Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity

26 Pages·2010·5.65 MB·English

Peroxisomes Are Signaling Platforms
for Antiviral Innate Immunity
EvelynDixit,1,2SteeveBoulant,3YijingZhang,1AmyS.Y.Lee,3,4CharlotteOdendall,1BennettShum,5NirHacohen,5
ZhijianJ.Chen,6,7SeanP.Whelan,3,4MarcFransen,8MaxL.Nibert,3,4GiulioSuperti-Furga,2andJonathanC.Kagan1,*
1HarvardMedicalSchoolandDivisionofGastroenterology,Children’sHospitalBoston,Boston,MA02115,USA
2CeMM-ResearchCenterforMolecularMedicineoftheAustrianAcademyofSciences,1090Vienna,Austria
3DepartmentofMicrobiologyandMolecularGenetics,HarvardMedicalSchool,Boston,MA02115,USA
4TrainingPrograminVirology,DivisionofMedicalSciences,HarvardUniversity,Boston,MA02115,USA
5BroadInstituteofMITandHarvard,Cambridge,MA02142,USA
6DepartmentofMolecularBiology
7HowardHughesMedicalInstitute
UniversityofTexasSouthwesternMedicalCenter,Dallas,TX75390,USA
8KatholiekeUniversiteitLeuven,FaculteitGeneeskunde,DepartementMoleculaireCelbiologie,LIPIT,CampusGasthuisberg(O&N1),
3000Leuven,Belgium
*Correspondence:[email protected]
DOI10.1016/j.cell.2010.04.018
SUMMARY tionacrossalipidbilayer.However,animportantgapexistsin
ourknowledgeofhowstimulifromthecytosolareabletoinitiate
Peroxisomes have long been established to play specificsignalingevents.
a central role in regulating various metabolic activi- Howcommonistheuseoforganellesinsignaltransduction
tiesinmammaliancells.Theseorganellesactincon- from cytosolic receptors? An example of this situation can be
cert with mitochondria to control the metabolism of foundinthestudyofvirus-hostinteractions.Theabilitytodetect
lipids and reactive oxygen species. However, while cytosolicvirusesdependsontheRIG-I-likereceptor(RLR)family
ofproteins,whicharesolubleRNAhelicasesthatdetectviruses
mitochondria have emerged as an important site of
containing RNA (and in some cases DNA) genomes (Ablasser
antiviral signal transduction, a role for peroxisomes
etal.,2009;Chiuetal.,2009;Yoneyamaetal.,2004).Thebest
in immune defense is unknown. Here, we report
characterizedRLRs,RIG-IandMDA-5,detect5-triphosphate-
0
that the RIG-I-like receptor (RLR) adaptor protein
containingshortdouble-strandedRNA(dsRNA)andlongdsRNA,
MAVSislocatedonperoxisomesandmitochondria.
respectively(Katoetal.,2008;Pichlmairetal.,2006).RLRscan
We find that peroxisomal and mitochondrial MAVS eitherdetectviralRNAdirectlyorafterRNApolymeraseIII-medi-
act sequentially to create an antiviral cellular state. atedtranscriptionofmicrobialDNA(Ablasseretal.,2009;Chiu
Uponviralinfection,peroxisomalMAVSinducesthe etal.,2009;Katoetal.,2008).Micedeficientineitherofthese
rapid interferon-independent expression of defense RLRsaresensitivetodifferentclassesofviruses,underscoring
factors that provide short-term protection, whereas boththeirspecificityofactionandtheirimportanceinimmune
mitochondrial MAVS activates an interferon-depen- defense(Gitlinetal.,2006;Katoetal.,2006).
Although RIG-I and MDA-5 have specificities for different
dentsignalingpathwaywithdelayedkinetics,which
ligands,bothinduceacommonsignalingpathwaythattriggers
amplifies and stabilizes the antiviral response. The
the expression of type I interferons (IFNs) and IFN-stimulated
interferon regulatory factor IRF1 plays a crucial role
genes (ISGs). Many ISGs function as direct antiviral effec-
inregulatingMAVS-dependentsignalingfromperox-
tors, acting to prevent viral genome replication, viral particle
isomes. These results establish that peroxisomes
assembly, or virion release from infected cells. Generally, it is
areanimportantsiteofantiviralsignaltransduction. thought that RLRs induce the expression of IFNs that act in
both autocrine and paracrine manners to amplify ISG expres-
INTRODUCTION sion. However, ISGs can also be induced directly upon viral
infection, without the need for IFN signaling (Collins et al.,
A fundamental feature of eukaryotic cells is the use of mem- 2004; Mossman et al., 2001). At the receptor-proximal level,
brane-bound organelles to compartmentalize activities and RLR-dependent responses are regulated by the adaptor pro-
serveasscaffoldsforsignaltransduction.Thebest-character- tein MAVS (also called IPS-1, Cardif, or VISA) (Nakhaei et al.,
ized signaling pathways involve membrane-bound receptors 2009).Uponviraldetection,MAVSbindstoRLRsandpromotes
that respond to extracellular or lumenal stimuli. In these theactivationofNF-kB,AP-1,andvariousinterferonregulatory
instances, the spatial separation of an extracellular stimulus factors(IRFs),whichacttoinduceISGsandcreateanantiviral
from the cytosol mandates the use of organelles as signaling state in the cell. Although much has been learned about the
platforms,astransmembranereceptorsmusttransmitinforma- genetics of RLR signaling, less is known about where within
668 Cell141,668–681,May14,2010ª2010ElsevierInc.
thecellsignaltransductionoccurs.IdentifyingthesitesofRLR etal.,2001;Horngetal.,2001)wasnotdetectedonperoxisomes
signal transduction is critical to understanding how antiviral (Figure 1A). To confirm that the peroxisomal staining was dis-
networks are integrated into the general cellular infrastructure tinctfrommitochondria,wealsostainedcellswithMitoTracker.
