ppp-RNA Activates Antiviral Signal through Redistribution of IPS-1 Mediated by MFN1

17 Pages·2010·11.47 MB·English

Virus-Infection or 59ppp-RNA Activates Antiviral Signal
through Redistribution of IPS-1 Mediated by MFN1
KazuhideOnoguchi1,2,3,KojiOnomoto1,ShioriTakamatsu1,2,MichihikoJogi1,2,AzumiTakemura1,Shiho
Morimoto1, Ilkka Julkunen4, Hideo Namiki3, Mitsutoshi Yoneyama5,6, Takashi Fujita1,2*
1DepartmentofMolecularGenetics,InstituteforVirusResearch,KyotoUniversity,Kyoto,Japan,2GraduateSchoolofBiostudies,KyotoUniversity,Yoshida-KonoeSakyo,
Kyoto,Japan,3GraduateSchoolofScienceandEngineering,WasedaUniversity,Tokyo,Japan,4DepartmentofVaccinationandImmuneProtection,NationalInstitutefor
HealthandWelfare,Helsinki,Finland,5DivisionofMolecularImmunology,MedicalMycologyResearchCenter,ChibaUniversity,Chuo-ku,Chiba,Japan,6PRESTO,Japan
ScienceandTechnologyAgency,Saitama,Japan
Abstract
In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses
including production of typeI andIII interferon (IFN) and thesubsequent expression ofIFN-inducible genes. Interferon-b
promoterstimulator1(IPS-1,alsoknownasMAVS,VISAandCardif)isadownstreammoleculeofRLRandisexpressedon
theoutermembraneofmitochondria.WhileitisknownthatthelocationofIPS-1isessentialtoitsfunction,itsunderlying
mechanismisunknown.Ouraiminthisstudywastodelineatethefunctionofmitochondriasoastoidentifymoreprecisely
itsroleininnateimmunity.Indoingsowediscoveredthatviralinfectionaswellastransfectionwith59ppp-RNAresultedin
theredistributionofIPS-1toformspeckle-likeaggregatesincells.WefurtherfoundthatMitofusin1(MFN1),akeyregulator
ofmitochondrialfusionandaproteinassociatedwithIPS-1ontheoutermembraneofmitochondria,positivelyregulates
RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced
redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1
through theirfusionprocesses.
Citation:OnoguchiK,OnomotoK,TakamatsuS,JogiM,TakemuraA,etal.(2010)Virus-Infectionor59ppp-RNAActivatesAntiviralSignalthroughRedistribution
ofIPS-1MediatedbyMFN1.PLoSPathog6(7):e1001012.doi:10.1371/journal.ppat.1001012
Editor:C.ChengKao,IndianaUniversity,UnitedStatesofAmerica
ReceivedFebruary17,2010;AcceptedJune18,2010;PublishedJuly22,2010
Copyright:(cid:2)2010Onoguchietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:TheMinistryofEducation,Culture,Sports,ScienceandTechnologyofJapan(http://www.mext.go.jp/english/),theMinistryofHealth,Labourand
WelfareofJapan(http://www.mhlw.go.jp/english/index.html),thePRESTOJapanScienceandTechnologyAgency(http://www.jst.go.jp/kisoken/presto/index_e.
html),theUeharaMemorialFoundation(http://www.ueharazaidan.com/),theMochidaMemorialFoundationforMedicalandPharmaceuticalResearch(http://
www.mochida.co.jp/zaidan/l),andNipponBoehringerIngelheim(http://www.boehringer-ingelheim.co.jp/com/Home/index.jsp).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction in the redistribution of IPS-1, as well as in virus-induced IFN
production. Our study highlights the novel mitochondrial
Type I and III interferons (IFNs) play central roles in innate regulatory function of specifically sorting IPS-1 and providing a
immune responses to viral infections [1,2,3,4]. In a variety of signaling platformfor antiviral responses.
tissues, IFN production is triggered by a cytoplasmic sensor,
retinoicacidinduciblegeneI(RIG-I)-likeReceptor(RLR),which Results
specificallysensesviralRNAandinducesantiviralsignaling[5,6].
