Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity

  • An Erratum to this article was published on 22 March 2016

Abstract

Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

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Figure 1: Mice deficient in CCP5 or CCP6 are susceptible to infection with DNA viruses.
Figure 2: CCP5 and CCP6 are required for the activation of IRF3 and induction of Ifnb.
Figure 3: cGAS is a substrate for CCP5 and CCP6.
Figure 4: cGAS is glutamylated differentially by TTLL4 and TTLL6.
Figure 5: TTLL6-mediated polyglutamylation of cGAS suppresses its DNA-binding ability.
Figure 6: TTLL4-mediated monoglutamylation of cGAS blocks its synthase activity.
Figure 7: Glutamylation and deglutamylation of cGAS tightly modulates antiviral immunity.

Change history

  • 29 February 2016

    In the version of this article initially published online, the title of the legend to Figure 1 ("Mice deficient in CCP5 or CCP5 are susceptible to infection with DNA viruses") was incorrect. The title should be "Mice deficient in CCP5 or CCP6 are susceptible to infection with DNA viruses." The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank S. Meng, J. Cheng, M. Ding, J. Wang, X. Gao, X. Zhang, L. Zhou, X. Wu, J. Hao, D. Liu, J. Jia, C. Jiang and . Teng for technical support; J. Bennink (National Institute of Allergy and Infectious Diseases) for vesicular stomatits virus and VACV strains; H. Peng (Institute of Biophysics, Chinese Academy of Sciences) for Vero cells and HSV-1f virus. Supported by the National Natural Science Foundation of China (31530093, 91419308, 31300645, 31471386 and 31570892), the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDA01010407 and XDA01020203), the Youth Innovation Promotion Association of Chinese Academy of Sciences (S.W.) and the China Postdoctoral Science Foundation (2015M571141 to P.X.).

Author information

P.X. designed and performed experiments, analyzed data and wrote the paper; B.Y. and S.W. performed experiments and analyzed data; X.Z. generated mutant mice; Y.D. and Z.X. performed some experiments; Y.T. initiated the study and analyzed data; and Z.F. initiated the study, and organized, designed and wrote the paper.

Correspondence to Yong Tian or Zusen Fan.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ccp5−/− and Ccp6−/− mice display reduced antiviral activity against DNA viruses instead of RNA viruses.

(a) Knockout strategies for Ccp25 knockout mouse generation using CRISPR/Cas9 technology. The targeting exon was shown in upper panel and the knockout allele was sequenced in lower panel. (b) WT and Ccp knockout mice were intranasally inoculated with VACV (1×106 pfu for each mouse), followed by survival curve calculation. n=10 for each strain. (c) Brains from the indicated mice intravenously injected with HSV (1×106 pfu for each mouse) were homogenized on the indicated days, followed by viral titer examination. n=6 for each strain. (d, e) WT, Ccp5−/− and Ccp6−/− mice were intranasally inoculated with VACV (1×106 pfu for each mouse), followed by analysis of serum IFNs through ELISA. n=6 for each strain. (f) Brains from WT, Ccp5−/− and Ccp6−/− mice intravenously injected with VACV (1x106 pfu for each mouse) were homogenized on the indicated days, followed by viral titer examination. n=7 for each strain. (g–i) The indicated mice were intravenously injected with HSV (1×107 pfu for each mouse), followed by RT-PCR analysis of Ifnb at the indicated times. Peritoneal macrophages, peripheral CD11chigh dendritic cells and lung fibroblasts were examined. n=7 for each strain. Data are shown as means±SD. , P<0.01; **, P<0.001. Data are representative of at least three independent experiments.

Supplementary Figure 2 CCP5 and CCP6 are involved in the innate immune response to DNA viruses.

(a) WT, Ccp5−/− and Ccp6−/− BMDMs were transfected with the indicated types of DNA or RNA (1 μg/ml) for 18 h, followed by IFN-β determination (upper panel) and IRF3 dimerization examination (lower panel). (b) Ccp6−/− BMDMs rescued with CCP6-wt or CCP6-mut were incubated with HSV (MOI=1) for 8 h, followed by IRF3 dimerization examination. (c) Ccp6+/+ and Ccp6−/− BMDMs rescued with the indicated plasmids were infected with HSV-GFP (MOI=1) for 24 h, followed by microscopy examination. Scale bar, 400 μm. (d) WT BMDMs overexpressed with CCP5 or CCP6 for 24 h were infected with HSV-GFP (MOI=1) for 24 h, followed by microscopy examination (left panel). GFP positive cells were calculated (right panel). Scale bar, 400 μm. (e, f) WT, Ccp5−/− and Ccp6−/− BMDMs treated with 10 μM CoCl2 (e) or 2 μM phenanthroline (f) for 6 h were infected with HSV-GFP (MOI=1) for 24 h, followed by microscopy examination. Scale bar, 400 μm. (g) WT, Ccp5−/− and Ccp6−/− BMDMs treated with 10 μM CoCl2 or 2 μM phenanthroline for 6 h were incubated with VSV (MOI=1) for 8 h, followed by RT-PCR analysis of Ifnb. Data are shown as means±SD. , P<0.05; **, P<0.01; ***, P<0.001. Data are representative of at least three independent experiments.