withinwhichtheyoperate. AlthoughnocostainingwasdetectedbetweenPTS1andMito-
The first clue that cytosolic RLR signaling may occur from Tracker, MAVS was detected on both PTS1-positive peroxi-
organelles came from studies of the MAVS adaptor. MAVS somesandMitoTracker-positivemitochondria(Figure1B).Sim-
contains a C-terminal transmembrane domain that anchors it ilarresultswereobtainedwhenepitope-taggedMAVSinmurine
to the mitochondrial outer membrane (Seth et al., 2005). It is macrophages(FigureS1availableonline)orendogenousMAVS
fromthislocationthatMAVSisthoughttoengageactiveRLRs inhumanhepatocyteswereexamined(Figure1C).Asaninde-
and induce signal transduction. Whether mitochondria are the pendent means of assessing MAVS localization, hepatocytes
only organelles that promote RLR-mediated signaling has not were biochemically fractionated to separate peroxisomes and
beenaddressed. mitochondria, whichwere respectivelydistinguished byPex14
Mitochondriahavelongbeenappreciatedtohaveanintimate andmtHSP70(Figure1D).BothMAVSandFis1(aproteinthat
functional relationship with peroxisomes (Hettema and Motley, occupiesbothorganelles[Kochetal.,2005])weredetectedin
2009).Botharemembrane-boundorganellesfoundinmammalian fractionscontainingeitherperoxisomesormitochondria.Collec-
cells and are involved in the metabolism of lipids and reactive tively,onthebasisofstudiesinbothhumanandmousecells,
oxygenspecies.However,whilemitochondriaarewell-established thesedataestablishthatperoxisomesareabonafidereservoir
sites of both antiviral signaling and antiviral apoptosis, peroxi- oftheRLRadaptorproteinMAVS.
somesarethoughttofunctionsolelyasmetabolicorganelles. OnepossiblereasonMAVSispresentonperoxisomesisthat
Recently,severalmitochondrialproteinshavebeenfoundto newlysynthesizedMAVSmightfirstpassthroughperoxisomes
residealsoonperoxisomes.Includedinthisgrouparetheouter en route to mitochondria. To address this possibility, we used
membraneproteinsFis1andMff,whichregulatethemorphology human fibroblasts from a patient lacking a functional Pex19
ofbothorganelles(Gandre-BabbeandvanderBliek,2008;Koch protein.Pex19controlsperoxisomebiogenesis,andthusPex19-
etal.,2005).Interestingly,Fis1,Mff,andMAVSallhavesimilar deficientcellscontainnoperoxisomesorperoxisomalremnant
domainstructures:eachcontainsanN-terminaleffectordomain structures (Matsuzono et al., 1999; Sacksteder et al., 2000).
and a C-terminal localization motif, which consists of a trans- Notably,MAVSwasdeliveredtomitochondriainPex19-deficient
membrane domain and a short lumenal tail containing basic cells (Figure 1E), indicating that the pathway to mitochondria
aminoacids.Thatotherso-called‘‘tail-anchored’’mitochondrial doesnotrequireaperoxisomalintermediate.Moreover,MAVS
outermembraneproteinsoperatefromperoxisomesraisedthe localizedtobothperoxisomesandmitochondriainPex19-defi-
possibilitythatMAVSalsofunctionsfromtheseorganelles. cientcellsthatexpressedPex19aftertransient transfectionor
WehavediscoveredthatMAVSdoesindeedresideonperox- retroviral gene transfer (Figure 1E). It is therefore unlikely that
isomes and can induce antiviral signaling from this organelle. localizationofMAVStoperoxisomesistheresultofabiosynthetic
OurworksupportsamodelwherebyperoxisomalMAVSinduces pathwayfordeliveringoutermembraneproteinstomitochondria.
the immediate expression of antiviral factors that function to
containanascentinfection.Long-termcontainmentoftheinfec- ASystematicStrategytoSeparateFunctions
tion,however, requiresthe function of mitochondrial MAVSas ofPeroxisomalandMitochondrialMAVS
well.Thesedatademonstratethatperoxisomesarenotsimply Our finding that MAVS is located on peroxisomes raised the
metabolic organelles, but rather serve as critical subcellular possibilitythattheseorganellesserveasasiteofantiviralsignal
hubsthatpromoteMAVS-dependentantiviralimmunity. transduction. We first considered using Pex19-deficient cells
to address sufficiency of mitochondrial MAVS in antiviral sig-
RESULTS naling,butsinceperoxisomesarerequiredforbiochemicalpro-
cesses that occur in mitochondria, Pex19-deficient cells have
MAVSIsLocatedonBothMitochondria profound defects in mitochondrial function (Wanders, 2004).
andPeroxisomes Wethereforeusedthealternativeapproachofgeneticallysepa-
MAVShasasimilardomainorganizationtoothertail-anchored rating the putative mitochondrial and peroxisomal functions of
membraneproteinsthatfunctionfrommitochondriaandperox- MAVS. This was accomplished by replacing the previously
isomes (Gandre-Babbe and van der Bliek, 2008; Koch et al., definedMAVSlocalizationmotif(Sethetal.,2005)withasetof
2005). We therefore sought to determine whether MAVS also domainsthatinsteaddirecttheproteintoasinglecompartment
resides on peroxisomes. The subcellular localization of MAVS (Figure 2A). Using the localization motif of the peroxin Pex13
was examined in mouse embryonic fibroblasts (MEFs) whose (Fransen et al., 2001), we created a protein called MAVS-Pex.
peroxisomesweremarkedbyaDsRedallelecontainingatype BydeletingtheMAVSlocalizationmotif,wealsocreatedacyto-
1 peroxisomal targeting signal (PTS1). In addition to staining solicallele(MAVS-Cyto)(Sethetal.,2005).Becausethefidelity
structures that appeared to be mitochondria, MAVS was of mitochondrial sorting signals is not always transferrable to
detected on PTS1-positive peroxisomes scattered throughout other proteins (Ingelmo-Torres et al., 2009), we lastly created
thecell(Figure1A).AsimilarstainingpatternwasseenforMff twodifferentallelesofMAVScontainingasortingsignalderived
(Figure 1A), which functions fromboth peroxisomes and mito- fromtwoproteinsresidingonthemitochondrialoutermembrane
chondria(Gandre-BabbeandvanderBliek,2008).Incontrast, protein, either OMP25 or Fis1 (Koch et al., 2005; Nemoto and
the Toll-like receptor (TLR) adaptor protein TIRAP (Fitzgerald DeCamilli,1999).
Cell141,668–681,May14,2010ª2010ElsevierInc. 669
A C
PTS1 MAVS merge PTS1 MAVS merge
PTS1 MFF merge
D
Fraction: 3 8 9 14 17 18 19 21
IB: Pex14
PTS1 TIRAP merge
IB: mtHSP70
IB: Fis1
IB: MAVS
B
PTS1 Mitotracker MAVS merge
E
mtHSP70 MAVS merge PTS1 MAVS merge
Pex19 deficient fibroblasts Pex19 transient expression
mtHSP70 MAVS merge PTS1 MAVS merge
Pex19 stable expression Pex19 stable expression
Figure1. MAVSresidesonmitochondriaandperoxisomes
(A)MEFsweretransfectedwiththeperoxisomalmarkerDsRed-PTS1andFlag-MAVS,myc-MFF,orFlag-TIRAP.Cellswerestainedwithanti-MAVS,anti-myc,or
anti-Flagantibodies,respectively.Allimagesforallpanelsarerepresentativeofatleastthreeindependentexperimentsinwhichover500cellswereexaminedper
conditionand>95%ofthecellsdisplayedsimilarstaining.