Once RLR is activated, its signal is relayed through physical Dynamic redistribution of IPS-1 in virus-infected or
interactiontoIFN-bpromoterstimulator1(IPS-1,alsoknownas 59ppp-RNA-transfected cells
MAVS,VISAorCardif)[7,8,9,10].IPS-1 interactswithmultiple ToexaminethelocalizationofIPS-1duringviralinfections,we
signal transducers and protein kinases that activate transcription generated HeLa cell lines stably expressing FLAG-tagged IPS-1
factors to induce IFN and other cytokine genes [11]. IPS-1 is (IPS-1-HeLaclones,Fig.1).Althoughthetemporaryexpressionof
expressed on the mitochondrial outer membrane and this wildtype(wt)IPS-1resultsinconstitutivesignaling[7,8,9,10],the
localization is essential for signaling to occur [9]. However the stable cell lines did not exhibit the constitutive activation of
reason for this underlying mechanism is unknown. Here, we downstream target genes. However, upon infection with the
investigatedthecellulardistributionofIPS-1invirus-infectedcells. Sendaivirus(SeV),thecellsexhibitedincreasedexpressionofIFN
We observed that IPS-1 is usually distributed evenly in all and chemokine genes (IFNB1, IL29, IL28A, IL28B and CXCL11)
mitochondria in uninfected cells, however upon viral infection or andinterferon-stimulatedgenes(DDX58,IFIH1,DHX58,IFIT1-3,
the introduction of 59ppp-RNA, which mimics viral RNA and OASL) (Fig. 1Band C). Furthermore, the IPS-1-HeLa clones
[12,13,14], a redistribution of IPS-1 occurred, resulting in a exhibited diminished susceptibility to Encephalomyocarditis virus
speckle-like pattern on mitochondria. Furthermore, we demon- (EMCV) replication (1 to 2 log) (Fig. 1D). The low basal activity
stratedthatamitochondrialGTPase,Mitofusin1(MFN1),which andelevatedsignalingafterSeV-infectionsuggestthatFLAG-IPS-
regulatesmitochondrialfissionandfusion[15],playsacriticalrole 1isunderaregulatorycontrolsimilartothatofendogenousIPS-1.
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RedistributionofIPS-1
foci of NP in the cytoplasm and induced foci of RIG-I to form
Author Summary
(Fig. 5A) [16]. RIG-I was evenly distributed in the cytoplasm,
Virus-infections,suchasinfluenzaandchronichepatitisC, howeversomeofthefocico-localizedwiththoseofNP(Fig.5A).A
areprominentdiseasesandoutbreaksofnewlyemerging similarformationoffociandco-localizationwithviralnucleopro-
viruses are serious problems for modern society. Higher teincomplexwasobservedwithotherviruses(Ko.O.unpublished
animals, including humans, are genetically equipped with observations). IPS-1 accumulated on the periphery of the foci of
mechanisms, collectively known as innate immunity, to RIG-I (Fig. 5B) and NP (Fig. 5C). We speculate that activated
counteract viral infections. RIG-I-like receptor (RLR), a
RIG-I recruits IPS-1, because RIG-I and IPS-1 interacted with
cytoplasmic sensor, contributes to immune regulation by
eachotherthroughCARD-CARDinteraction[7,8].IPS-1didnot
detectinginfectionsbyRNAvirusesandtriggeringaseries
co-localizewithRIG-InorNPpresumablybecausemitochondria
of responses which results in the activation of innate
do not penetrate these foci nor is IPS-1 released from
antiviral genes. Furthermore, it has been demonstrated
mitochondria. Immunoelectron microscopy using the anti-NP
that IPS-1, the adaptor protein of RLR, is expressed on
antibody clearly identified the NP foci (Fig. 6A), and anti-FLAG
mitochondrial outer membrane. Mitochondrion is an
organelle of prokaryotic cell origin; it regulates energy staining (Fig. 6B) showed that mitochondrial IPS-1 accumulated
production, and is involved in cell growth and cell death. on theperiphery of NPfoci inNDVinfectedcells.
Why IPS-1 is located on the mitochondrial outer mem-
brane and how mitochondria are involved in antiviral A dominant negative mutant of RIG-I does not induce
signalingareyettobeexplainedclearly.Inthisreport,we IPS-1 redistribution
discoveredthatmitochondrialfusionproteinMFN1playsa
To determine if the observed redistribution of IPS-1 is
novel function to mediate IPS-1 redistribution, which
functionally relevant, we used a point mutant of RIG-I (K270A),
appears tobe acritical stepin RLRsignaling.
which normally recognizes ligand RNA but functions as a
dominant negative inhibitor (Fig. 7A) [14]. It was observed that
NDVinfectioninducedfociofbothwtandK270ARIG-Itoform
Like endogenous IPS-1, FLAG-tagged IPS-1 is expressed on
(Fig. 7B), however wt but not K270A promoted the speckled
mitochondria in uninfected cells as shown by co-staining with
staining pattern of IPS-1 (Fig. 7C). The results indicate that the
MitoTracker (Fig. 2A, Mock). However, compared to the even
redistributionofIPS-1isstronglycorrelatedwiththeactivationof
cytoplasmicstaininginthemock-infectedcells,thestainingpattern
antiviral signaling.
