Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 15;27(1):108760.
doi: 10.1016/j.isci.2023.108760. eCollection 2024 Jan 19.

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells

Dayana B Hristova  1 , Marisa Oliveira  1 , Emma Wagner  1 , Alan Melcher  2 , Kevin J Harrington  2 , Alexandre Belot  3 , Brian J Ferguson  1
Affiliations

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells

Dayana B Hristova et al. iScience. .

Abstract

To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to anti-viral innate immunity, but also to autoinflammation and anti-tumour immunity through the initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signaling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent anti-viral innate immune system. Here, we show that DNA-PK is essential for cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.

Keywords: Molecular biology; Virology.

PubMed Disclaimer

Conflict of interest statement

The authors have no interests to declare.

Figures

None
Graphical abstract
Figure 1
Figure 1
The interferon response to dsDNA stimulation in HFFs is dependent on cGAS, STING and TBK1 (A) cGAS knockout cell line pools were generated by CRISPR/Cas9 with two sgRNAs targeting different exons of the MB21D1 gene (sg1 and sg2) and were immunoblotted with anti-cGAS antibody. WT (cGAS+/+) and cGAS−/− cells were stimulated with stimulated with htDNA or poly(I:C) and analyzed by immunoblotting with the indicated antibodies. (B) A cGAS−/− clonal cell line (cGAS−/− C1) was derived from cGAS−/− sg1 pool and immunoblotted with anti-cGAS antibody. WT (cGAS+/+) and cGAS−/− C1 cells were stimulated with htDNA and analyzed by qRT-PCR 6 h later for the transcription of IFNB, CXCL10 and ISG54. (C) STING knockout clonal cell lines (STING−/− C1 and C2) were generated by CRISPR/Cas9 using an gsRNA targeting the TMEM173 gene and were immunoblotted with an anti-STING antibody. WT (STING+/+) and STING−/− cells were stimulated with htDNA and analyzed by qRT-PCR 6 h later for the transcription of IFNB and CXCL10 and ISG54. (D) WT HFFs were pre-treated with the TBK1/IKKε inhibitor BX795, stimulated by transfection with htDNA, and analyzed by qRT-PCR 6 h later for the transcription of IFNB and CXCL10. Data is representative of at least two biological repeats and presented as mean ± SEM. Data was analyzed by two-tailed Student’s T test with n = 3; ∗∗∗p < 0.001.
Figure 2
Figure 2
DNA-PKcs is required for STING-dependent sensing in human fibroblasts (A) DNA-PKcs is activated during intracellular DNA stimulation. Representative images of cells treated with etoposide, htDNA or carrier controls, fixed and stained by immunofluorescence with an antibody recognising phosphoserine 2056 on DNA-PKcs. Cells are counterstained with DAPI, scale bar = 20 μm. (B) DNA-PKcs knockout cell lines were generated by CRISPR/Cas9 with two guide RNAs targeting different exons of the PRKDC gene (sg1 and sg2) and were immunoblotted with anti-DNA-PKcs antibody. (C) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were stimulated with htDNA or ctDNA and analyzed by qRT-PCR 6 h later for the transcription of IFNB, ISG54 and CXCL10. (D) WT, DNA-PKcs−/−, STING−/− or cGAS−/− cells were stimulated with htDNA and IFNβ activity in the supernatant was measured 24 h later by bioassay. (E) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were stimulated with ctDNA (top panel) or htDNA (bottom panel) or Poly(I:C) (PIC) for the indicated times and analyzed by immunoblotting with the indicated antibodies. Red arrows show the position of the phospho-STING specific band. Data is representative of at least two biological repeats and presented as mean ± SEM. Data was analyzed by two-tailed Student’s T test with n = 3; ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
DNA-PKcs kinase activity is dispensable for DNA sensing Cells were pre-treated with AZD7648 for 1 h prior to transfection with htDNA or poly(I:C) and (A) immunoblotted for the indicated antibodies or (B) analyzed by qRT-PCR for IFNB and CXCL10 6 h post stimulation. Data is representative of at least two biological repeats and presented as mean ± SEM. Data was analyzed by two-tailed Student’s T test with n = 3; NS = non-significant.
Figure 4
Figure 4
DNA-PKcs is required for the innate sensing of DNA viruses in human cells (A) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were infected with MVA at the indicated MOIs and analyzed by immunoblotting with the indicated antibodies. Red arrow shows the position of the phospho-STING specific band. (B) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were infected with VACV TBio 6517 at MOI 0.01 and the production of infectious virions was quantified 24 or 48 h later by plaque assay on BSC-1 cells. (C) HFFs were infected with WT (S17) or dl1043 HSV-1 (ΔICP0) at MOI 5 and immunoblotted with the indicated antibodies. (D) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were infected with dl1043 HSV-1 (ΔICP0) at MOI 5 and immunoblotted with the indicated antibodies). (E) WT (DNA-PKcs+/+) and DNA-PKcs−/− cells were infected with MVA or dl1043 HSV-1 (ΔICP0) at the indicated times and analyzed by qRT-PCR for IFNB and CXCL10 6 h post infection. Data is representative of at least two biological repeats and presented as mean ± SEM. Data was analyzed by two-tailed Student’s T test with n = 3; ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 5
Figure 5
The human DNA-PKcs L3062 mutation enhances STING signaling during DNA stimulation and DNA virus infection (A) Primary skin fibroblasts from healthy donor or a patient harboring the DNA-PKcs L3062 mutation were stimulated with HT-DNA and (A) immunoblotted with the indicated antibodies or (B) analyzed by qRT-PCR for IFNB and CXCL10 6 h post stimulation. (C) Primary skin fibroblasts from healthy donor or a patient harboring the DNA-PKcs L3062 mutation were infected with MVA or HSV-1ΔICP0 at MOI 5 and analyzed by qRT-PCR for IFNB and CXCL10 6 h post infection. Data is representative of at least two biological repeats and presented as mean ± SEM. Data was analyzed by two-tailed Student’s T test with n = 3; ∗∗∗p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Mansur D.S., Smith G.L., Ferguson B.J. Intracellular sensing of viral DNA by the innate immune system. Microb. Infect. 2014;16:1002–1012. - PubMed
    1. Iwasaki A., Medzhitov R. Control of adaptive immunity by the innate immune system. Nat. Immunol. 2015;16:343–353. - PMC - PubMed
    1. Unterholzner L., Keating S.E., Baran M., Horan K.A., Jensen S.B., Sharma S., Sirois C.M., Jin T., Latz E., Xiao T.S., et al. IFI16 is an innate immune sensor for intracellular DNA. Nat. Immunol. 2010;11:997–1004. - PMC - PubMed
    1. Ferguson B.J., Mansur D.S., Peters N.E., Ren H., Smith G.L. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. Elife. 2012;1 - PMC - PubMed
    1. Sun L., Wu J., Du F., Chen X., Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013;339:786–791. - PMC - PubMed
-