Supplementary Figure 3 cGAS interacts with CCP5 and CCP6 but not with other members of the CCP family.

(a) cGAS peptide sequences identified by mass spectrometry. (b) GST-tagged WT (FL) or catalytic domain truncated (Δ160–424) CCP5 were incubated with BMDM lysates for 4 h, followed by a GST pulldown assay. Precipitates were immunoblotted with the indicated antibodies. (c) WT BMDMs were stained with antibodies against β-tubulin, cGAS and CCP6, followed by confocal microscopy. Scale bar, 10 μm. (d) WT BMDMs treated with 10 μM CoCl2 or 2 μM phenanthroline for 6 h were immunoprecipitated with antibody against CCP6, followed by immunoblotting with the indicated antibodies. (e) GST-tagged WT (FL) or catalytic domain truncated (Δ230–401) CCP6 were incubated with BMDM lysates for 4 h, followed by a GST pulldown assay. Precipitates were immunoblotted with the indicated antibodies. (f) Schematic representation of putative glutamylation sites on cGAS. (g, h) Structural illustration of two putative glutamylation sites on cGAS (PDB code 4K97). Putative glutamylation sites were marked in red and the catalytic residues were marked in purple. Data are representative of at least three independent experiments.

Supplementary Figure 4 cGAS associates with TTLL4 and TTLL6.

(a, b) WT BMDMs co-transfected with CCP5 (a) or CCP6 (b) and cGAS-wt or cGAS-mut for 24 h were immunoprecipitated with antibody against Flag, followed by immunoblotting with the indicated antibodies. (c, d) Ccp5−/− (c) and Ccp6−/− (d) BMDMs transfected with cGAS-wt or cGAS-mut for 24 h were immunoprecipitated with antibody against Flag, followed by immunoblotting with the indicated antibodies. (e, f) GST-tagged CCP5-mut (e) or CCP6-mut (f) was incubated with lysates from cells overexpressed with WT or mutant cGAS for 24 h, followed by a GST pulldown assay. (g) BMDMs, DCs and lung fibroblasts from WT mice were subjected to RNA extraction and RT-PCR analysis of the indicated genes. (h) GST-tagged WT (FL) or truncated cGAS was incubated with BMDM lysates for 4 h, followed by a GST pulldown assay. (i, j) GST-tagged wt or mutant cGAS was incubated with recombinant TTLL4 (i) or TTLL6 (j) for 4 h, followed by a GST pulldown assay. (k) Knockout schemes for Ttll4 and Ttll6 knockout mouse generation using CRISPR/Cas9 technology. The targeting exon was shown (upper panel) and the knockout allele was sequenced (lower panel). Data are shown as means±SD. Data are representative of at least three independent experiments.

Supplementary Figure 5 CCP5 or TTLL4 does not affect the DNA-binding ability of cGAS.

(a) Confocal microscopy of Ccp5+/+ and Ccp5−/− BMDMs transfected with FITC-conjugated 50-mer dsDNA (left panel). Pearson’s Correlation Coefficient between cGAS and FITC-DNA was calculated (right panel). Colocalized dots were annotated by white arrow heads. Scale bar, 10 μm. (b) ChIP assay of 200-mer dsDNA transfected CCP5+/+ and CCP5−/− BMDMs expressing WT or mutant cGAS. (c) ChIP assay of 200-mer dsDNA transfected Ttll4+/+ and Ttll4−/− BMDMs expressing WT or mutant cGAS. (d) In vitro DNA pulldown of WT or mutant cGAS glutamylated by TTLL4. (e) Ttll6+/+ and Ttll6−/− BMDMs were incubated with HSV-GFP (MOI=1) for 24 h, followed by microscopy examination. Cells were counterstained with DAPI for nucleus (left panel). GFP positive cells were calculated (right panel). Scale bar, 400 μm. (f) Cgas−/− BMDMs rescued with cGAS-wt or cGAS-E272A were incubated with HSV (MOI=1) for 8 h, followed by detection of Ifnb. (g) Cgas−/− BMDMs rescued with cGAS-wt or cGAS-E272A were incubated with HSV-GFP (MOI=1) for 24 h, followed by microscopy examination. Cells were counterstained with DAPI for nucleus (left panel). GFP positive cells were calculated (right panel). Scale bar, 400 μm. Data are shown as means±SD. , P<0.01; **, P<0.001. Data are representative of at least four independent experiments.

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Xia, P., Ye, B., Wang, S. et al. Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity. Nat Immunol 17, 369–378 (2016). https://doi.org/10.1038/ni.3356

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