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. 2020 Sep 1;117(35):21568-21575.
doi: 10.1073/pnas.1922330117. Epub 2020 Aug 17.

KAT5 acetylates cGAS to promote innate immune response to DNA virus

Ze-Min Song  1 , Heng Lin  1 , Xue-Mei Yi  1 , Wei Guo  1 , Ming-Ming Hu  1 , Hong-Bing Shu  2
Affiliations

KAT5 acetylates cGAS to promote innate immune response to DNA virus

Ze-Min Song et al. Proc Natl Acad Sci U S A. .

Abstract

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA-triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus-induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus.

Keywords: DNA virus; KAT5; acetylation; cGAS; innate immune response.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
KAT5 positively regulates DNA-virus–triggered signaling. (A) KAT5 promotes cGAS- but not MITA- or TBK-mediated ISRE activation. HEK293 cells were transfected with the indicated plasmids for 20 h before luciferase assays were performed. (B) Overexpression of KAT5 promoted HSV-1–induced transcription of downstream genes. The stable KAT5-overexpressing and control THP-1 cell lines were left uninfected or infected with HSV-1 (multiplicity of infection [MOI] = 1) for 10 h before qPCR analysis. (C) Effects of KAT5 knockdown on HSV-1–induced transcription of downstream genes. The KAT5 knockdown and control THP-1 cell lines were left uninfected or infected with HSV-1 (MOI = 1) for the indicated times before qPCR analysis. Knockdown efficiency of KAT5 was confirmed by immunoblots (Right). (D) Effects of KAT5 knockdown on transcription of downstream genes induced by transfected DNA. The KAT5-RNAi stable knockdown and control THP-1 cell lines were transfected with the indicated dsDNA for 4 h before qPCR analysis. (E) Effects of KAT5 knockdown on HSV-1–induced phosphorylation of MITA-S366, TBK1-S172, and IRF3-S396. The KAT5 knockdown and control THP-1 cell lines were left uninfected or infected with HSV-1 (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. ns, no significance. *P < 0.05, **P < 0.01 (unpaired t test). Data shown are mean ± SD from one representative experiment performed in triplicates (A–D).
Fig. 2.
Fig. 2.
KAT5 functions at the cGAS level. (A) Effects of KAT5 knockdown on cGAMP synthesis induced by HSV-1 infection. The KAT5-knockdown and control THP-1 cells (1 × 108) were left uninfected or infected with HSV-1 (MOI = 2) for 3 h, and then the cell extracts containing cGAMP were separated by chromatography using a C18 column. The abundance of cGAMP was quantitated by mass spectrometry. Quantification of cGAMP was shown in the histograph (Right). **P < 0.01 (unpaired t test). Data shown are mean ± SD of three technical repeats. (B) Effects of KAT5 knockdown on cGAMP-induced transcription of downstream genes. The KAT5-knockdown and control THP-1 cells were left untreated or treated with 2′3′-cGAMP (40 nM) for the indicated times before qPCR analysis. ns, no significance (unpaired t test). Data shown are mean ± SD from one representative experiment performed in triplicate. (C) Effects of KAT5 knockdown on cGAMP-induced phosphorylation of TBK1-S172 and IRF3-S396. The KAT5-knockdown and control THP-1 cells were left untreated or treated with 2′3′-cGAMP (40 nM) for the indicated times before immunoblotting analysis with the indicated antibodies. (D) Interaction of KAT5 and cGAS in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 20 h. Cell lysates were immunoprecipitated with control IgG or anti-HA. The lysates and immunoprecipitates were subjected to immunoblotting analysis with the indicated antibodies. (E) Interaction of KAT5 and cGAS in vitro. GST, GST-cGAS, and His-KAT5 proteins were purified from Escherichia coli. Recombinant GST or GST-cGAS was mixed with His-KAT5. The GST or GST-cGAS protein was pulled down with glutathione Sepharose beads and then analyzed by immunoblotting with the indicated antibodies. (F) Association of endogenous KAT5 with cGAS. The mouse lung fibroblasts were left uninfected or infected with HSV-1 (MOI = 1) for the indicted times before cells were harvested for immunoprecipitation with control IgG or anti-KAT5. The lysates and immunoprecipitates were subjected to immunoblotting analysis with the indicated antibodies.
Fig. 3.
Fig. 3.
