MitochondrialDNA stress primes the antiviral innate immune response

Mitochondrial DNA stress primes the antiviral innate immune response

A.Phillip West1,William Khoury-Hanold2,Matthew Staron2,Michal C.Tal2{,Cristiana M.Pineda1,Sabine b87cb1ade2bd960591c67714ng1,

Megan Bestwick1{,Brett A.Duguay3,Nuno Raimundo1{,Donna A.MacDuff4,Susan M.Kaech2,5,James R.Smiley3,

Robert E.Means1,Akiko Iwasaki2,5&Gerald S.Shadel1,6

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 pro-tein TFAM(transcription factor A,mitochondrial)regulates nucleoid architecture,abundance and b87cb1ade2bd960591c67714plete mtDNA depletion profoundly impairs oxidative phosphorylation,triggering calcium-dependent stress signalling and adaptive metabolic responses3.How-ever,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.Mechanis-tically,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 demon-strate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signal-ling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

To explore the cellular responses to mtDNA stress in the absence of oxidative phosphorylation deficiency,we employed a TFAM hetero-zygous knockout(Tfam1/2)mouse model.Cells and tissues from these animals exhibit modest or no significant differences in mtDNA-encoded transcripts and oxygen consumption rates,despite an approximately 50%depletion of mtDNA(Extended Data Fig.1a–c)5,6.In addition to mtDNA depletion,Tfam1/2mouse embryonic fibroblasts(MEFs)have reduced oxidative mtDNA damage repair capacity and markedly altered mtDNA packaging,organization and distribution(Fig.1a)6.Nucleoids in Tfam1/2MEFs were less numerous and exhibited a larger size distri-bution(Fig.1a and Extended Data Fig.1d).Thus,Tfam1/2cells provide a robust model to characterize cellular responses triggered by moderate mtDNA stress.

Gene expression profiling of Tfam1/2MEFs revealed an unexpec-ted enrichment of interferon-stimulated genes(ISGs)and antiviral sig-nalling factors(Fig.1b).Of the45most overexpressed genes,39were ISGs,including many with direct antiviral activity(Ifi44,Ifit1,Ifit3, Oasl2,Rtp4)7,8.We also observed increased expression of cytoplasmic RNA and DNA sensors,such as Ddx58and Ifih1and p200family pro-teins Ifi203,Ifi204and Ifi205,as well as transcription factors Irf7,Stat1 and Stat2,ISGs that function to positively reinforce the antiviral res-ponse.Direct measurement of basal ISG mRNA and protein expression in Tfam1/2MEFs validated the microarray results(Fig.1c,d).Finally, Tfam1/2MEFs expressed three-to fourfold more Ifnb and Ifna4upon transfection with the IFIH1agonist poly(I:C)(Fig.1e),consistent with enhanced type I interferon responses.

Poly(I:C) 9 h

p

r

e

s

s

i

o

n

(

f

o

l

d

)

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

f

o

l

d

)

+/–T

f

a

m

I

?

4

4

U

s

p

1

8

I

?

t

1

I

?

t

3

I

r

f

7

C

x

c

l

1

S

t

a

t

1

I

s

g

1

5

I

?

h

1

D

d

x

5

8

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

f

o

l

d

)

IRF3

IFIH1

NLRX1

DDX58

STAT1

α-Tubulin

TBK1

W

T

1

T

f

a

m

+

/

–

1

T

f

a

m

+

/

–

2

W

T

2

a

Mito.

Merge

WT Tfam+/–

b

c

d

TFAM

e

I?44

Trim30

Usp18

I?t1

I?t3

Oasl2

Rtp4

Irf7

Cxcl10

Ube2l6

Rnf213

Bst2

Stat1

Isg15

Lgals3bp

Rnf213

Parp12

Samd9l

Apol9a

Gbp2

Irgm2

Xaf1

I?205

Parp9

I?203

Stat2

I?h1

Oas1g

Apol9b

Sp100

Gbp6

Ddx58

I?t4

Gbp3

I?204

Tor3a

Dtx3l

Oas1b

Ccl5

Parp14

Agrn

Tgtp1

Cmpk2

Nell2

Eif2ak2

High

Fold change log

Low

T

f

a

m

+

/

–

1

T

f

a

m

+

/

–

2

W

T

1

W

T

2

Figure1|Tfam1/2cells exhibit mtDNA stress,elevated ISG expression and augmented type I interferon responses.a,Confocal microscopy images of MEFs stained with anti-DNA(DNA)and anti-HSP60(Mito.)antibodies. b,Heat maps of microarray analyses.Genes in Tfam1/2MEFs exhibiting statistically significant(P,0.05),twofold or greater increases over wild type (WT)are shown.c,d,Quantitative real-time-PCR(qRT–PCR)(c)and western blots(d)of basal ISG expression in two littermate wild-type and Tfam1/2 MEF lines.e,qRT–PCR analysis of type I interferon expression in MEFs9h after cytosolic delivery of poly(I:C).Error bars indicate6s.e.m.of triplicate technical replicates and are representative of three independent experiments. ***P,0.001.

1Department of Pathology,Yale School of Medicine,New Haven,Connecticut06520,USA.2Department of Immunobiology,Yale School of Medicine,New Haven,Connecticut06520,USA.3Li Ka Shing Institute of Virology,Department of Medical Microbiology and Immunology,University of Alberta,Edmonton,Alberta T6G2S2,Canada.4Department of Pathology and Immunology,Washington University School of Medicine,St Louis,Missouri63110,USA.5Howard Hughes Medical Institute,Chevy Chase,Maryland20815-6789,USA.6Department of Genetics,Yale School of Medicine,New Haven, Connecticut06520,USA.{Present addresses:Institute for Stem Cell Biology and Regenerative Medicine,Stanford University School of Medicine,Stanford,California94305,USA(M.C.T.);Department of Chemistry,Linfield College,McMinnville,Oregon97128,USA(M.B.);Institute for Cellular Biochemistry,Universita¨tsmedizin Go¨ttingen,37073Go¨ttingen,Germany(N.R.).

G2015Macmillan Publishers Limited.All rights reserved

23A P R I L2015|V O L520|N A T U R E|553

To ensure that the mtDNA stress and ISG expression phenotypes were not unique to Tfam1/2MEFs,we employed inducible TFAM de-pletion models(TF D).Analogous to Tfam1/2cells,TF D MEFs and bone-marrow-derived macrophages(BMDMs)displayed mtDNA stress phenotypes,augmented ISG expression,and heightened type I inter-feron responses to poly(I:C)(Extended Data Fig.1d–i).Collectively, these data indicate that TFAM depletion induces mtDNA nucleoid stress that triggers antiviral‘priming’,characterized by basally elevated ISG expression and potentiated type I interferon production.