(B)MEFsexpressingFlag-MAVSaswellasEGFP-PTS1andPex19fromabicistronicconstructwerestainedwithanti-MAVSantibodyandMitoTrackertovisu-
alizemitochondria.
(C)Huh-7hepatocytesweretransfectedwithDsRed-PTS1andendogenousMAVSwasdetectedwithanti-MAVSantisera.
(D)PeroxisomeswereseparatedfrommitochondriaonaNycodenzgradientwithHepG2hepatocytelysates.Selectedfractionsofthegradientwereanalyzedby
immunoblottingwithPex14,mtHSP70,Fis1,orMAVSantisera.
(E)Pex19-deficienthumanfibroblastswerestainedforendogenousMAVSbeforeandafterintroductionofafunctionalPex19alleleasindicated.Mitochondria
werestainedwithanti-mtHSP70antibody.PeroxisomeswerevisualizedbytransfectionwithabicistronicconstructencodingEGFP-PTS1andPex19.
SeealsoFigureS1.
UsingretroviralgenetransferofMAVS-KOMEFs,wecreated focal microscopy. Full-length MAVS (MAVS-WT) was located
cell lines expressing comparable levels of each MAVS allele on both mitochondria and peroxisomes (data not shown), and
(Figures2Band2C)anddeterminedtheirlocalizationsbycon- MAVS-Cyto was found on neither organelle (Figure 2D and
670 Cell141,668–681,May14,2010ª2010ElsevierInc.
A B Figure 2. Targeting of MAVS to Distinct
MAVS-WT 1 10 CARD 77 500514TM535540 130 MMAAVVSS--WMiTmic SofuIbtcseTllrualnasrmCeommbpraarntmeeDnotmsbayinReplacement
97 MMAAVVSS--CMyittoo (A)SchematicofWTandmutantMAVSallelesto
MAVS-Pex
MAVS-Mimic 1 10 CARD 77 500OMP25 537 65 MAVS-KO bmeitotcehsotenddrifao.r signaling from peroxisomes and
32 (B)StablecelllinesexpressingtheMAVSalleles
MAVS-Cyto 1 10 CARD 77 500 0100 101 102 103 ltiisotnedofinM(AA)VwSe-KreOgceenlelsra.tReedsbuyltirnegtrtorvainraslgterannicsdceulcls-
10 77 500 CGenotype: WT Mimic Cyto Mito Pex KO etioxpnreisssdeirdeactMedAVbSyaalnlelIeRaEnSd.GSFhPow,wnhaorseeotrvaenrslalaid-
MAVS-Mito 1 CARD Fis1526 histogramsofstablepopulationsofeachcellline
IB: MAVS expressing equivalent levels of the bicistronic
10 77 500 IB: Actin mRNAsencodingMAVSandGFP.
MAVS-Pex 1 CARD Pex13 598 (C)Lysatesfromstablecelllinesdescribedin(B)
andparentalMAVSKOMEFswereanalyzedby
D immunoblottingwithanti-MAVSantibody.
(D)MicrographsofMAVSchimericcelllinesindi-
PTS1 MAVS merge mtHSP70 MAVS merge
x cated were stained with anti-MAVS antibody.
e
P Mitochondria were stained with anti-mtHSP70
S-
V antibody.Peroxisomeswerevisualizedbytrans-
A
M fection with DsRed-PTS1. Note that MAVS-Pex
resides on peroxisomes, MAVS-Mito on mito-
PTS1 MAVS merge mtHSP70 MAVS merge chondria, MAVS-Mimic on both organelles, and
o
Mit MAVS-Cytolocalizesonneitherofthetwoorgan-
S- elles.Allimagesforallpanelsarerepresentative
AV ofatleastthreeindependentexperimentswhere
M
over500cellswereexaminedperconditionand
>95%ofthecellsdisplayedsimilarstaining.
PTS1 MAVS merge mtHSP70 MAVS merge
mic SeealsoFigureS2.
Mi
S-
V
A
M
both RIG-I and MDA-5 signaling path-
PTS1 MAVS merge mtHSP70 MAVS merge
o ways (Loo et al., 2008), allowing direct
yt
C examination of both RLRs in a single
S-
AV experiment.
M
Cells were infected with reovirus, and
extractswereexaminedatvarioustimes
for expression of viperin, a well-charac-
Figure S2). As expected, MAVS-Pex was found exclusively on terized ISG (Chin and Cresswell, 2001; Severa et al., 2006).
peroxisomes(Figure2DandFigureS2).Oftheallelescontain- MAVS-WT-, -Mimic-, or -Mito-expressing cells induced viperin
ing the putative mitochondrial targeting sequences, the allele expression in response to infection (Figure 3A). This response
harboringtheFis1transmembranedomainwasfoundprimarily wasMAVSdependent,asMAVS-KOcellsshowednochangein
on mitochondria, whereas the one containing the OMP25 viperin expression. MAVS-Cyto cells were unableto induce vi-
transmembranedomainwaslocatedonbothmitochondriaand perinexpression,confirmingthatmembranelocalizationisneces-
peroxisomes(Figure2DandFigureS2).Wethereforerefertothe saryforMAVSfunction(Sethetal.,2005).Interestingly,despite
mitochondria-specificalleleasMAVS-Mitotoindicateitsexclu- the fact that MAVS-Pex is found onlyon peroxisomes, MAVS-
sive localization to mitochondria and the allele found on both Pexcellsinducedviperinexpressionafterinfection(Figure3A).
organelles as MAVS-Mimic to indicate its ability to copy the An examination of the kinetics of ISG induction indicated
localization pattern of MAVS-WT. Collectively, this set of thatcellscontainingMAVSonperoxisomes(MAVS-WT,-Mimic,
MAVS-expressingMEFlinesdiffersonlyinthesubcellularposi- and -Pex) induced viperin expression within 4 hr of infection.
tioningofthesignaling domainofMAVSandtherebyprovides Incontrast,exclusivelocalizationtomitochondria(MAVS-Mito)
anidealsystemtodeterminetherelativerolesofmitochondrial resultedinviperinexpressionwithdelayedkinetics(Figure3A).
