Abstract
Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids1. The abundant mtDNA-binding protein TFAM (transcription factor A, mitochondrial) regulates nucleoid architecture, abundance and segregation2. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses3. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined4. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signalling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.
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Change history
22 April 2015
The bar graph in Fig. 3d was corrected.
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Acknowledgements
We thank N. Chandel for Tfamfl/fl mice, D. Martin for Tfam+/− MEFs, J. Schoggins and S. Virgin for cGas−/− MEFs, G. Barber for Sting−/− MEFs, G. Sen for IFIT3 antibodies, J. Rose for VSV antibodies and recombinant vaccinia virus, K. Bahl and J. Schell for advice with VSV infections, and S. Ding for advice with HSV-1 gene expression analysis. This work was supported by a joint grant from the United Mitochondrial Disease Foundation and Mitocon, NIH R01 AG047632 and P01 ES011163 (G.S.S.), NIH R01 AI054359 and R01 AI081884 (A.I.), Canadian Institutes for Health Research grant MOP37995 and a Canada Research Chair in Molecular Virology (J.R.S.), American Cancer Society Postdoctoral Fellowship PF-13-035-01-DMC (A.P.W.), NIH T32 AI055403 (W.K.-H.), NIH F31 AG039163 (M.C.T.), NIH NRSA F32 DK091042 (M.B.), Alberta Innovates-Health Solutions and a Queen Elizabeth II Graduate Scholarship (B.A.D.), and a United Mitochondrial Disease Foundation Postdoctoral Fellowship (N.R.).
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Contributions
A.P.W. designed and performed experiments, analysed data, interpreted results and wrote the paper; W.K.H. provided viral stocks, advice on viral infection protocols, and performed in vivo HSV-1 infections; M.S. performed LCMV and influenza infections; M.C.T. aided in experimental design and assisted with viral infections; C.M.P. performed experiments and analysed data; M.B. performed steady-state mitochondrial transcript analysis; N.R. assisted with gene expression array analysis; D.A.M. generated cGas−/− MEFs; B.A.D. and J.R.S. generated and provided HSV-1 UL12 constructs and HSV-1 ΔUL12 viruses; S.M.K. provided reagents and facilities for LCMV infections and interpreted results; S.M.L. and R.E.M. provided reagents and advice and performed viral infections; A.I. supplied reagents, designed experiments, and interpreted results; G.S.S. designed experiments, interpreted results and wrote the paper.
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Extended data figures and tables
Extended Data Figure 1 TFAM deficiency induces mtDNA depletion, nucleoid stress, elevated ISG expression and augmented type I interferon responses, but does not drastically alter oxygen consumption and mitochondrial transcription rates.
a, Quantitative PCR analysis of relative mtDNA copy number from wild-type (WT) and Tfam+/− MEFs. b, Basal oxygen consumption rate of wild-type and Tfam+/− MEFs as determined by Seahorse Bioscience XF96 Extracellular Flux assay. c, qRT–PCR of mtDNA-encoded rRNA (16s) and mRNA (ND6, Cytb, Cox1) transcripts in wild-type and Tfam+/− MEFs. d–f, Untransfected Tfam+/− (d) or wild-type MEFs transfected with control (siCtrl) or Tfam (siTfam) siRNAs (d–f) were stained with anti-HSP60 (Mito.) and anti-DNA (DNA) antibodies. Nucleoid area from multiple independent images was calculated, stratified into groups, and graphed as percentage of the total number of nucleoids counted for each sample (d). Inset panels are 3× magnification to enhance viewing of mitochondrial network and nucleoid architecture (e). TFAM and ISG mRNA expression were measured by qRT–PCR (f). g–i, Tfamflox/flox ER-cre− or Tfamflox/flox ER-cre+ BMDMs were incubated in 4OHT for 96 h to induce TFAM depletion. Immunofluorescence staining was performed as described above (g). ISG mRNA and protein expression was monitored by qRT–PCR and western blotting (h). qRT–PCR analysis of type I interferon and Il6 expression in 4OHT-treated Tfamfl/fl ER-cre−/+ BMDMs 2 h post-cytosolic delivery of interferon-stimulatory DNA (ISD) or poly(I:C) (PIC) (i). Error bars indicate ±s.e.m. of triplicates and data are representative of three independent experiments. P < 0.05; P < 0.01; P < 0.001; NS, not significant.
