitonin-permeabilized L929 cells stably expressing shRNA against GFP, cGAS, or STING. IFN-b RNA was measured by qRT-PCR at the IRF3 IRF3 0 Plasmid: - vector cGAS STING MAVS HSV1 Mock DNA HSV1 (IRF3)2 IRF3 (IRF3)2 IRF3 Relative cGAMP Abundance duction. In contrast to cGAS, IFI16 and DDX41 did not induce IFN-b even when HT-DNA was cotransfected. cGAS is required for IFN-b 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-b 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-b in response to HT-DNA was severely compromised relative to another cell line expressing a control shRNA against green fluorescent protein (shGFP; Fig. 3A). Expression of cGAS in the L929–sh-cGAS cells restored IFN-b 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-b. In contrast, expression of cGAS or delivery of cGAMP failed to induce IFN-b in L929-shSTING cells, whereas expression of STING or MAVS restored IFN-b 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 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 15 FEBRUARY 2013 VOL 339 SCIENCE www.sciencemag.org IFNβ RNA (fold) shGFP sh-cGAS shSTING cGAMP: 0hr 2hr 4hr Mock DNA HSV-1 Mock DNA HSV-1 shGFP sh-cGAS 0 10 20 30 40 50 Retention time (min) indicated times after cGAMP delivery. (D and E) L929 cells stably expressing shRNA against GFP or cGAS were infected with HSV1 (DICP34.5) (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 heatresistant 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. 1800 1500 1200 200 150 100 50 0 on February 14, 2013 www.sciencemag.org Downloaded from
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