and peroxisomal localization in MAVS-dependent signal TheseresultssuggestthatlocalizationofMAVStoeitherperox-
transduction. isomesormitochondriaissufficienttoinduceantiviralsignaling
butthatperoxisomalresidenceallowsformorerapidexpression
MAVS-DependentSignalingOccursfromBoth of ISGs. Interestingly, rapid expression of ISGs by MAVS-Pex
PeroxisomesandMitochondria appeared to be transient, as viperin expression decreased at
ToaddressthefunctionofperoxisomalMAVS,wemonitoredthe latertimesofinfection(Figure3A).
expression of antiviral factors in response to infection with To determine whether peroxisomal signaling by MAVS
reovirus. We chose reovirus because it is a known inducer of requires signaling by both RIG-I and MDA-5, we performed
Cell141,668–681,May14,2010ª2010ElsevierInc. 671
A Figure 3. Peroxisomal MAVS Mediates ISG
Genotype: WT Cyto Mito Pex Mimic KO Expression,butDoesNotInduceTypeIIFN
Reovirus Secretion
T3D (hpi): 0 4 9 16 0 4 9 16 0 4 9 16 0 4 9 16 0 4 9 16 0 4 9 16 (A) MAVS-expressing MEFs and MAVS-KO cells
IB: Viperin were infected with reovirus. At indicated times,
cell-associatedISGexpressionwasdeterminedby
IB: Actin immunoblottingwithananti-viperinantibody.
(B)Similarto(A)exceptforinfectionwithinfluenza
Lane: 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 virusstrainDNS1inlieuofreovirus.
(CandD)Cellculturemediafrom(A)and(B)were
B
testedfortypeIIFNactivityusingabioassay.Error
Genotype: WT Cyto Mito Pex Mimic KO
barsshowthestandarddeviationoftriplicateinfec-
0 4 9 18 0 4 9 18 0 4 9 18 0 4 9 18 0 4 9 18 0 4 9 18 tions.
(E)MAVS-expressingMEFsandparentalMAVS-KO
IB: Viperin
cellsweretreatedwith100IU/mlIFNb.Atindicated
times, cell-associated ISG expression was deter-
IB: Actin
mined by immunoblotting with anti-viperin anti-
Lane: 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 body. Note that all cell lines respond similarly to
IFNb,indicatingintacttypeIIFNsignaling.
C D Alldataaretheresultofatleasttwoindependent
600 40
Reovirus (hpi): Influenza virus (hpi): experiments.SeealsoFigureS3.
350 0 0
(IU/ml)10200 491264 (IU/ml) 2300 491264
N- N-
IF 10 IF prediction, reovirus infection induced
10
peroxisomal aggregation and the forma-
0 0 N.DN.DN.DN.D N.DN.DN.D N.DN.DN.DN.D N.D N.DN.DN.DN.D tion of peroxisomal tubules (Figures S3A
WT Cyto Mito Pex Mimic KO WT Cyto Mito Pex Mimic KO and S3B). The tubes formed ranged from
approximately 2 mm in length to over 5
E mm and depended on MAVS localization
Genotype: WT Cyto Mito Pex Mimic KO to peroxisomes. Cells expressing MAVS-
0 4 9 24 0 4 9 24 0 4 9 24 0 4 9 24 0 4 9 24 0 4 9 24 WT exhibited this activity, and cells ex-
IB: Viperin pressingMAVS-Pexhavegreatlyexagger-
atedbehaviorintheseassays,withnearly
IB: Actin
all cells displaying peroxisomes over 5
Lane: 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 mminlength(FiguresS3AandS3B).Cells
expressingMAVS-MitoorMAVS-Cytoex-
hibited little or no change in peroxisome
similar experiments using influenza virus, which activates the morphology. These data suggest that RLRs engage peroxi-
RIG-Ipathwayexclusively(Gitlinetal.,2006;Katoetal.,2006). somalMAVStoinduceperoxisomaltubulesandthattheextent
Asimilarpatternofviperinexpressionwasobservedwithinflu- of tubulation is determined by the concentration of MAVS on
enzaaswithreovirus,althoughthekineticdifferencesbetween these organelles. These independent assays demonstrate that
MAVS-Pexand-Mitowereevenmorepronouncedwithinfluenza MAVS-dependentsignalingoccurslocally(ontheperoxisome).
(Figure 3B). Thus, RIG-I signaling alone is sufficient to induce
MAVS-dependent signaling from peroxisomes. In sum, these PeroxisomalMAVSTriggersanIFN-Independent
data indicate that peroxisomal MAVS induces rapid but tran- SignalingPathwaythatPromotesISGExpression
sientviperinexpression,whereasmitochondrialMAVSinduces The different kinetics of viperin induction by peroxisomal and
delayed but stable viperin expression. Signaling from both mitochondrial MAVS suggest that more than one mechanism
organelles thuscontributes tothe rapidand stableexpression of RLR-induced ISG expression may operate in virus-infected
ofviperinthatisobservedinMAVS-WTcells. cells. ISG expression can be induced directly, or it can be
Whiletheabovestudiesprovidestronggeneticevidencefor induced indirectly through the action of secreted type I IFNs
MAVS signaling from peroxisomes in a population of cells, (Collins et al., 2004; Mossman et al., 2001). To determine
they do not allow us to examine individual cells for compart- whetherIFNscontributetoexpressionofISGsinducedbymito-
ment-specificsignalingevents.Toaddressthis,wetookadvan- chondrial or peroxisomal MAVS, we monitored the rate of IFN
tageofthefactthatvarioussignalingpathwaysinducemorpho- productionbyreovirus-infectedcells.Asexpected,IFNproduc-
logical changes in the organelles where signaling occurs, tion was dependent on membrane-localized MAVS, and all
includingRLR-dependentactivitiesonmitochondria(Castanier mitochondrial MAVS proteins (MAVS-WT, -Mimic, and -Mito)
et al., 2010; Yasukawa et al., 2009). We reasoned that if triggered IFN production, though with delayed kinetics in the
signalingwasactuallyoccurringontheseorganelles,thentheir caseofMAVS-Mito(Figure3C).Thesedataindicatethatinthe
morphologymaychangeduringviralinfection.Insupportofthis case of the mitochondria-localized MAVS proteins, IFN
672 Cell141,668–681,May14,2010ª2010ElsevierInc.
A B
10 100
Reovirus (hpi): Reovirus (hpi):
0 75 0
8 4 4
ml) 9 ml) 1500 9
(IU/ 6 (IU/ 8
IFN- 4 IFN- 46
2
0 02 N.D. N.D. N.D.N.D.N.D.N.D. N.D.N.D.N.D. N.D.N.D.N.D.N.D.N.D.N.D. N.D.N.D.