Since mitochondrial stress can trigger the release of mtDNA into the cytosol to engage the NLRP3inflammasome,we assayed for extra-mitochondrial mtDNA in TF D cells9,10.Analysis of pure cytosolic ex-tracts revealed a three-to fourfold increase of specific mtDNA fragments from the D-loop regulatory region,indicating liberation of immunosti-mulatory mtDNA into the cytosol(Extended Data Fig.2)11.Confocal and electron microscopy of TF D cells revealed significantly elongated, interconnected mitochondrial networks consistent with a hyperfused phenotype(Fig.1a and Extended Data Figs1e,g and3a,b).Since mi-tochondrial fission facilitates proper nucleoid distribution and removal of damaged mtDNA,we examined whether mitochondrial hyperfusion in TF D cells governed mtDNA stress-induced ISG expression12,13.Knock-down of mitofusin1(Mfn1)induced fission and largely abrogated ISG expression in TF D MEFs(Extended Data Fig.3c–e).Moreover,deple-tion of the mtDNA quality-control enzyme endo/exonuclease(59–39), endonuclease G-like(EXOG)exacerbated ISG expression in Tfam1/2 MEFs(Extended Data Fig.3f)14.Collectively,these data indicate that TFAM depletion promotes accumulation of aberrant mtDNA,which accesses the cytosol to engage innate immune signalling.

We next examined the involvement of the cytosolic DNA sensor cGAS in mtDNA stress signalling,as it mediates ISG expression in response to exogenous and endogenous immunostimulatory DNA species15–17. Knockdown of cGAS in Tfam1/2MEFs or TFAM depletion in cGas2/2 MEFs largely abrogated ISG expression(Fig.2a).Furthermore,ISG mRNAs in TF D cells were reduced70–90%in the absence of STING, indicating cGAS–STING signalling is the predominant driver of mtDNA stress-induced ISG expression(Fig.2b).STING signals via the TBK1–IRF3/7axis to trigger antiviral gene expression,and knockdown of ei-ther TBK1or IRF3robustly blocked ISG expression in Tfam1/2MEFs (Fig.2c,d)18,19.Consistent with IRF3activating ISG transcription,we observed enhanced nuclear accumulation of IRF3in TF D cells(Fig.2e). Finally,using cGas2/2MEFs reconstituted with hemagglutinin(HA)-tagged,murine cGAS,we observed prominent re-localization of cGAS from nuclear and/or cytoplasmic pools to the vicinity of aberrant mtDNA nucleoids in TF D MEFs(Fig.2f,g).Taken together,these results indi-cate that mtDNA stress facilitates cGAS-dependent sensing of cyto-plasmic mtDNA,resulting in STING–TBK1–IRF3signalling to trigger ISG expression.

To establish functional significance of mtDNA stress-induced anti-viral priming,we challenged MEFs with herpes simplex virus1(HSV-1) or vesicular stomatitis virus(VSV)that express green fluorescent pro-tein(GFP)for easy detection.In contrast to wild-type cells,which dis-played robust viral GFP expression post-infection,Tfam1/2MEFs were markedly resistant to HSV-1and VSV(Fig.3a).In addition,Tfam1/2 MEFs exhibited heightened type I interferon and ISG expression upon viral challenge,consistent with potentiated type I interferon responses in these cells(Extended Data Fig.4a).Similar results were obtained upon challenge with the rodent gammaherpesvirus MHV-68(Fig.3b and Ex-tended Data Fig.4b).Furthermore,TF D BMDMs displayed augmented antiviral gene expression and markedly lower HSV-1-and VSV-encoded mRNA and protein6–24h post-infection(Extended Data Fig.4c–f).

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

IRF3

Histone H3

Nuclear extract

TFAM

IRF3

HSP60

Whole-cell extract

MEF Tfam?/?

BMDM + 4OHT

+/++/–cre–cre+

MEF Tfam?/?

BMDM + 4OHT

+/++/–cre–cre+

c d

e

b

a

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

R

e

l

a

t

i

v

e

e

x

p

r

e

s

s

i

o

n

(

%

)

Tfam+/–

Tfam+/–

Tfam+/– MEF

Tfam

+/– MEF

20

40

60

80

100

P

e

r

c

e

n

t

cGAS–HA reconstituted MEF

siCtrl

Nuceloids >450 nm2

with cGAS colocalization

siTfam

f

g

Figure2|mtDNA stress triggers ISG expression

in a cGAS-and STING-dependent fashion.

a,b,ISG expression in Tfam1/2MEFs transfected

with the indicated short interfering RNAs(siRNAs;

top panels),or wild-type(WT),cGas2/2(a),

and Sting2/2(b)MEFs transfected with TFAM

siRNAs(bottom panels).Ctrl,control.c,d,ISG

expression in Tfam1/2MEFs transfected with the

indicated siRNAs for96h.e,Western blots of

whole-cell and nuclear extracts of wild-type and

Tfam1/2MEFs or Tfam fl/fl ER-cre1/2(cre1/2)

BMDMs exposed to4-hydroxytamoxifen(4OHT)

for96h.f,g,cGAS2/2MEFs reconstituted with

cGAS–HA were transfected with the indicated

siRNAs for96h,then stained with anti-DNA

(DNA),anti-HSP60(Mito.)and anti-HA(cGAS–

HA)antibodies and imaged.cGAS co-localization

scoring was performed as described in the

Methods.Error bars indicate6s.e.m.of triplicate

technical(a–d)or biological(g)replicates and are

representative of three independent experiments.

*P,0.05;**P,0.01;***P,0.001. RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

554|N A T U R E|V O L520|23A P R I L2015

Finally,we found that Tfam 1/2mice exhibit basally elevated ISG expres-sion,which confers resistance to acute infection by lymphocytic chor-iomeningitis virus (LCMV)Armstrong (Extended Data Fig.5a and Fig.3c).

To probe a direct requirement for mtDNA stress in antiviral prim-ing in TFAM-deficient cells,we used dideoxycytidine (ddC),a deox-yribonucleoside analogue that specifically inhibits mtDNA replication and decreases mtDNA nucleoid size 2,20.Treatment of wild-type MEFs with ddC resulted in reduced mtDNA copy number and decreased average nucleoid size without altering basal ISG expression (Extended Data Fig.5b–d).In contrast,ddC drastically diminished mtDNA stress (that is,enlarged nucleoids measuring greater than 450nm 2)in Tfam 1/2and TF D MEFs (Fig.3d and Extended Data Fig.5e),which was accom-panied by attenuation of antiviral priming and basal ISG expression (Fig.3e and Extended Data Fig.5d,f).Moreover,ddC ablated the viral resistance phenotype of Tfam 1/2MEFs (Fig.3f).We observed similar decreases in type I interferon production and a reduction in the viral resistance phenotype in ddC-treated TF D BMDMs (Extended Data Fig.5g,h,blue bars).These results demonstrate that mtDNA stress directly potentiates antiviral innate immunity.

