Mitochondrial dna stress primes the antiviral innate immune response

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. Access through your institution Buy or subscribe This is a

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REPLICATION STRESS TRIGGERS A PRO-INFLAMMATORY ENDOSOMAL PATHWAY OF NUCLEOID DISPOSAL Article 08 February 2024 VRK2 IS INVOLVED IN THE INNATE ANTIVIRAL RESPONSE BY PROMOTING

MITOSTRESS-INDUCED MTDNA RELEASE Article 30 March 2021 THE POXVIRUS F17 PROTEIN COUNTERACTS MITOCHONDRIALLY ORCHESTRATED ANTIVIRAL RESPONSES Article Open access 30 November 2023 ACCESSION

CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE63767 DATA DEPOSITS Microarray data have been submitted to the NCBI Gene Expression Omnibus under accession number GSE63767. CHANGE

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374–378 (2003) CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank N. Chandel for _Tfam__fl/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.). AUTHOR INFORMATION Author

notes * Michal C. Tal, Megan Bestwick & Nuno Raimundo Present address: Present addresses: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of

Medicine, Stanford, California 94305, USA (M.C.T.); Department of Chemistry, Linfield College, McMinnville, Oregon 97128, USA (M.B.); Institute for Cellular Biochemistry, Universitätsmedizin

Göttingen, 37073 Göttingen, Germany (N.R.)., AUTHORS AND AFFILIATIONS * Department of Pathology, Yale School of Medicine, New Haven, 06520, Connecticut, USA A. Phillip West, Cristiana M.

Pineda, Sabine M. Lang, Megan Bestwick, Nuno Raimundo, Robert E. Means & Gerald S. Shadel * Department of Immunobiology, Yale School of Medicine, New Haven, 06520, Connecticut, USA

William Khoury-Hanold, Matthew Staron, Michal C. Tal, Susan M. Kaech & Akiko Iwasaki * Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of

Alberta, Edmonton, Alberta T6G 2S2, Canada, Brett A. Duguay & James R. Smiley * Department of Pathology and Immunology, Washington University School of Medicine, St Louis, 63110,

Missouri, USA Donna A. MacDuff * Howard Hughes Medical Institute, Chevy Chase, 20815-6789, Maryland, USA Susan M. Kaech & Akiko Iwasaki * Department of Genetics, Yale School of Medicine,

New Haven, 06520, Connecticut, USA Gerald S. Shadel Authors * A. Phillip West View author publications You can also search for this author inPubMed Google Scholar * William Khoury-Hanold

View author publications You can also search for this author inPubMed Google Scholar * Matthew Staron View author publications You can also search for this author inPubMed Google Scholar *

Michal C. Tal View author publications You can also search for this author inPubMed Google Scholar * Cristiana M. Pineda View author publications You can also search for this author inPubMed

 Google Scholar * Sabine M. Lang View author publications You can also search for this author inPubMed Google Scholar * Megan Bestwick View author publications You can also search for this

author inPubMed Google Scholar * Brett A. Duguay View author publications You can also search for this author inPubMed Google Scholar * Nuno Raimundo View author publications You can also

search for this author inPubMed Google Scholar * Donna A. MacDuff View author publications You can also search for this author inPubMed Google Scholar * Susan M. Kaech View author

publications You can also search for this author inPubMed Google Scholar * James R. Smiley View author publications You can also search for this author inPubMed Google Scholar * Robert E.

Means View author publications You can also search for this author inPubMed Google Scholar * Akiko Iwasaki View author publications You can also search for this author inPubMed Google

Scholar * Gerald S. Shadel View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Gerald S. Shadel. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing financial interests. 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, _Tfam__flox/flox__ ER-cre__−_ or _Tfam__flox/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 _Tfam__fl/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 _Tfam__fl/fl__ ER-cre__−_ or _Tfam__fl/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, _Tfam__fl/fl__ ER-cre__−_ or _Tfam__fl/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, _Tfam__fl/fl__ ER-cre__−_ or _Tfam__fl/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. POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT

SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 04 July 2014 * Accepted:

15 December 2014 * Published: 02 February 2015 * Issue Date: 23 April 2015 * DOI: https://doi.org/10.1038/nature14156 SHARE THIS ARTICLE Anyone you share the following link with will be

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