ofIPS-1becamenoticeablyspeckledinSeV-infectedcells(Fig.2A,
SeV). Quantification of the fluorescence image revealed that
Mitofusin 1, but not Mitofusin 2, plays a critical role in
mitochondriaheavilystainedwithMitoTrackerbutlightlystained
with anti-FLAG antibody were produced in SeV-infected cells. RIG-I-induced antiviral signaling
Thisredistributionwasalsoobservedwithanothermitochondrial RIG-I was originally identified by screening an expression
marker, endoplasmic reticulum-associated amyloid b-peptide- cDNA library [17]. In addition to the cDNA encoding RIG-I,
bindingprotein(ERAB)(Fig.2B),anddifferentviruses(Newcastle there were several other candidate cDNA clones which enhance
disease virus (NDV), Sindbis virus, EMCV, Influenza virus, and virus-responsive reporter activity. Two of the independent clones
Vesicular stomatitis virus(VSV)) (Fig.3). encodedafull-lengthprotein,Mitofusin1(MFN1).HumanMFN1
We also examined the distribution of IPS-1 in 59ppp-RNA- is composed of 741 amino acids and domains of GTPase and
transfected cells. Unlike synthetic single stranded RNA (59OH- transmembranes(Fig.8A).MFN1togetherwithitsrelatedprotein
RNA),59ppp-RNAisachemicalligandforRIG-Iandisknownto Mitofusin 2 (MFN2) is expressed on the outer membrane of
mimic viral signaling [12,13,14]. Interestingly, as with a viral mitochondria and regulates mitochondrial dynamics [18,19].
infection, 59ppp-RNA induced a redistribution of IPS-1, suggest- Hyper- and hypo-functioning of either MFN1 or MFN2 result
ingthattheredistributionwastriggeredthroughRIG-Isignaling. in elongated/aggregated and fragmented mitochondria, respec-
ItisworthnotingthatEMCV,whichselectivelyactivatesanother tively. GTPase activity was previously shown to be essential for
RLR, melanoma differentiation-associated gene 5 (MDA5), also mitochondrial morphological change, particularly the fragmenta-
caused the redistribution of IPS-1, suggesting that this effect is tionofmitochondriainducedbyaGTP-binding-deficientmutant
common to RLRs. We suspected that IPS-1-HeLa cells exhibit of MFN1(MFN1T109A)[18].
enhancedredistributionofIPS-1duetoenhancedsignaling(.10 Consistentwiththescreeningresults,overexpressionofMFN1,
IFN-b mRNA accumulation, Fig. 1B). This led us to analyze the butnotMFN2,augmentedIFN-bpromoteractivity(Fig.8B).The
distribution pattern of endogenous IPS-1 in HeLa cells, and we GTPase activity is involved in this MFN1 function, since MFN1
observed that the distribution pattern of endogenous IPS-1 T109A significantly inhibited the signaling induced by NDV or
changed in SeV-infected cells, although exclusive staining by 59ppp-RNA(Fig.8CandD).Itisworthnotingthatoverexpression
mitochondrialmarkerwasnotobserved(Fig.4A).SimilartoIPS- ofMFN1,whichresultsinelongatedmitochondria,isnotbyitself
1-HeLacells,weobservedthathepatocellularcarcinomaSKHep1 sufficient to deliver the signal. To confirm that the increased
cells NDV, SeV, Influenza virus, or Sindbis virus infection also signaling observed by MFN1 overexpression was correlated with
inducedaspeckledstainingpatterninendogenousIPS-1(Fig.4B), RIG-Iactivation,wetransfectedcellswithacombinationofRIG-I
anddisplayedenhancedIRF-3dimerizationwhencomparedwith and MFN1. The RIG-I/MFN1 combination showed enhanced
HeLa cells (our unpublished data). This suggests that the IFN-b promoter activity, but the RIG-I K270A mutant/MFN1
redistribution is not simply an artifact due to the overexpression combination failed to do so (Fig. 8E). MEFs derived from mice
of FLAG-IPS-1. withdisruptedMfn1orMfn2genewasusedtoconfirmthespecific
involvementofMFN1invirus-inducedantiviralsignaling(Fig.9A
Localization of viral nucleocapsid, RIG-I, and IPS-1 and B). The results indicated that MFN1, but not MFN2, is
InordertoactivateRLRsignaling,weusedNDVtoinfectcells essential for thesignal transduction mediatedby RIG-I.