The observation that ddC-treated wild-type BMDMs displayed reduced Ifnb and increased viral gene expression upon challenge with HSV-1,despite normal responses to cytosolic nucleic acids (Extended Data Fig.5h,i,grey bars),indicates that virus-induced mtDNA stress may boost host antiviral responses,consistent with reports linking viral in-fection to mtDNA dysregulation 21,22.The alphaherpesvirusprotein UL12.5,encoded by HSV-1and HSV-2,localizes to mitochondria and pro-motes rapid mtDNA depletion in human cells,which we confirmed in MEFs (Extended Data Fig.6a)22–24.Since mtDNA depletion and nu-cleoid stress are often coupled,we explored nucleoid architecture and

abundance kinetically during HSV-1infection.Notably,3h after chal-lenge with HSV-1,mtDNA stress was readily apparent,with nucleoids less evenly distributed and enlarged (Fig.4a).After 6h,,10%of nu-cleoids measured larger than 450nm 2,and there was a significant de-crease in total nucleoid intensity (Fig.4b).After 12h,we observed pronounced mtDNA depletion.The mtDNA stress observed 3to 6h after HSV-1challenge closely mirrored that of TFAM-deficient cells (Fig.4b),as did TFAM protein levels (Fig.4c).MHV-68and HSV-2triggered mtDNA stress similar to HSV-1,indicating that mtDNA stress is a common cellular perturbation during herpesvirus infection (Ex-tended Data Fig.6b,c).However,induction of mtDNA stress and TFAM depletion were not a general consequence of viral infection,as cells infected with VSV,influenza,LCMV or vaccinia possessed normal mtDNA architecture,TFAM expression and copy number (Fig.4a–c and Extended Data Fig.6c,d).

Finally,we sought to determine whether HSV-1-induced mtDNA dysregulation is necessary to fully engage antiviral signalling.Transduc-tion of MEFs and BMDMs with replication-incompetent retroviruses encoding only the mitochondria-targeted HSV-1UL12M185gene pro-duct was sufficient to cause mitochondrial hyperfusion,nucleoid en-largement and mtDNA loss,indicative of mtDNA stress (Fig.4d and Extended Data Fig.7a)24.UL12M185expression was also sufficient to trigger TFAM depletion and antiviral priming (that is,augmented ISG mRNA and protein expression)(Fig.4e and Extended Data Fig.7a).To explore the effect of HSV-1-induced mtDNA stress on innate anti-viral responses,we employed a recombinant,UL12-deficient HSV-1strain (D UL121UL98–FLAG)that is severely impaired in its ability to induce mtDNA stress but replicates similarly to a matched UL12-sufficient strain (Extended Data Fig.7b,c)25.Infection with D UL12HSV-1resulted in attenuated TBK1phosphorylation and type I interferon

R e l a t i v e e x p r e s s i

o n (%)

+ ddC

Mock

P e r c e n t a g e o f n u c l e o i d s 2

22

M H V 68 g e n o m e a b u n d a n c e x 102 (A .U .)

R e l a t i v e e x p r e s s i o n (%)WT Tfam +/–Phase

Phase

P e r c e n t a g e o f m a x .WT

Tfam +/–

Phase

Phase

a

TFAM

HSP60IFIT3MHV68

GFP

WT

Tfam +/–

M 6 h 18 h M 6 h 18 h

MHV68-GFP b

Kidney

VSV-VS S V GFP P F WT

Tfam +/–

Tfam +/– + ddC Phase

Phase Phase Phase

Phase Phase

d

e

f

Tfam +/–

mtDNA nucleoid area

Tfam +/–Liver

c

LCMV Armstrong 4 d post i.p. injection

P e r c e n t a g e o f m a x .R e l a t i v e e x p r e s s i o n (%)

Figure 3|mtDNA stress potentiates viral resistance.a ,Viral GFP expression in MEFs infected with HSV-1-GFP or VSV-GFP at multiplicity of infection (MOI)0.5for 24h.

b ,MHV68-GFP abundance and ISG expression in MEFs infected at MOI 0.5.A.U.,arbitrary units.

c ,LCMV Armstrong glycoprotein (GP)an

d nucleoprotein (NP)gen

e expression 4days after intraperitoneal (i.p.)infection o

f wild-type (WT)and Tfam 1/2mice;n 54.d ,e ,Nucleoid area (d )or ISG expression (e )of MEFs exposed to ddC for 96h.f ,ddC-exposed MEFs were infected with HSV-1-GFP or VSV-GFP at MOI 0.1and imaged after 24h.Error bars represent 6s.e.m.of triplicate technical (b ,e )or quadruplicate biological (c )replicates and are representative of two independent experiments.**P ,0.01;***P ,0.001.

LETTER RESEARCH

G 2015

Macmillan Publishers Limited.All rights reserved

23A P R I L 2015|V O L 520|N A T U R E |555

and ISG expression between 3to 6h post-infection,despite comparable early HSV-1gene expression (Fig.4f,g).However,after 24h,D UL12HSV-1genome abundance was roughly threefold higher compared to the UL12-sufficient control,consistent with impaired antiviral innate immunity (Fig.4h).Finally,D UL12HSV-1elicited less robust antiviral innate immune responses in the vagina and more readily spread to dorsal root ganglia of wild-type mice due to a deficit in mtDNA stress-dependent antiviral priming (Extended Data Fig.7d,e).These results reveal that herpesvirus-induced mtDNA stress is necessary to effectively engage ISG expression and antiviral priming,and suggest that cellular mon-itoring of mtDNA homeostasis represents an additional sensory mech-anism to robustly engage antiviral innate immunity.

In closing,our work uncovers a novel cellular response to mtDNA stress that engages the antiviral innate immune response.Specifically,we show that mtDNA stress,induced by herpesvirus infection and me-diated by loss of the mtDNA packaging protein TFAM,triggers a cGAS–STING–IRF3-dependent pathway to upregulate ISGs and potentiate type I interferon responses to viral infection (Extended Data Fig.8).Our results support a model whereby viral-mediated disruption of mtDNA homeostasis serves as a cell-intrinsic indicator of infection that works in parallel with canonical virus sensing to enhance antiviral innate immunity.Conversely,pathologic type I interferon signatures promote autoimmune diseases such as systemic lupus erythematosus,and altered ISG expression correlates with radiation-resistant and

R e l a t i v e e x p r

e s s i o n × 102

(f o l d )

HSV-1 (UL12–FLAG)

HSV-1 (

ΔUL12 + UL98–FLAG)

R e l a t i v e e x p r e s s i o n (f o l d )

H S V -1 g e n o m e a b u n d a n c e x 103 (A .U .)