KAT5 mediates acetylation of the N-terminal domain of cGAS. (A) Overexpression of KAT5 increased acetylation of cGAS but not RIG-I. HEK293 cells were transfected with the indicated plasmids for 20 h, and then the cells were treated with the deacetylase inhibitors TSA and NAM for 8 h before coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. (B) Effects of KAT5 and its mutant on acetylation of cGAS in vitro. GST-cGAS was purified from E. coli. FLAG-KAT5 and FLAG-KAT5(QG/EE) were purified from HEK293. The indicated purified proteins were incubated for 2 h, followed by immunoblotting analysis with the indicated antibodies. (C) Effects of KAT5 on acetylation of cGAS and its truncated mutants. The experiments were similarly performed as in A. (D) Effects of KAT5 on ISRE activation mediated by cGAS and its mutants. The HEK293 cells stably expressing MITA were transfected with the indicated plasmids for 24 h before luciferase assays were performed. (E) Effects of KAT5 on acetylation of cGAS and its N-terminal lysine mutants. The experiments were similarly performed as in A. (F) Effects of KAT5 knockdown on HSV-1–induced acetylation of cGAS. The KAT5-knockdown or control THP-1 cell lines were infected with HSV-1 (MOI = 2) for the indicated times before coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies.
Fig. 4.
Fig. 4.
Acetylation of cGAS by KAT5 increases its DNA-binding affinity. (A) Effects of KAT5 and KAT5(QG/EE) on the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids for 20 h. The cell lysates were then incubated with biotinylated HSV120 and streptavidin-Sepharose beads. The bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. (B) Binding of dsDNA by cGAS and cGAS(4R). The experiments were similarly performed as in A. (C) Binding affinities of acetylated or unacetylated cGAS to dsDNA. GST-cGAS and GST-cGAS(4R) were purified from E. coli. FLAG-KAT5 was purified from HEK293. GST-cGAS or GST-cGAS(4R) were subjected to acetylation reaction with FLAG-KAT5. The reactions were then mixed with Cy5-labeled HSV60 before MST measurements. Aliquots of the reactions were also analyzed by immunoblotting with the indicated antibodies. (D) Effects of KAT5 knockdown on dsDNA-induced cGAS aggregation. The KAT5-knockdown and control HT1080-FLAG-cGAS cell lines were transfected with HT-DNA for 5 h before immunofluorescent analysis of cGAS (green) (Left). A total of more than 180 cells from each sample were quantified for cGAS foci (Right). **P < 0.01 (unpaired t test). Data shown are representative of three experiments with similar results.
Fig. 5.
Fig. 5.
KAT5 potentiates viral DNA-triggered innate immune response in vivo. (A) Genotyping of Kat5-mutated mice. The genomic DNA was extracted from wild-type, Kat5+/SA, or Kat5SA/SA knock-in mice and analyzed by PCR and DNA sequencing. (B) Inhibition of DNA-virus–induced transcription of downstream antiviral genes in Kat5SA/SA BMDMs. Wild-type and Kat5SA/SA BMDMs were left uninfected or infected with HSV-1 or MCMV for 6 h before qPCR analysis. **P < 0.01 (unpaired t test). Data shown are mean ± SD from one representative experiment performed in triplicate. (C) Inhibition of transfected DNA-induced transcription of downstream antiviral genes in Kat5SA/SA BMDMs. Wild-type and Kat5SA/SA BMDMs were transfected with the indicated dsDNA for 4 h before qPCR analysis. *P < 0.05, **P < 0.01 (unpaired t test). Data shown are mean ± SD from one representative experiment performed in triplicate. (D) Inhibition of DNA-virus–induced phosphorylation of Tbk1-S172 and Irf3-S396 in Kat5SA/SA BMDMs. Wild-type and Kat5SA/SA BMDMs were left uninfected or infected with HSV-1 for the indicated times before immunoblotting analysis with the indicated antibodies. (E) Serum levels of IFN-β, CXCL10, and IL-6 following DNA virus infection in wild-type and Kat5SA/SA mice. The mice (n = 3 or 4 per strain, 8 wk old, male) were infected with HSV-1 (3 × 107 plaque-forming units [PFU]/mouse) or MCMV (1 × 104 PFU/mouse) for 6 h before enzyme-linked immunosorbent assay. *P < 0.05, **P < 0.01 (unpaired t test). Data shown are mean ± SD from one representative experiment. (F) Viral titers in brain following HSV-1 infection in wild-type and Kat5SA/SA mice. Wild-type and Kat5SA/SA mice (n = 3 per strain, 8 wk old, male) were infected with HSV-1 at 3 × 107 PFU per mouse for 5 d. HSV-1 viral titers in the brains of infected mice were quantified by plaque assays. **P < 0.01 (unpaired t test). Data shown are mean ± SD from one representative experiment. (G) Effects of Kat5 mutation on HSV-1–induced death of mice. Wild-type and Kat5SA/SA mice (n = 7 per strain, 8 wk old, female) were infected intraperitoneally with HSV-1 at 5 × 107 PFU per mouse, and the survival of mice was monitored daily for 10 d. P = 0.0025 (log-rank test).
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