Extended Data Figure 2 TFAM deficiency promotes accumulation of cytosolic mtDNA.
a, Wild-type (WT) or Tfam+/− MEFs were subjected to digitonin fractionation as described in the Methods and whole-cell extracts (WCE), pellets (Pel) or cytosolic extracts (Cyt) were blotted using the indicated antibodies. b, DNA was extracted from digitonin extracts of wild-type and Tfam+/− MEFs or Tfamfl/fl ER-cre− or Tfamfl/fl ER-cre+ BMDMs incubated in 4OHT for 72 h. Cytosolic mtDNA was quantitated via qPCR using a mitochondrial Dloop primer set (mt-Dloop3). Normalization was performed as described in the Methods. c, Samples were prepared as described in b, and cytosolic mtDNA was quantitated via qPCR using the indicated primer sets. Error bars indicate ±s.e.m. of triplicates and data are representative of three independent experiments. P < 0.01, P < 0.001.
Extended Data Figure 3 Mitochondrial hyperfusion regulates the accumulation of mtDNA nucleoid stress in TFD MEFs.
a, b, Wild-type (WT) MEFs were transfected with control or Tfam siRNAs for 96 h. Cells were fixed and processed for electron microscopy analysis (a). Mitochondrial perimeter measurements were obtained from multiple independent images, stratified into groups, and graphed as a percentage of the total number of mitochondria counted for each sample (b). c–e, Wild-type MEFs were transfected with control, Mfn1 and/or Tfam siRNAs for 96 h. Cells were fixed and stained with an anti-HSP60 antibody (Mito.) and an anti-DNA antibody (DNA) for confocal microscopy (c). Nucleoid area from multiple independent images was calculated as previously described (d). RNA was extracted for ISG expression analysis by qRT–PCR (e). f, Wild-type and Tfam+/− MEFs were transfected with the indicated siRNAs for 96 h and ISG expression analysed by qRT–PCR. Error bars indicate ±s.e.m. of triplicates and data are representative of two independent experiments. P < 0.01; P < 0.001.
Extended Data Figure 4 mtDNA stress in TFD MEFs and BMDMs potentiates type I interferon responses to viral infection and enhances viral clearance.
a, b, Wild-type (WT) and Tfam+/− MEFs were infected with VSV-GFP (a) or MHV68-GFP (b) and, after the indicated times, cytokine and ISG mRNA expression was determined by qRT–PCR, or cytokine secretion was determined by ELISA. c–f, Tfamfl/fl ER-cre− or Tfamfl/fl ER-cre+ BMDMs were incubated in 4OHT for 96 h to induce TFAM depletion. Cells were infected with HSV-1-GFP (c, e, f) or VSV-GFP (d, e), incubated for the indicated times, and viral gene expression was determined by qRT–PCR (c, d) and western blotting (e), or cytokine and ISG mRNA expression was determined by qRT–PCR (f). Error bars indicate ±s.e.m. of triplicates and data are representative of two independent experiments. P < 0.01; P < 0.001; A.U., arbitrary units; ND, not detected; NS, not significant.
Extended Data Figure 5 Tissues from Tfam+/− mice display elevated ISG expression, and ddC abrogates mtDNA stress, ISG expression and viral resistance phenotypes of TFD cells.
a, RNA was extracted from the liver and kidneys of 8-week-old wild-type (WT) and Tfam+/− mice (n = 2 each) and subjected to qRT–PCR analysis for basal ISG expression. b-d, Relative mtDNA copy number (b), mtDNA nucleoid area (c) and ISG expression (d) of wild-type and Tfam+/− MEFs exposed to ddC for 96 h. e, f, mtDNA nucleoid area (e) and ISG expression (f) of wild-type MEFs transfected with control or Tfam siRNAs for 96 h in the presence or absence of ddC. g–i, Tfamfl/fl ER-cre− or Tfamfl/fl ER-cre+ BMDMs were incubated in 4OHT for 96 h to induce TFAM depletion in the presence of ddC. ddC was washed out and cells allowed to recover overnight before infection. Cells were infected with VSV-GFP (g) or HSV-1-GFP (h) at MOI 1, or wild-type BMDMs were transfected with poly(I:C) or interferon-stimulatory DNA (ISD) (i), and incubated for the indicated times. Ifnb expression or viral gene expression was determined by qRT–PCR. Error bars indicate ±s.e.m. of triplicates and data are representative of two independent experiments. P < 0.05; P < 0.01; P < 0.001; NS, not significant.