R e l a t i v e e x p r e s s i o n (f o l d )

Empty RV

HSV-1 UL12 M185 RV

M o c k H

S V -1 3 h H S V -1 6 h V S V 6 h M o c

k

P e r c e n t a g e o f n u

c l e o i

d s

<200 nm 2

200–450 nm 2>450 nm

2

VSV HSV-1TFAM HSP60

M o c k H S V -1V S V M o c k

UL12 M185TFAM IFIT3α-Tubulin

HSV-1 UL12 M185 RV

Empty RV

++

+/–

WT Tfam +/–

mtDNA nucleoid area

Mock

HSV-1 3 h

HSV-1 6 h

HSV-1 12 h

VSV 6 h

Empty RV

HSV-1 UL12M185 RV 12 h HSV-1 UL12M185 RV 48 h

a

b

c

d

e

***

p-TBK1TBK1Calnexin UL12–FLAG HSV-1 (ΔUL12 + UL98–FLAG)

HSV-1 (UL12–FLAG)

6 h 6 h UL12.5–FLAG

UL98–FLAG 3 h 3 h M M ––––f

g

h

4 h post-infection

HSV-1 UL30 DNA

RNA

Figure 4|HSV-1induces mtDNA stress and TFAM depletion sufficient to trigger ISG expression and necessary to fully engage antiviral immunity.a –c ,Wild-type (WT)MEFs were mock infected or infected with HSV-1-GFP or VSV-GFP at multiplicity of infection (MOI)10for the indicated times,and imaged after staining with anti-DNA (DNA),anti-HSP60(Mito.),and anti-HSV (HSV-1)or GFP (VSV)antibodies (a ).mtDNA nucleoid area was

calculated as described in the Methods (b ).Extracts were blotted as indicated (c ).d ,e ,Wild-type MEFs were transduced with HSV-1UL12M185–FLAG-expressing or empty retroviruses (RV)and cells were stained with anti-DNA

(DNA),anti-HSP60(Mito.)and anti-FLAG antibody (UL12M185)(d ),and protein or ISG expression examined after 24h (e ).f ,g ,Protein and RNA expression in BMDMs infected with HSV-1(UL12–FLAG)or UL12-deficient HSV-1(D UL121UL98–FLAG)at MOI 2for the indicated times.h ,HSV-1genome abundance in L929cells that were infected as in f ,g .A.U.,arbitrary units.Error bars indicate 6s.e.m.of triplicate technical replicates and are representative of two independent experiments.***P ,0.001;NS,not significant.

RESEARCH LETTER

G 2015

Macmillan Publishers Limited.All rights reserved

556|N A T U R E |V O L 520|23A P R I L 2015

metastatic phenotypes in some cancers26,27.Mitochondrial and mtDNA dysregulation have been noted in systemic lupus erythematosus,and perturbations in TFAM and/or mtDNA homeostasis are frequently observed in cancer28–30.Therefore,further investigation into this path-way will not only expand our knowledge of innate antiviral defence,but may also broaden our understanding of how mitochondria contribute to the pathogenesis of human diseases and ageing beyond their well characterized roles in metabolism,apoptosis and reactive oxygen spe-cies production.

Online Content Methods,along with any additional Extended Data display items and Source Data,are available in the online version of the paper;references unique to these sections appear only in the online paper.

Received4July;accepted15December2014.

Published online2February;corrected online22April2015(see full-text

HTML version for details).

1.Spelbrink,J.N.Functional organization of mammalian mitochondrial DNA in

nucleoids:history,recent developments,and future challenges.IUBMB Life62, 19–32(2010).

2.Kasashima,K.,Sumitani,M.&Endo,H.Human mitochondrial transcription factor

A is required for the segregation of mitochondrial DNA in cultured cells.Exp.Cell

Res.317,210–220(2011).

3.Ryan,M.T.&Hoogenraad,N.J.Mitochondrial–nuclear communications.Annu.

Rev.Biochem.76,701–722(2007).

4.Wallace,D.C.A mitochondrial paradigm of metabolic and degenerative diseases,

aging,and cancer:a dawn for evolutionary medicine.Annu.Rev.Genet.39,

359–407(2005).

b87cb1ade2bd960591c67714rsson,N.G.et al.Mitochondrial transcription factor A is necessary for mtDNA

maintenance and embryogenesis in mice.Nature Genet.18,231–236(1998). 6.Woo,D.K.et al.Mitochondrial genome instability and ROS enhance intestinal

tumorigenesis in APC Min/1mice.Am.J.Pathol.180,24–31(2012).

7.Rusinova,I.et al.Interferome v2.0:an updated database of annotated interferon-

regulated genes.Nucleic Acids Res.41,D1040–D1046(2013).

8.Schoggins,J.W.&Rice,C.M.Interferon-stimulated genes and their antiviral

effector functions.Curr.Opin.Virol.1,519–525(2011).

9.West,A.P.,Shadel,G.S.&Ghosh,S.Mitochondria in innate immune responses.

Nature Rev.Immunol.11,389–402(2011).

10.Shimada,K.et al.Oxidized mitochondrial DNA activates the NLRP3

inflammasome during apoptosis.Immunity36,401–414(2012).

11.Nicholls,T.J.&Minczuk,M.In D-loop:40years of mitochondrial7S DNA.

Exp.Gerontol.56,175–181(2014).

12.Ban-Ishihara,R.,Ishihara,T.,Sasaki,N.,Mihara,K.&Ishihara,N.Dynamics of

nucleoid structure regulated by mitochondrial fission contributes to cristae

reformation and release of cytochrome c.Proc.Natl b87cb1ade2bd960591c67714A110,

11863–11868(2013).

13.Malena,A.,Loro,E.,Di Re,M.,Holt,I.J.&Vergani,L.Inhibition of mitochondrial

fission favours mutant over wild-type mitochondrial DNA.Hum.Mol.Genet.18, 3407–3416(2009).

14.Cymerman,I.A.,Chung,I.,Beckmann,B.M.,Bujnicki,J.M.&Meiss,G.EXOG,a

novel paralog of Endonuclease G in higher eukaryotes.Nucleic Acids Res.36,

1369–1379(2008).

15.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.Science339,786–791 (2013).

16.Ablasser,A.et al.TREX1deficiency triggers cell-autonomous immunity in a cGAS-

dependent manner.J.Immunol.192,5993–5997(2014).

17.Cai,X.,Chiu,Y.-H.&Chen,Z.J.The cGAS–cGAMP–STING pathway of cytosolic DNA

sensing and signaling.Mol.Cell54,289–296(2014).

18.Atianand,M.K.&Fitzgerald,K.A.Molecular basis of DNA recognition in the

immune system.J.Immunol.190,1911–1918(2013).19.Goubau,D.,Deddouche,S.&Reis e Sousa,C.Cytosolic sensing of viruses.Immunity

38,855–869(2013).

20.Pohjoisma¨ki,J.L.O.et al.Alterations to the expression level of mitochondrial

transcription factor A,TFAM,modify the mode of mitochondrial DNA replication in cultured human cells.Nucleic Acids Res.34,5815–5828(2006).

21.Wiedmer,A.et al.Epstein–Barr virus immediate-early protein Zta co-opts

mitochondrial single-stranded DNA binding protein to promote viral and inhibit mitochondrial DNA replication.J.Virol.82,4647–4655(2008).

22.Saffran,H.A.,Pare,J.M.,Corcoran,J.A.,Weller,S.K.&Smiley,J.R.Herpes simplex

virus eliminates host mitochondrial DNA.EMBO Rep.8,188–193(2007).