C WT + BFAWT Mito + BFMAito Pex + BFPAex Mimic + BMFimAic WT + abWT Mito + abMito Pex + abPex Mimic + aMbimic
Genotype: WT Mito Pex Mimic
Brefeldin A: + + + - - - + + + - - - + + + - - - + + + - - -
Reovirus (hpi): 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9
IB: Viperin
IB: Actin
Lane: 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
D
Genotype: WT Mito Pex Mimic
IFN antibodies: + + + - - - + + + - - - + + + - - - + + + - - -
Reovirus (hpi): 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9 0 4 9
IB: Actin
Lane: 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Figure4. PeroxisomalMAVSDirectlyInducesViperinExpression
(A)MAVS-expressingMEFsandMAVS-KOcellswerepretreatedwith20mg/mlBFAbeforeinfectionwithreovirusinpresenceofthedrug.Atindicatedtimes,cell
supernatantsweretestedfortypeIIFNactivityviaabioassay.
(B)Similarto(A)excepttypeIIFNactivitywasblockedbyadditionof250NU/mlanti-IFNband500NU/mlanti-IFNaantibodiesafterinfectionwithreovirus.
(CandD)Celllysatesfrom(A)and(B)weretestedforISGexpressionbyimmunoblottingwithanti-viperinantibody.NotethatIFNactivityisnotrequiredforviperin
expressionmediatedbyperoxisomalMAVS.
Alldataaretherepresentativeofatleastthreeindependentexperiments.Errorbarsshowstandarddeviationoftriplicateinfections.
expressioncoincideswithISGinduction.Surprisingly,nodetect- was largely resistant to these treatments (Figures 4C and 4D).
able IFNs were produced by MAVS-Pex cells. Similar results These data therefore indicate that the subcellular positioning
were obtained with cells infected with influenza virus of MAVS determines the type of signaling pathway activated
(Figure3D),thoughtherelativeamountsofIFNsproducedwith during viral infection. Peroxisomal MAVS induces the rapid
these two viruses differed dramatically, reflecting unique anddirectexpressionofviperin,whichisfollowedbymitochon-
aspectsofeachviruslifecycle.However,ourinabilitytodetect drialMAVStriggeringviperinexpressiondirectly,aswellasindi-
aroleforIFNsinpromotingviperinexpressioninMAVS-Pexcells rectlythroughtheIFN-mediatedfeed-forwardloop.
was not due to an inability of the cells to respond to IFNs,
becauseadditionofrecombinantIFNbwassufficienttoinduce TheGlobalTranscriptionalResponsetoReovirus
viperinexpressioninallcellsexamined(Figure3E). InfectionIsMediatedbytheCollectiveActions
Theobservationthatviperincanbeexpressedintheabsence ofMAVS-DependentPeroxisomal
oftypeIIFNinductionsuggeststhatIFNsmaynotcontributeto andMitochondrialSignaling
ISGexpressioninducedbyperoxisomalMAVS.Wetestedthis Basedonthesetofcandidategenesexamined,ourdatasug-
possibilitybyinfectingcellsunderconditionsinwhichthefunc- gests that peroxisomal and mitochondrial MAVS each induce
tionsofIFNsareprevented,byeitherdisruptingproteinsecre- acomplementarysetofgenesthatarecollectivelyinducedby
tionwithbrefeldinA(BFA)orbyutilizingneutralizingantibodies MAVS-WT.Todeterminewhetherthisisthecase,microarrays
againstsecretedIFNs.Bothtreatmentsdisruptedtheactivityof wereperformedonreovirus-infectedcells.Infectionswereper-
typeIIFNsproducedduringreovirusinfection(Figures4Aand formedfor3,9,or16hr,andRNAwascollected forgenome-
4B)andinhibited theexpressionof viperinbycellsexpressing wideexpressionanalysis.Atalltimesexamined,similarexpres-
mitochondrial MAVS (MAVS-WT, -Mimic, and -Mito) (Figures sionprofileswereobservedwhenMAVS-WTandMAVS-Mimic
4Cand4D).ThesedataindicatethatsignalingbyIFNspromotes cellswerecompared,confirmingthatsimilaritiesinMAVSlocal-
viperinexpression.However,becausethesetreatmentsdidnot izationarepredictiveofsimilaritiesinMAVSfunction(Figures5A
completely abolish viperin expression, an IFN-independent and5B).ThesecellsinducedtheexpressionofnumerousISGs,
pathwayofviperininductionmustalsoexist.Interestingly,MAVS IFNs,andchemokines(Figure5C).Notably,wewereunableto
signaling from peroxisomes primarily utilized the IFN-indepen- detect the expression of the proinflammatory cytokines TNFa,
dent pathway, as viperin expression within MAVS-Pex cells IL-1b, or IL-6 (data not shown). MAVS-Cyto cells were most
Cell141,668–681,May14,2010ª2010ElsevierInc. 673
A C
3h 3h 9h 16h
c c c
MKOito WT Mimi Mito Pex Cyto KO WT Mimi Mito Pex Cyto KO WT Mimi Mito Pex Cyto KO
Cyto
Ifnb1
Mimic Ifna4
Pex Ifna5
Ifna8
WT Viperin
OoocxT Pdcd1lg1
KMitCytMimiPeW OCxacsll110
9h Mx2
Il33
Trim21
Cyto Ifit3
Gbp2
Mito Daxx
Pex Trex1
Irf5
KO Ifit2
WT Usp18
Mimic Ifi205
Ccl4
ooxOTc Irf1
CytMitPeKWMimi IGl1b5p5
16h Oasl2
Gbp4
WT
Mimic
Pex
Mito
Cyto
KO
TcxooO
WMimiPeMitCytK 1 .05
B
WT vs Mimic 3h WT vs Mito 3h WT vs Pex 3h WT vs Cyto 3h Mito vs Pex 3h
WT vs Mimic 9h WT vs Mito 9h WT vs Pex 9h WT vs Cyto 9h Mito vs Pex 9h
WT vs Mimic 16h WT vs Mito 16h WT vs Pex 16h WT vs Cyto 16h Mito vs Pex 16h
674 Cell141,668–681,May14,2010ª2010ElsevierInc.
similartoMAVS-KOcells(Figures5Aand5B),furtherconfirming cells. These data establish that signaling from either peroxi-
thatmembranelocalizationofMAVSiscriticalforitsfunctionin somesormitochondriaissufficienttoinduceafunctionalanti-
antiviralsignaling.Interestingly,MAVS-Pex-or-Mito-expressing viralresponse,butsignalingfrombothorganellesisnecessary
cells displayed a transcriptome that each partially overlapped formaximalcontainmentofreovirusreplication.
withthatofMAVS-WTcells,butweredistinctfromoneanother Vesicular stomatitis virus (VSV) is one of many viruses that
(Figures 5B and 5C). For example, at 16 hr after infection, interferewithtypeIIFNexpressionaspartoftheirpathogenic
MAVS-Mito cells upregulated genes encoding chemokines, lifecycle(Figures6Band6C)(FerranandLucas-Lenard,1997).