Extended Data Figure 6 Alpha- and gammaherpesviruses induce mtDNA stress, but influenza, LCMV, and vaccinia do not.
a, Relative mtDNA copy number of wild-type (WT) MEFs 24 h post-infection with VSV-GFP, HSV-1-GFP or mock infection at the indicated MOIs. b, Wild-type MEFs were infected with MHV68-GFP at MOI 0.5. After the indicated times cells were stained and subjected to confocal microscopy or the relative mtDNA copy number was determined. c, Wild-type MEFs were infected with HSV-2, influenza-GFP or LCMV-GFP at MOI 10. After 6 h, cells were stained and subjected to confocal microscopy. d, Wild-type MEFs were infected with vaccinia virus at MOI 10 (for microscopy) or 1. After the indicated times cells were stained and subjected to confocal microscopy or the relative mtDNA copy number was determined. Error bars indicate ±s.e.m. of triplicates and data are representative of two independent experiments. P < 0.05; P < 0.01; P < 0.001; A.U., arbitrary units; ND, not detected; NS, not significant.
Extended Data Figure 7 HSV-1 UL12 M185 expression is sufficient to trigger mtDNA stress, TFAM depletion and antiviral priming in BMDMs; infection with UL12-deficient HSV-1 fails to induce mtDNA stress, elicits lower vaginal type I interferon responses and spreads more readily to dorsal root ganglia.
a, Wild-type (WT) BMDMs were transduced with HSV-1-UL12-M185-expressing- or empty retroviruses (RV) and relative mtDNA abundance, protein expression, and ISG mRNA expression determined. b, Wild-type MEFs were infected with HSV-1 (UL12–FLAG) or UL12-deficient HSV-1 (ΔUL12 + UL98–FLAG) at MOI 10 for 3 h and analysed by confocal microscopy. c, Wild-type MEFs were infected with HSV-1 (UL12–FLAG) or UL12-deficient HSV-1 (ΔUL12 + UL98–FLAG) at MOI 2 for 24 h and mtDNA abundance was determined by qPCR. d, The vaginas of wild-type mice (n = 3 per condition) were inoculated with 106 plaque-forming units of HSV-1 (UL12–FLAG) or UL12-deficient HSV-1 (ΔUL12 + UL98–FLAG) and 24 h post-infection, vaginal RNA was extracted and gene expression analysed by qRT–PCR. e, Mice (n = 3 per condition) were infected as previously described and 6 days post-infection, DNA from dorsal root ganglia was isolated for mtDNA and HSV-1 genome abundance measurements by qPCR. Error bars indicate ±s.e.m. of triplicates and data are representative of two independent experiments. P < 0.05; P < 0.01; P < 0.001; NS, not significant.
Extended Data Figure 8 Model illustrating mtDNA stress-dependent antiviral priming.
TFAM depletion, induced genetically or during herpesvirus infection, triggers mtDNA stress, characterized by nucleoid loss and enlargement. This results in the release of fragmented mtDNA that recruits and activates peri-mitochondrial cGAS to generate the second messenger cyclic GMP-AMP (cGAMP) and activate endoplasmic-reticulum-resident STING. STING then activates TBK1, which phosphorylates IRF3 to induce dimerization and nuclear translocation. Active IRF3 elevates basal gene expression of ISGs with antiviral signalling and effector functions. Signalling molecules encoded by ISGs, such as IRF7, ISG15, STAT1 and STAT2, cooperate with IRF3 to potentiate the RIG-I-like receptor (RLR), interferon-stimulatory DNA (ISD) and type I interferon (IFN-I) responses, while effector molecules encoded by ISGs, such as IFI44, IFIT1, IFIT3 and OASL2, augment viral resistance. Both outcomes collectively and robustly boost innate antiviral defences to dampen viral replication.
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West, A., Khoury-Hanold, W., Staron, M. et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520, 553–557 (2015). https://doi.org/10.1038/nature14156
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DOI: https://doi.org/10.1038/nature14156
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