23.Corcoran,J.A.,Saffran,H.A.,Duguay,B.A.&Smiley,J.R.Herpes simplex virus

UL12.5targets mitochondria through a mitochondrial localization sequence

proximal to the N terminus.J.Virol.83,2601–2610(2009).

24.Duguay,B.A.&Smiley,J.R.Mitochondrial nucleases ENDOG and EXOG

participate in mitochondrial DNA depletion initiated by herpes simplex virus1 UL12.5.J.Virol.87,11787–11797(2013).

25.Duguay,B.A.et al.Elimination of mitochondrial DNA is not required for herpes

simplex virus1replication.J.Virol.88,2967–2976(2014).

26.Crow,M.K.&Kirou,K.A.Interferon-a in systemic lupus erythematosus.Curr.Opin.

Rheumatol.16,541–547(2004).

27.Khodarev,N.N.et al.Signal transducer and activator of transcription1regulates

both cytotoxic and prosurvival functions in tumor cells.Cancer Res.67,

9214–9220(2007).

28.Lee,H.-T.et al.Leukocyte mitochondrial DNA alteration in systemic lupus

erythematosus and its relevance to the susceptibility to lupus nephritis.Int.J.Mol.

Sci.13,8853–8868(2012).

29.Lee,H.-M.,Sugino,H.,Aoki,C.&Nishimoto,N.Underexpression of mitochondrial-

DNA encoded ATP synthesis-related genes and DNA repair genes in systemic lupus erythematosus.Arthritis Res.Ther.13,R63(2011).

30.Wallace,D.C.Mitochondria and cancer.Nature Rev.Cancer12,685–698(2012). Acknowledgements We thank N.Chandel for Tfam fl/fl mice,D.Martin for Tfam1/2 MEFs,J.Schoggins and S.Virgin for cGas2/2MEFs,G.Barber for Sting2/2MEFs,G.Sen for IFIT3antibodies,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-1gene expression analysis.This work was supported by a joint grant from the United Mitochondrial Disease Foundation and Mitocon,NIH R01AG047632and P01

ES011163(G.S.S.),NIH R01AI054359and R01AI081884(A.I.),Canadian Institutes for Health Research grant MOP37995and a Canada Research Chair in Molecular Virology(J.R.S.),American Cancer Society Postdoctoral Fellowship

PF-13-035-01-DMC(A.P.W.),NIH T32AI055403(W.K.-H.),NIH F31AG039163 (M.C.T.),NIH NRSA F32DK091042(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 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-1infections;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 cGas2/2MEFs;B.A.D.and J.R.S.generated and provided HSV-1UL12constructs and HSV-1D UL12viruses;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.

Author Information Microarray data have been submitted to the NCBI Gene Expression Omnibus under accession number GSE63767.Reprints and permissions information is available at b87cb1ade2bd960591c67714/reprints.The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper.Correspondence and requests for materials should be addressed to G.S.S.(gerald.shadel@b87cb1ade2bd960591c67714).

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

23A P R I L2015|V O L520|N A T U R E|557

METHODS

Animal strains.Tfam1/2and Tfam fl/fl mice were previously described and main-tained on a C57BL/6background6,31.Tfam fl/fl mice were bred to Estrogen receptor (ER)–Cre transgenic mice from Jackson(stock no.004682)for inducible,4OHT-mediated deletion.All animal experiments were conducted in compliance with guidelines established by the Yale University Institutional Animal Care and Use Committee.

Antibodies and reagents.Rabbit anti-mouse TFAM polyclonal anti-sera was pre-viously described6,rabbit anti-VSV polyclonal anti-sera was a gift from J.Rose at Yale University,mouse anti-Viperin was a gift from P.Cresswell at Yale Univer-sity,and rabbit-anti IFIT3was a gift from G.Sen at Cleveland Clinic.The following antibodies were obtained commercially:goat anti-HSP60(N-20)and rabbit anti-calnexin(H-70)(Santa Cruz Biotechnology);mouse and rabbit anti-FLAG(F1804, F7425)(Sigma);mouse anti-DNA(CBL186)(Millipore);mouse anti-GFP(JL-8) (BD Biosciences);rabbit anti-HSV-1/2(ab9533)and anti-histone H3(ab1791) (Abcam);rat anti-HA-FITC(11988506001)(Roche);rabbit anti-NLRX1(17215-1-AP)(Proteintech);mouse anti-a-tubulin(DM1A)(Neomarkers);mouse anti-GAPDH(6C5)(Ambion);and rabbit anti-DDX58(D14G6),-IFIH1(D74E4),-STAT1 (9172),-IRF3(D83B9),-TBK1(D1B4)and anti-phospho-TBK1(D52C2)(Cell Signaling Technology).Mouse IFN a enzyme-linked immunosorbent assay(ELISA) and recombinant mouse IFN b was from PBL Assay Science,and mouse IL-6ELISA was from eBioscience.All primer sequences and siRNAs used are found in Ex-tended Data Tables1and2.

Cell culture.Primary wild-type,Tfam1/2,Sting2/2and cGas2/2MEFs were gen-erated from E12.5–14.5embryos,maintained in DMEM(Invitrogen)supplemen-ted with10%FBS(Atlanta Biological),and sub-cultured no more than five passages before experiments.Sting2/2MEFs were provided by G.Barber at the University of Miami32.L929cells were obtained from ATCC and maintained in DMEM supple-mented with10%FBS.siRNA transfection of MEFs was performed with25nM siRNA duplexes(Integrated DNA Technologies)and Lipofectamine RNAiMAX reagent(Invitrogen)according to the manufacturer’s instructions.ddC(Sigma) was resuspended in PBS,added to MEFs or BMDMs at a final concentration of 10–20m M,and replenished every48h.BMDMs were generated from bone mar-row of8–12-week-old littermate Tfam fl/fl ER-cre2and Tfam fl/fl ER-cre1mice and cultured on Petri plates in DMEM containing10%FBS plus30%L929culture media. To induce Cre-mediated deletion,1m M4OHT dissolved in DMSO(Sigma)was added to BMDM cultures on day6and incubated for an additional2–3days.Cells were then lifted from plates by incubating in cold PBS containing1mM EDTA,re-plated in fresh media containing10%L929conditioned media,and allowed to rest overnight before experimentation(for a total of72or96h of4OHT exposure). Transfection of interferon-stimulatory DNA(ISD)33and poly(I:C)(Sigma)into the cytosol of BMDMs was performed using Lipofectamine2000(Invitrogen).In brief,13106BMDMs were seeded in6-well dishes after4OHT treatment,and transfected the next day with4m g ISD per well or2.5m g per well of poly(I:C)com-plexed at a ratio of2:1Lipofectamine2000to nucleic acid.Poly(I:C)transfection into the cytosol of MEFs was performed as described previously34.