ISGs,IFNb,andseveralIFNafamilymembers(Figures5Band Undertheseconditions,theIFN-independentmeansofsignaling
5C).MAVS-Pexcellsalsoinducedtheexpressionofchemokines thatisinducedbyperoxisomalMAVSmaybeparticularlyimpor-
andISGs,butwithoutanydetectablechangesinIFNexpression tantincontrollinginfection.Consistentwiththisidea,MAVS-Mito
and with much faster kinetics (within 3 hr). Thus, on a global cellswereassusceptibletoVSVinfectionasMAVS-KOor-Cyto
scale,peroxisomalMAVSinducestherapidexpressionofISGs cells(Figure6D),suggestingthatintheabsenceofIFNproduc-
withoutinducingIFNexpression,whereasmitochondrialMAVS tion,themitochondrialsignalingpathwayisfunctionallydefec-
promotes IFN and ISG expression but with delayed kinetics. tive.Mostnotably,MAVS-Pexcellswerenearlyaseffectiveat
We confirmed these results by examining the expression of controlling VSV as MAVS-WT cells (Figure 6D). These results
severalcandidateIFNsandISGsusingnCounter,whichallows suggest that MAVS signaling from peroxisomes is the primary
for multiplex analysis of gene expression with the sensitivity meansofcontrollingvirusesthatinterferewithIFNexpression,
of quantitative RT-PCR (Geiss et al., 2008) (Figures S4A and thus underscoring the importance of this organelle in host
S4B).Overall,atalltimesexamined,mostgenesexpressedby defense.
either peroxisomal or mitochondrial MAVS were induced by
MAVS-WTor-Mimic(Figures5Band5C).Thesedatatherefore DownstreamRegulatorsofMAVSSignaling
support a model whereby the host transcriptional response is fromPeroxisomes
theresultofMAVSsignalingfrombothmitochondriaandperox- To identify downstream signaling regulators of peroxisomal
isomes. We do note however, that the magnitude of antiviral MAVS, we overexpressed each MAVS allele in 293T human
gene expression induced by cells expressing MAVS-WT or kidney epithelial cells. MAVS-WT, -Mimic, -Mito, and -Pex
MAVS-Mimicwasgreaterthanthemagnitudeinducedbycells each induced the activation of reporter genes controlled by
where MAVS was restricted to a single organelle, which sug- NF-kB and AP-1 (Figure 6E). Inaddition, an IRF1reporter and
gests that signaling from both organelles may be coordinated anISREthattypicallyreportsIRF3activitywereinduced,sug-
toensuremaximalantiviralgeneexpression. gestingaroleoftheseIRFsinMAVSsignalingfromperoxisomes
(Figure 6E). MAVS-Cyto did not activate any reporter. Within
PeroxisomalSignalTransductionCreatesaTransient thesecells,wefoundthatISREactivationbyeitherMAVS-WT
butFunctionalAntiviralState or MAVS-Pex was potentiated by overexpression of TRAF3
MAVS-dependentsignalingpromotesanantiviralstate,whichis and inhibited by expression of a dominant negative allele of
functionallydefinedastheabilityofcellstorestrictmultiplication TRAF6(FigureS5A),suggestingtheinvolvementoftheseknown
of viruses. To determine the significance of mitochondrial or RLRregulatorsinperoxisomalsignaling(Sahaetal.,2006;Yosh-
peroxisomal signaling pathways in this regard, we asked idaetal.,2008).Incontrast,expressionofadominant-negative
whether signaling from either organelle is sufficient to restrict allele of the antiviral factor FADD has a minimal affect on
viralreplication.WeaddressedthisbyinfectingMAVS-express- MAVSsignaling(FigureS5A),whichisconsistentwitharecent
ing cells with reovirus and measuring production of infectious report (Balachandran et al., 2007). Notably, overexpression of
virions over time. As expected, MAVS-WT and -Mimic cells NLRX1, a negative regulator that is uniquely located on mito-
weremostresistanttoinfection,andMAVS-KOand-Cytocells chondria(FigureS5B)(Mooreetal.,2008)didnotinterferewith
were most susceptible (Figure 6A). These data indicate that MAVS-Pex signaling, but did inhibit signaling by MAVS-WT
MAVSsignalingisrequiredtolimitreovirusreplication.Interest- (FigureS5A).
ingly, cells expressing MAVS-Pex or MAVS-Mito exhibited an ToconfirmtherolesofIRF1andIRF3inperoxisomalsignaling,
unusual biphasic behavior. Over the first 24 hr, these cells we enlisted VSV to study the IFN-independent means of ISG
restrictedviralreplicationaswellasMAVS-WT,butthiscapacity expression, since only the peroxisomal pathway functions to
diminished, and by 72 hr were most similar to the MAVS-KO control replication of this virus, although some ISGs were
Figure 5. Genome-wide Transcriptome Analysis Reveals a General Role for Peroxisomal and Mitochondrial MAVS in Antiviral Gene
Expression
(A)RNAfromMAVS-expressingMEFsandparentalMAVS-KOcellsafterinfectionwithreovirusfor3,9,or16hrwassubjecttomicroarrayanalysis.Thesimilarity
oftheoverallgeneexpressionprofilesmediatedbytheindicatedMAVSallelesisdisplayedasPearsoncorrelationcoefficient-basedheatmap.Samplesare
clusteredalongbothaxesbasedontheircorrelationvalue.Notethatat3hrafterinfection,MAVS-Pexcellsdisplayageneexpressionpatternthatismostsimilar
toMAVS-WTcells.
(B)Pairwisecomparisonsofindicatedcelllinesbasedon4089significantlyregulatedgenesdepictedinalog-logscalescatterplot.Eachdatapointindicates
agenewhoseexpressionlevelexhibitedachangeofgreaterthan2-fold.
