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Science 339 (6121): 826-830

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

Cyclic GMP-AMP Is an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA

Jiaxi Wu1,*, Lijun Sun1,2,*, Xiang Chen1, Fenghe Du1, Heping Shi3, Chuo Chen3, 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.
3 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.


Figure 1
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Fig. 1. DNA-dependent generation of a heat-resistant small molecule activates the STING pathway. (A) Illustration of an activity assay for cellular factors that activate the STING pathway. (B) Cytosolic extracts from mock or ISD-transfected L929-shSTING cells were incubated with PFO-permeabilized THP1 cells together with 35S-labeled IRF3. Dimerization of IRF3 was analyzed by native gel electrophoresis followed by autoradiography. (C) Similar to (B), except that in lanes 4 to 6, cytosolic extracts were heated at 95°C for 5 min to denature proteins and then the heat-resistant supernatant was incubated with PFO-permeabilized THP1 cells. (D) L929-shSTING cytosolic extracts were incubated with the indicated nucleic acids in the presence of ATP, and then the heat-resistant supernatant was assayed for its ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells. (E) THP1 cells stably expressing shRNA against GFP (control) or STING were permeabilized with PFO and then incubated with the heat-resistant supernatant from the reaction mixture containing DNA-supplemented L929 cytosolic extracts (lanes 2 and 5) or from DNA-transfected L929 cells (lanes 3 and 6). IRF3 activation was analyzed by native gel electrophoresis. (F) THP1 cells described in (E) were transfected with HT-DNA or poly(I:C) or infected with Sendai virus (SeV), followed by measurement of IRF3 dimerization. (G) Cytosolic extracts from the indicated cell lines were incubated with HT-DNA, and then heat-resistant supernatants were assayed for their ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells. Unless noted otherwise, all results in this and other figures were representative of at least two independent experiments.

 

Figure 2
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Fig. 2. Purification and identification of the heat-resistant STING activator. (A) Full scan nano-LC-MS spectra of active and inactive fractions from the C18 column. Arrows indicate an ion at +1 (675.11) and +2 (338.14) charge states present only in the active fraction. (B) Tandem mass (MS2) spectra after CID fragmentation of the ion with m/z = 338.14 (z = 2) from the MS1 scan shown in (A). Arrows indicate the m/z values of the expected fragmentation patterns of cyclic GMP-AMP (cGAMP, bottom). Asterisk indicates an ion (m/z = 506) that resulted from a neutral loss of a water molecule (18) from the ion with m/z = 524. (C) Fractions (B7 to B12) from the C18 column were analyzed for the presence of cGAMP by selective reaction monitoring of the expected ions and for their ability to stimulate IRF3 dimerization. (D) Comparison of the CID MS2 spectra of the purified STING activator and chemically synthesized cGAMP.

 

Figure 3
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Fig. 3. DNA transfection and DNA virus infection induce IFN-β through cGAMP. (A) Chemically synthesized cGAMP (100 nM) was delivered to digitonin-permeabilized L929 cells for the indicated times, then IFN-β RNA and secreted protein were measured by qRT-PCR (inset) and ELISA, respectively. Unless noted otherwise, the error bars in this and all other panels denote SEM (n = 3). (B) Similar to (A), except that different concentrations of cGAMP were delivered into L929 cells for 8 hours, followed by qRT-PCR analyses of IFN-β RNA. (C) Similar to (B), except that different concentrations of cGAMP and c-di-GMP were delivered into L929 cells, followed by ELISA assays for IFN-β. (D) L929 cells were infected with HSV-1{Delta}34.5 or VSV-{Delta}M51-GFP, transfected with HT-DNA, or mock-treated. An aliquot of the cell extracts was directly analyzed for IRF3 dimerization (top), whereas another aliquot was heated to denature proteins and the heat-resistant supernatant was assayed for its ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells (bottom). (E) The heat-resistant supernatant from (D) was fractionated by HPLC using a C18 column, and the presence of cGAMP in the fractions was measured by mass spectrometry using SRM. (F) L929 cells were transfected with HT-DNA (4 μg/ml) for the indicated time, then IFN-β RNA was measured by qRT-PCR and IRF3 dimerization was analyzed by native polyacrylamide gel electrophoresis (PAGE). Aliquots of the cell extracts were tested for the presence of cGAMP on the basis of its ability to induce IRF3 dimerization after delivery into Raw264.7 cells. (G) THP1 cells were infected with HSV-1{Delta}34.5 and vaccinia virus (VACV) for 6 hours, then the activation of endogenous IRF3 and generation of cGAMP activity were measured as described in (F).

 

Figure 4
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Fig. 4. cGAMP binds to STING and activates IRF3 in a STING-dependent manner. (A) Increasing concentrations of HT-DNA or cGAMP were delivered to indicated cells, and the induction of IFN-β was measured by qRT-PCR. Inset shows immunoblots of STING and β-tubulin in the cell lines. (B) Indicated cell lines were infected with HSV1{Delta}34.5 or permeabilized with digitonin and then incubated with cGAMP. Activation of endogenous IRF3 was analyzed by native gel electrophoresis (top). Aliquots of the cytosolic extracts were heated to denature proteins, and the supernatant was assayed for its ability to stimulate IRF3 in permeabilized Raw264.7 cells (bottom). (C) cGAMP, c-di-GMP, ISD, or poly(I:C) was delivered into L929 cells stably expressing a shRNA against GFP or STING for the indicated time, followed by analysis of IRF3 dimerization. (D) Recombinant STING protein was incubated with [32P]ATP or [32P]cGAMP in the presence or absence of the cold competitors as indicated. After UV cross-linking, the mixtures were resolved by SDS-PAGE followed by autoradiography.

 


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