Viral stocks and infections.VSV-G-GFP35,HSV-1-GFP36,MHV-68-GFP,HSV-237,vaccinia virus(strain WR)expressing bacteriophage T7RNA polymerase38,influ-enza A PR8NS1-GFP39,HSV-1(UL12–FLAG)and HSV-1(UL12D1UL98–FLAG)25 were maintained as described previously34,40,41.MEFs or BMDMs were infected at the indicated multiplicity of infection(MOI)in serum-free DMEM for1h,washed, and incubated for various times.Cells were then fixed and stained for microscopy, lysed for western blot,solubilized in buffer RLT Plus(Qiagen)for RNA extraction, or prepared for FACS analysis.FACS was performed by first trypsinizing MEFs, followed by labelling with LIVE/DEAD Fixable Far Red stain(Molecular Probes). Cells were then fixed with4%paraformaldehyde,washed,and analysed on a FACSCalibur flow cytometry machine(BD).FACS plots were first gated on live cells before analysing viral GFP fluorescence.Viral gene expression in BMDMs was determined using qRT–PCR as described below,except that after values were nor-malized against GAPDH cDNA using the2{DD C T method,all data points were subtracted by one to centre on zero.

LCMV Armstrong infection of wild-type and Tfam1/2mice was performed as described previously42.In brief,10-week-old female mice were infected with23 105plaque-forming units of virus intraperitoneally,and4days post-infection,mice were euthanized,tissues isolated,and total RNA prepared using RNeasy Plus kits (QIAGEN).After generating complementary DNA,samples were subjected to qPCR analysis as described below using published methods43,44.

In vivo HSV-1infection,dorsal root ganglia isolation and viral titration.Six-week old female mice were purchased from Charles River Laboratories and treated with Depo Provera(GE Healthcare)5days before infection45.The vaginal canals of Depo Provera treated mice were swabbed with a Calginate swab(Fisher)and106 plaque-forming units were delivered via pipette tip into the vagina.One day post-infection,vaginal tissue was isolated for RNA extraction.Infected mice were euthanized at indicated time points and dorsal root ganglia were dissected as pre-viously described46.Dorsal root ganglia were homogenized using a motorized pestle and total DNA was isolated using the DNeasy Blood&Tissue Kit(Qiagen)accord-ing to the manufacturer’s instructions.Relative HSV-1genome abundance was determined using primers specific for nuclear Tert and HSV-1TK(thymidine kinase).

cGAS–HA and UL12M185cloning and retroviral expression.A plasmid en-coding HSV-1UL12M185SPA containing a33FLAG tag at the carboxy-terminus was described previously24.This construct,or a plasmid encoding murine cGAS–HA(Invivogen),was sub-cloned into the pMXs-IRES-Puro vector and replication incompetent retroviruses were packaged using plat-E cells according to the man-ufacturer’s instructions(Cell Biolabs).SV40large T immortalized cGAS2/2MEFs were exposed to supernatants containing cGAS–HA retroviruses and incubated overnight.Two days post-transduction,3m g ml21puromycin was added to select a stable population of cells expressing cGAS–HA.Supernatants containing empty or UL12M185SPA retroviruses and4m g ml21polybrene were incubated with cells(53104MEFs or23105BMDMs)in12-well dishes for a period of8h.Viral supernatants were then washed off,fresh media was added to wells,and the cells were incubated for the duration of the experiment until lysis.

Quantitative PCR.To measure relative gene expression by qRT–PCR,total cellular RNA was isolated using RNeasy Plus RNA extraction kit(Qiagen).Approximately 400–2000ng RNA was normalized across samples and cDNA was generated using the High Capacity cDNA RT kit(Applied Biosystems).cDNA was then subjected to qPCR using Fast SYBR Green Master Mix(Applied Biosystems)and primers as indicated on the ViiA7Real Time PCR system(Life Technologies).Three technical replicates were performed for each biological sample,and expression values of each replicate were normalized against GAPDH cDNA using the2{DD C T method.For relative expression(fold),control samples were centred at1;for relative expression (%),control samples were centred at100%.Mitochondrial DNA copy number anal-ysis was performed as described using primers specific to nuclear Tert and the D-loop region of mtDNA(listed in Extended Data Table1)6.Relative HSV-1geno-me abundance was determined using primers specific for nuclear Tert and HSV-1 UL30or TK.Relative MHV68genome abundance was determined using primers specific for nuclear Tert and MHV68orf40.Relative vaccinia genome abundance was determined using primers specific for nuclear Tert and vaccinia virus DNA polymerase E9L.

Immunofluorescence microscopy.For all microscopy images containing mtDNA nucleoids and associated panels,cells were grown on coverslips and transfected, treated,and/or infected as described.After washing in PBS,cells were fixed with4% paraformaldehyde for20min,permeabilized with0.1%Triton X-100in PBS for 5min,blocked with PBS containing10%FBS for30min,stained with primary antibodies for60min,and stained with secondary antibodies for60min.Cells were washed with PBS between each step.Coverslips were mounted with Prolong Gold anti-fade reagent containing DAPI(49,6-diamidino-2-phenylindole;Molecular Probes).Cells were imaged on a Zeiss LSM510META with a633water-immersed objective.A digital scan zoom of3.0was used to enhance magnification.Images were pseudo-coloured and merged using ImageJ software(NIH).For microscopy images in Fig.3,MEFs were infected as described and fixed with4%paraformalde-hyde for20min.Viral GFP fluorescence and phase contrast images were captured using an Olympus IX-71inverted scope with a103(Fig.3a)or203(Fig.3f)objec-tive.Viral GFP images were pseudo-coloured using ImageJ.

For nucleoid area quantification,approximately10–15unique fields of view from 10distinct confocal images,comprising between200–400nucleoids,were captured at random.After incorporating scale information obtained from the LSM Image Browser(Zeiss),images were made binary and the area of each nucleoid was deter-mined using the‘Analyze Particles’feature of ImageJ.Nucleoids were divided into the three size cut-offs:,200nm2;200–450nm2;and.450nm2,and the percent-age of nucleoids falling within each category was plotted.The percentage of nu-cleoids.450nm2displaying cGAS co-localization was scored by calculating nucleoid area from5distinct images of siCtrl-and siTfam-transfected cGAS–HA recon-stituted MEFs as described above.Nucleoids larger than450nm2with a substan-tial cGAS co-localization signal were scored as positive.

Electron microscopy.MEFs grown in Petri dishes and on coverslips for orienta-tion were fixed in2.5%gluteraldehyde in0.1M sodium cacodylate buffer pH7.4 for1h.The cells were rinsed in sodium cacodylate and those in Petri dishes were scraped and spun down in2%agar.All samples were fixed in1%osmium tetroxide for1h,stained en masse in2%uranyl acetate in maleate buffer pH5.2for a further hour,rinsed and dehydrated in an ethanol series,and infiltrated with resin(Embed812 EMS)and baked over night at60u C.Hardened blocks were cut using a Leica UltraCut UCT.60-nm sections were collected on formver/carbon-coated grids and contrast stained using2%uranyl acetate and lead citrate.Samples were viewed on an FEI Tencai Biotwin TEM at80Kv.Images were taken using Morada CCD and iTEM (Olympus)software.

RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

For mitochondrial perimeter quantification,approximately10–15unique electron microscopy images of each genotype were captured at random.After incorporating scale information from iTEM software,the perimeter along the outer membrane of each mitochondrion was traced and quantified using ImageJ.Mitochondria were divided into the three size cutoffs:,2m m;2–5m m;and.5m m,and the percentage of mitochondria falling within each category was plotted.

Oxygen consumption analysis.Cells were plated in XF96plates(SeaHorse Bio-sciences)at10,000cells per well and the next day cellular O2consumption was de-termined in a SeaHorse Bioscience XF96extracellular flux analyser according to the manufacturer’s instructions.Cells were maintained at37u C in normal growth medium without serum.

Nuclear fractionation and western blotting.Whole-cell extracts were solubilized in SDS lysis buffer(20mM Tris-HCl,1%SDS,pH7.5,containing protease and phosphatase inhibitors),boiled for5min,and DNA was sheared by sonication.For nuclear extraction,PBS-washed cell pellets were resuspended in10pellet volumes of RSB buffer(10mM NaCl,1.5mM CaCl2,10mM Tris-HCl pH7.5),swelled on ice for10min,homogenized with a motorized Teflon pestle,and the homogenate was centrifuged at980g for10min to pellet nuclei.Pellets were washed five times in PBS,SDS was then added to a final concentration of1%,and extracts were boiled for5min before sonicating to shear DNA and normalizing protein concentration. Western blotting was performed using standard protocols,and HSP60was used as whole-cell extract loading controls,while histone H3was probed as a nuclear load-ing control.

Detection of mtDNA in cytosolic extracts.Digitonin extracts from MEFs and BMDMs were generated largely as described previously47.Wild-type and Tfam1/2 MEFs(73106)or Tfam fl/fl ER-cre1/2BMDMs exposed to4OHT for72h(13 107)were each divided into two equal aliquots,and one aliquot was resuspended in 500m l of50m M NaOH and boiled for30min to solubilize DNA.50m l of1M Tris-HCl pH8was added to neutralize the pH,and these extracts served as normal-ization controls for total mtDNA.The second equal aliquots were resuspended in roughly500m l buffer containing150mM NaCl,50mM HEPES pH7.4,and15–25m g ml21digitonin(EMD Chemicals).The homogenates were incubated end over end for10min to allow selective plasma membrane permeabilization,then centri-fuged at980g for3min three times to pellet intact cells.The first pellet was saved as the‘Pel’fraction for western blotting.The cytosolic supernatants were transferred to fresh tubes and spun at17000g for10min to pellet any remaining cellular debris, yielding cytosolic preparations free of nuclear,mitochondrial and endoplasmic re-cticulum contamination.DNA was then isolated from these pure cytosolic frac-tions using QIAQuick Nucleotide Removal Columns(QIAGEN).Quantitative PCR was performed on both whole-cell extracts and cytosolic fractions using nuclear DNA primers(Tert)and mtDNA primers(Dloop1-3,Cytb,16S and Nd4),and the C T values obtained for mtDNA abundance for whole-cell extracts served as nor-malization controls for the mtDNA values obtained from the cytosolic fractions. This allowed effective standardization among samples and controlled for any var-iations in the total amount of mtDNA in control and TFAM-deficient b87cb1ade2bd960591c67714ing this digitonin method,no nuclear Tert DNA was detected in the cytosolic fractions, indicating nuclear lysis did not occur.

Bioinformatic analyses.Total cellular RNA from wild-type and Tfam1/2litter-mate MEFs was prepared using RNeasy Plus RNA extraction kits(QIAGEN)and used for the expression microarray procedure in conjunction with the Emory Uni-versity Integrated Genomics Core.RNA integrity was first verified by an Agilent Bioanalyzer and then amplified,labelled,and hybridized onto Mouse Gene1.0ST arrays(Affymetrix)using standard protocols recommended by the manufacturer, starting from2m g of total RNA.Data were normalized by the RMA method using GeneSpring software(Agilent)for each biological sample in duplicate.Student’s t-test was used to determine statistically significant changes in expression in Tfam1/2MEFs relative to wild type,with a cut-off P value of0.0548.Heat maps were generated using MultiExperiment Viewer49.

Statistical analyses.Error bars displayed throughout the manuscript represent s.e.m.unless otherwise indicated,and were calculated from triplicate or quadru-plicate technical replicates of each biological sample.For in vivo experiments,error bars were calculated from the average of triplicate technical replicates of3–4mice per point.Sample sizes were chosen by standard methods to ensure adequate power, and no randomization or blinding was used for animal studies.No statistical me-thod was used to predetermine sample size.Statistical significance was determined using unpaired Student’s t-tests;*P,0.05;**P,0.01;***P,0.001;NS,not sig-nificant(P.0.05).Data shown are representative of2–3independent experiments, including microscopy images,western blots and viral challenges.

31.Weinberg,F.et al.Mitochondrial metabolism and ROS generation are essential for

Kras-mediated tumorigenicity.Proc.Natl b87cb1ade2bd960591c67714A107,8788–8793(2010).

32.Ishikawa,H.&Barber,G.N.STING is an endoplasmic reticulum adaptor that

facilitates innate immune signalling.Nature455,674–678(2008).

33.Stetson,D.B.&Medzhitov,R.Recognition of cytosolic DNA activates an IRF3-

dependent innate immune response.Immunity24,93–103(2006).

34.Tal,M.C.et al.Absence of autophagy results in reactive oxygen species-dependent

amplification of RLR signaling.Proc.Natl b87cb1ade2bd960591c67714A106,2770–2775(2009).

35.Dalton,K.P.&Rose,J.K.Vesicular stomatitis virus glycoprotein containing the

entire green fluorescent protein on its cytoplasmic domain is incorporated

efficiently into virus particles.Virology279,414–421(2001).

36.Desai,P.&Person,S.Incorporation of the green fluorescent protein into the herpes

simplex virus type1capsid.J.Virol.72,7563–7568(1998).

37.Shin,H.&Iwasaki,A.A vaccine strategy that protects against genital herpes by

establishing local memory T cells.Nature491,463–467(2012).

38.Fuerst,T.R.,Niles,E.G.,Studier,F.W.&Moss,B.Eukaryotic transient-expression

system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.Proc.Natl b87cb1ade2bd960591c67714A83,8122–8126(1986).

39.Pang,I.K.,Pillai,P.S.&Iwasaki,A.Efficient influenza A virus replication in the

respiratory tract requires signals from TLR7and RIG-I.Proc.Natl b87cb1ade2bd960591c67714A 110,13910–13915(2013).

40.Yordy,B.,Iijima,N.,Huttner,A.,Leib,D.&Iwasaki,A.A neuron-specific role for

autophagy in antiviral defense against herpes simplex virus.Cell Host Microbe12, 334–345(2012).