(C)Heatmapofselectedgenesbasedontheirexpressionratiosacrossallsixcelllinesandduringalltimepointsuponreovirusinfection.
Genesarecoloredaccordingtoalog2-basedcolorbardepictedunderneatheachheatmap.SeealsoFigureS4.
Cell141,668–681,May14,2010ª2010ElsevierInc. 675
A Figure 6. Peroxisomal MAVS Elicits a Functional
1.0×1009 MAVS WT AntiviralResponse
virus titer (pfu/ml)11..00××11000078 MMMMMAAAAAVVVVVSSSSS---- KCMPMeOyiitmxtooic (it(MnABimfE))eeFcMMsst,eAAwvdVVierSSuwr--eseiWtxhtinipTtfer,reerecso-stMvsweiidirnemugrwseiciMdt-aheteExtVFaepSsnrrmeVaMsinnsaOdietndIagMnobfcAyMeV3plO.SllsaI-AqKoatuOfnetd3hMa.esMAEsinfAFatdesyVir.cSw8a-tKeherOrde,
1.0×1006 0h 16h 24h 48h 72h RNAwasisolated andanalyzed forISG andtypeIIFN
time post infection expressionwithnCounter.
(C) Same as (B) except MAVS-Pex, MAVS-Mito, and
B
200 MAVS-CytoMEFswereanalyzed.
transcript count124680000000000000000 WMKOiTmic transcript fold change11505000 WMKOiTmic (wt(IePDERimmee)F)xrep21Me,st9,yAi,M3nNvVTfipeitFSroMcuc--,ktseeSeBolxtldCsir,pteVwrCwoerirsystevhtsrwAeoeiVncP)e.Sgtt1roreRVaMrlneduaEss(ectciuFteiaofelsentnnrsrtmatlaMrynwosinOdetle)erIrapderooneafrbsprn0efyooeM.n0rrcpttmA1atelae.VlraqdsAMSliuztwtAeeto(hiVWdgtaehSseTt-sitoan,hKandeyOMRir.cIeSicamwnRetieiillEtclldhas,,
luciferaseactivityandareshownasfoldincreaserelative
0 Ifit1 Ifit2 ViperinOas1g IRF7 Ifnb1 Ifna4 0 Ifit1 Ifit2 ViperinOas1g IRF7 Ifnb1 Ifna4 tocellstransfectedwithemptyvector.Errorbarsshow
C standarddeviationoftriplicatetransfections.
4000 20
PMeitxo ge PMeitxo SeealsoFigureS5.
transcript count123000000000 Cyto transcript fold chan 11505 Cyto adonfednpteIRrISoFxG3isaeorxmepacrelesMnstAiroaVnlSrae.ngdulamtoarysaocftIFdNow-inndsetrpeeanm-
0 0
Ifit1 Ifit2 ViperinOas1g IRF7 Ifnb1 Ifna4 Ifit1 Ifit2 ViperinOas1g IRF7 Ifnb1 Ifna4
CellType-SpecificActionsof
D
PeroxisomalandMitochondrialMAVS
ml)1.0×1007 MMAAVVSS -WMiTmic Theprototypicalinnate-immuneadaptorMyD88
virus titer (pfu/11..00××11000035 MMMMAAAAVVVVSSSS--- KCMPeOyitxtoo rtWtrheaighsnuesltachdtreieivprsetiroosTtniLhtayRelrrsoeaifsgdpnaorapenlistnsopegrosnapisnnreoddsteifiinfneidssruecnauetlnssccoeldelldaiftifryse.ppreelTansoyt.
1.0×1001
0h 4h 8h 12h 24h 28h 32h addressthisforMAVS,weexaminedthefunc-
time post infection
tionofperoxisomalandmitochondrialMAVSin
E
50 15 macrophages. Each MAVS allele was ex-
NF-B activation(fold induction)12340000 AP1 activation(fold induction)105 pmTsihrmaeecsilrsaoleorpdchtaoailnigztaeihmtsaiomtnisooorobtlaafstleiezerdeavdcefhdrboomMinnAeMMVmSAEaVFrpSsror-owK(tcOe-odinmemrpiwviacearedes.
Figures S2 and S5C). In response to reovirus
0 0
ctrl WT MimicPex Mito Cyto ctrl WT MimicPex Mito Cyto infection, macrophages that contained mito-
50 25 chondrial MAVS (MAVS-WT or -Mito) induced
IRF1 activation(fold induction)12340000 ISRE activation(fold induction)1120505 t(gtFrioeaignnnu.esrceeUrxiSpnplt5irskDeees).nsMiMcoonAEdVFiinnsSg,-rCeIrFsyepNtooosvniw(rsFuaeisgs-tuiuonrneferae7cboCtleve)dirtauonsmdiniandIScfuerGcoces--
phages expressed inflammatory cytokines
0 0
ctrl WT MimicPex Mito Cyto ctrl WT MimicPex Mito Cyto such as IL-1b (Figure 7D), IL-6, IL-12b, and
TNFa(FigureS5Danddatanotshown).Another
difference between MEFs and macrophages
induced by VSV in cells expressing MAVS-Mito (Figure 6C). was that MAVS-Mito-expressing macrophages exhibited no
MEFs derived from various IRF-KO mice were infected with kineticdelayinreovirus-inducedgeneexpression.Theseresults
VSVandassessedfortheirabilitytoinduceISGs.WhereasWT suggestthatliketheTLRadaptorMyD88,thefunctionofMAVS
cellsinducedtheexpressionofseveralISGs(andnoIFNs),cells iscontrolledinacelltype-specificway.
lackingIRF1orIRF3wereincapableofinducingISGexpression PeroxisomalMAVSinducedtheexpressionofsomegenesto
(Figures 7A and 7B). A few ISGs (e.g., OAS1g) also required thesamelevelsobservedwiththeWTallele,suchasA20,IL-1b,
another family member, IRF5. These data indicate that IRF1 Cox2, CXCL2 (MIP-2a), CCL4 (MIP-1b), and Fos (Figure 7D),
676 Cell141,668–681,May14,2010ª2010ElsevierInc.
whereasotherswereinducedmorethan3-foldbutstilllessthan Our studies revealed another important similarity between
inWTcells,e.g.,viperin,IFIT1,andIFIT2(FigureS5D).Ofnote, theRLRandTLRnetworks,thatofcelltype-specificfunctions
peroxisomalMAVSwasunabletoinducetheexpressionofany foradaptorproteins.Infibroblasts,MAVSfunctionedtoinduce
IFNgeneinmacrophages(Figure7C).Thus,despitecelltype- expression of IFNs and ISGs, but not inflammatory cytokines.