41.Cardenas,I.et al.Placental viral infection sensitizes to endotoxin-induced pre-term

labor:a double hit hypothesis.Am.J.Reprod.Immunol.65,110–117(2011). 42.Marshall,H.D.et al.Differential expression of Ly6C and T-bet distinguish effector

and memory Th1CD41cell properties during viral infection.Immunity35,

633–646(2011).

43.Welsh,R.M.&Seedhom,M.O.Lymphocytic choriomeningitis virus(LCMV):

propagation,quantitation,and storage.Curr.Protoc.Microbiol.Unit15A.1, b87cb1ade2bd960591c67714/10.1002/9780471729259.mc15a01s8(2008).

44.McCausland,M.M.&Crotty,S.Quantitative PCR technique for detecting

lymphocytic choriomeningitis virus in vivo.J.Virol.Methods147,167–176(2008).

45.Parr,M.B.et al.A mouse model for studies of mucosal immunity to vaginal

infection by herpes simplex virus b87cb1ade2bd960591c67714b.Invest.70,369–380(1994).

46.Malin,S.A.,Davis,B.M.&Molliver,D.C.Production of dissociated sensory neuron

cultures and considerations for their use in studying neuronal function and

plasticity.Nature Protocols2,152–160(2007).

47.Holden,P.&Horton,W.A.Crude subcellular fractionation of cultured mammalian

cell lines.BMC Res.Notes2,243(2009).

48.Raimundo,N.et al.Mitochondrial stress engages E2F1apoptotic signaling to

cause deafness.Cell148,716–726(2012).

49.Saeed,A.I.et al.TM4:a free,open-source system for microarray data management

and analysis.Biotechniques34,374–378(2003).

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure1|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 Tfam1/2MEFs.b,Basal oxygen consumption rate of wild-type and Tfam1/2MEFs as determined by Seahorse Bioscience XF96Extracellular Flux assay.c,qRT–PCR of mtDNA-encoded rRNA(16s)and mRNA(ND6,Cytb,Cox1)transcripts in wild-type and Tfam1/2MEFs.d–f,Untransfected Tfam1/2(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 are33magnification 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-cre2or Tfam flox/flox ER-cre1 BMDMs were incubated in4OHT for96h 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 Il6expression in

4OHT-treated Tfam fl/fl ER-cre2/1BMDMs2h post-cytosolic delivery of interferon-stimulatory DNA(ISD)or poly(I:C)(PIC)(i).Error bars

RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure2|TFAM deficiency promotes accumulation of cytosolic mtDNA.a,Wild-type(WT)or Tfam1/2MEFs 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 Tfam1/2MEFs or Tfam fl/fl ER-cre2or Tfam fl/fl ER-cre1BMDMs incubated in 4OHT for72h.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 indicate6s.e.m.of triplicates and data are representative of three independent experiments.**P,0.01,***P,0.001.

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure3|Mitochondrial hyperfusion regulates the accumulation of mtDNA nucleoid stress in TF D MEFs.a,b,Wild-type(WT) MEFs were transfected with control or Tfam siRNAs for96h.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,Mfn1and/or Tfam siRNAs for96h.Cells were fixed and stained with an anti-HSP60antibody(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 Tfam1/2MEFs were transfected with the indicated siRNAs for96h and ISG expression analysed by qRT–PCR.Error bars indicate6s.e.m.of triplicates and data are representative of two independent experiments.**P,0.01;***P,0.001.

RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure4|mtDNA stress in TF D MEFs and BMDMs potentiates type I interferon responses to viral infection and enhances viral clearance.a,b,Wild-type(WT)and Tfam1/2MEFs 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-cre2or Tfam fl/fl ER-cre1BMDMs were incubated in4OHT for96h 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 indicate6s.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.

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure5|Tissues from Tfam1/2mice display elevated ISG expression,and ddC abrogates mtDNA stress,ISG expression and viral resistance phenotypes of TF D cells.a,RNA was extracted from the liver and kidneys of8-week-old wild-type(WT)and Tfam1/2mice(n52each)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 Tfam1/2MEFs exposed to ddC for96h.e,f,mtDNA nucleoid area(e) and ISG expression(f)of wild-type MEFs transfected with control or Tfam siRNAs for96h in the presence or absence of ddC.g–i,Tfam fl/fl ER-cre2or Tfam fl/fl ER-cre1BMDMs were incubated in4OHT for96h 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 MOI1,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 indicate6s.e.m.of triplicates and data are representative of two independent experiments.*P,0.05;**P,0.01;***P,0.001; NS,not significant.

RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure6|Alpha-and gammaherpesviruses induce mtDNA stress,but influenza,LCMV,and vaccinia do not.a,Relative mtDNA copy number of wild-type(WT)MEFs24h 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 MOI0.5.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 MOI10.After6h,cells were stained and subjected to confocal microscopy.d,Wild-type MEFs were infected with vaccinia virus at MOI10(for microscopy)or1.After the indicated times cells were stained and subjected to confocal microscopy or the relative mtDNA copy number was determined.Error bars indicate6s.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.

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure7|HSV-1UL12M185expression is sufficient to trigger mtDNA stress,TFAM depletion and antiviral priming in BMDMs; infection with UL12-deficient HSV-1fails 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(D UL121UL98–FLAG)at MOI10for3h and analysed by confocal microscopy.c,Wild-type MEFs were infected with HSV-1(UL12–FLAG)

or UL12-deficient HSV-1(D UL121UL98–FLAG)at MOI2for24h and mtDNA abundance was determined by qPCR.d,The vaginas of wild-type mice (n53per condition)were inoculated with106plaque-forming units of HSV-1 (UL12–FLAG)or UL12-deficient HSV-1(D UL121UL98–FLAG)and

24h post-infection,vaginal RNA was extracted and gene expression analysed by qRT–PCR.e,Mice(n53per condition)were infected as previously described and6days post-infection,DNA from dorsal root ganglia was isolated for mtDNA and HSV-1genome abundance measurements by qPCR.

Error bars indicate6s.e.m.of triplicates and data are representative of

two independent experiments.*P,0.05;**P,0.01;***P,0.001;

NS,not significant.

RESEARCH LETTER

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Figure8|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 IRF3to induce dimerization and nuclear translocation.Active IRF3elevates basal gene expression of ISGs with antiviral signalling and effector functions.Signalling molecules encoded by ISGs,such as IRF7,ISG15,STAT1and STAT2, cooperate with IRF3to 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,IFIT3and OASL2, augment viral resistance.Both outcomes collectively and robustly boost innate antiviral defences to dampen viral replication.

LETTER RESEARCH

G2015Macmillan Publishers Limited.All rights reserved

Extended Data Table 1|Oligonucleotides used in

qPCR RESEARCH LETTER

G 2015Macmillan Publishers Limited.All rights reserved

Extended Data Table 2|Dicer substrate siRNAs

used

All siRNAs were predesigned by Integrated DNA Technologies and transfected at 25nM final concentration.LETTER RESEARCH

G 2015Macmillan Publishers Limited.All rights reserved

本文来源：https://www.bwwdw.com/article/sf4e.html