specific activities of MAVS, a fundamental feature of the RLR In contrast, IFNs, ISGs, and cytokines were all induced by
signalingnetworkappearstobethatperoxisomalMAVSfunc- MAVSsignalinginmacrophages.Thus,MAVScanbegrouped
tionstopromoteanIFN-independentmeansofgeneexpression. withtheTLRadaptorMyD88asimmuneregulatorsthatinduce
celltype-specifictranscriptionalresponses.Whatisthebenefit
of cell type-specific actions of innate immune signaling path-
DISCUSSION ways?Onebenefitmaylieintheprimaryfunctionsofthecells
responding to a given virus. For example, macrophages are
The best-characterized sensors of cytosolic viruses are mem- dedicated sentinels of the innate immune system. As such,
bersoftheRLRfamily,whichenlisttheadaptorproteinMAVS withinthesecells,infectiontriggersMAVS-dependentinflamma-
to initiate antiviral signaling (Kawai and Akira, 2007). MAVS is tory cytokine production and antiviral factors. Fibroblasts, in
one of a growing group of tail-anchored membrane proteins, contrast, aretissue-resident cells thatare primarily involved in
which contain a C-terminal transmembrane domain (Gandre- organhomeostasis—aconditionthatisdisruptedunderinflam-
Babbe and van der Bliek, 2008; Koch et al., 2005; Seth et al., matory conditions. Thus, designing MAVS to induce antiviral
2005). This anchor was originally reported to promote MAVS factors but not inflammatory cytokines in fibroblasts may aid
recruitment to the mitochondrial outer membrane, providing a thesecellsinmaintaining homeostasis underinfectious condi-
landmarkofwhereRLRsignalingcanoccur(Sethetal.,2005). tions.Wespeculatethatthediversificationofadaptorfunctions
This discovery established that cytosolic detection systems, ininnateimmunitymaybeageneralmechanismtotailorsignal-
likeextracellulardetectionsystems(e.g.,TLRs),usemembranes ingpathwaystotheneedsoffunctionallydiversecelltypes.
asscaffoldsforsignaltransduction.IntheTLRnetwork,however, OurfindingthatperoxisomallocalizationofMAVSisrequired
signaling occurs from a variety of different organelles, not just forrapidbuttransientinductionofantiviralISGswhereasmito-
one(BartonandKagan, 2009).Wereport herethatin addition chondrialMAVSpromotesISGexpressionwithdelayedkinetics
tomitochondria,theantiviralsignalingproteinMAVSislocated in fibroblasts isespecially intriguing. The kinetic differences of
onperoxisomesinseveralhumanandmurinecelltypes. ISGexpressionwereexplainedbytheobservationthatperoxi-
Thecentralfindingofthisstudy—thatperoxisomesareasiteof somal MAVS induced a cell-intrinsic means of ISG induction,
signaltransduction—wasestablishedwithacomplementaryset which occurred in the absence of detectable IFN expression.
ofassaysthatmeasured(1)messengerRNAs(mRNAs)encoding Mitochondrial MAVS induced cell-intrinsic ISG expression as
ISGsandIFNs,(2)proteinlevelsofISGsandIFNs,(3)theinduc- well, but maximal induction occurred through the actions of
tion of a functional antiviral state in cells, and (4) infection- secretedIFNs.Ourstudiesdidnotrevealanobviousdifference
induced changes in peroxisome morphology. In each of these inthedownstreamregulatorsactivatedbyperoxisomalversus
assays,wefoundthatperoxisomesareasiteofMAVS-depen- mitochondrial MAVS, but the studies performed in 293T cells
dent signaling. Moreover, we obtained these results by using suggest that the selective positioning of negative regulators
severalunrelatedRNAvirusesasphysiologicaltriggersofRLR (e.g., NLRX1) may contribute to organelle-specific responses.
signaling,whichsuggeststhatperoxisomalsignalingisafunda- Futureworkwillberequiredtoaddressthispoint.
mentalcomponentoftheRLRnetwork. The functional importance of RLR signaling from peroxi-
The RLR signaling network now joins the TLRs as pattern somes was best revealed by experiments with VSV, which
recognition systems that signal from multiple organelles. Both interferes with IFN expression and renders the mitochondrial
systems require that a transmembrane protein be positioned pathwayineffective.Asaresult,eventhoughMAVSispresent
on specific organelles—the receptors themselves in the case onmitochondriaandperoxisomesinWTcells,afunctionalanti-
of TLRs and the MAVS adaptor in the case of RLRs (Akira viralresponseagainstVSVisonlyinducedbytheperoxisomal
et al., 2006; Barton and Kagan, 2009). Interestingly, when pathway.WealsoexploitedVSVinfectiontodissecttheperox-
considering these two networks, the function of diversifying isomal signaling pathway using cells derived from genetically-
signalinglocaleappearstobedistinct.InthecaseofTLRs,differ- deficient mice. While we found that IRF3 plays a role in ISG
entialreceptorplacementdiversifiesthetypesofpathogensthat expression, this factor is also involved in the regulation of IFN
canbedetected:TLRsfoundonendosomesrecognizeviruses, expression(Satoetal.,2000)andmaythereforebeconsidered
whileTLRsfoundontheplasmamembranetypicallyrecognize a more general regulator of antiviral gene expression. Indeed
bacteria. In contrast, differential MAVS placement does not IRF3 is also involved in IFN expression induced by non-RLRs
diversifythetypesofvirusesdetectedbyRLRs,butdiversifies (Kawai and Akira, 2007). IRF1, on the other hand, is needed
thetypesofsignalingpathwaysthatareactivated.Inthecase for expression of all ISGs that we examined in VSV-infected
of reovirus and influenza virus infection of fibroblasts, peroxi- cells and is not required for IFN expression (Tamura et al.,
somal MAVS triggers the rapid expression of ISGs, whereas 2008). IRF1 may thus uniquely control IFN-independent
mitochondrialMAVStriggersdelayedISGandIFNexpression. signalingeventsthatleadtoISGexpressionandantiviralimmu-
Thisdiversificationisfunctionallyimportant,asourdataindicate nity.OurexperimentswithVSValsorevealedaprobablebenefit
that MAVS signaling must occur from both organelles to limit of utilizing both IFN-dependent and IFN- independent mecha-
reovirusreplication. nisms of ISG induction: for pathogens that disrupt the
Cell141,668–681,May14,2010ª2010ElsevierInc. 677