becauseitisavailableananti-nucleocapsidprotein(NP)antibody, We examined other regulatory proteins for mitochondrial
aprobefortheviralRNA-NPcomplex.NDVinfectionresultedin fission/fusion mechanism. Optic atrophy protein 1 (OPA1) is
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Figure1.StableHeLacellclonesexpressingFLAGtaggedIPS-1.A,ExpressionofFLAG-IPS-1wasexaminedincontrolandIPS-1-expressing
HeLaclones(#2,17,and21)byimmunoblottingusinganti-FLAGantibody.B,ExpressionofIFNB1incontrolandIPS-1-HeLacellswasexaminedby
quantitativerealtimePCR(qRT-PCR).Openandfilledbarsindicatemock-treatedandSeV-infectedcellsfor12h,respectively.Datarepresentmeans
6s.d.(n=3).C,ExpressionprofilesofcytokineandchemokinegenesincontrolandIPS-1-HeLacells.TotalRNAextractedfromindicatedcellsmock-
treatedorSeV-infectedfor12hwassubjectedtoanalysisusingaDNAmicroarray.RelativemRNAlevelsusingacontrolexpressionof1.0areshown.
D,ReplicationofEMCVincontrolandIPS-1-HeLaclones.TheindicatedcellcloneswereinfectedwithEMCVataMOIof1or10.Theviraltiterinthe
culturemediumat24hpost-infectionwasdeterminedwiththeplaqueassay.Datarepresentmeans6s.d.(n=3).
doi:10.1371/journal.ppat.1001012.g001
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RedistributionofIPS-1
Figure2.RedistributionofIPS-1inSeV-infectedcells.A,TheIPS-1-HeLaclone#2wasmock-treatedorinfectedwithSeVfor12handstained
with MitoTracker (Mitochondria) and anti-FLAG antibody (FLAG-IPS-1). Nuclei were visualized by staining with DAPI throughout this study. The
fluorescentimagewasquantifiedintheareaindicatedbyblueline(rightmostpanel).Quantificationresultsfrommock-orSeV-infectedcellsare
shownatthebottom.FluorescenceofDAPIcorrespondstoareainthenucleus.ThemitochondriaheavilystainedwithMitoTrackerbutlightlystained
withanti-FLAGareshownbyarrows.B,IPS-1-HeLacellsweremock-treatedorinfectedwithSeVfor12h.Cellswerestainedwithanti-FLAGantibody
(FLAG-IPS-1)andanti-ERABantibody(ERAB).
doi:10.1371/journal.ppat.1001012.g002
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Figure 3. Redistribution of IPS-1 induced by virus-infection and 59ppp-RNA-transfection. IPS-1-HeLa cells were infected with the
indicatedvirusesortransfectedwith59OH-RNAor59ppp-RNAchemicallysynthesizedbyinvitrotranscriptionusingT7RNApolymerase.At12hpost-
infectionor-transfection,thecellswerestainedwithanti-FLAGantibodyandMitoTracker(Mitochondria).Arrowheadsshowdeadcellswithshrunk
nucleiinSindbisvirus-infectedcells.
doi:10.1371/journal.ppat.1001012.g003
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RedistributionofIPS-1
Figure4.RedistributionofendogenousIPS-1invirus-infectedcells.A,HeLacellswereinfectedwithMockorSeVfor12h.Thecellswere
stainedwithanti-IPS-1antibodyandMitoTracker(Mitochondria).B,SKHep1cellswereinfectedwithNDV,SeV,Influenzavirus,orSindbisvirusfor
12h.Thecellswerestainedwithanti-IPS-1antibodyandMitoTracker(Mitochondria).
doi:10.1371/journal.ppat.1001012.g004
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Figure 5. Localization of viral nucleocapsid, RIG-I, and IPS-1. A, HeLa cells were infected with NDV for 12h and stained with anti-RIG-I
antibody(RIG-I)andanti-NPantibody(NDVNP).BandC,IPS-1-HeLacellswereinfectedwithNDVfor12handstainedwithanti-FLAGantibodyand
anti-RIG-Iantibodyoranti-NPantibody.
doi:10.1371/journal.ppat.1001012.g005
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Figure6.LocalizationofIPS-1andmitochondria.A,IPS-1-HeLacellsinfectedwithNDVfor9hwerefixed,stainedwithanti-NPantibody,and
subjectedtoultrathinsectioningasshownintheMethods.Theareaenclosedbyaredrectangleisenlarged.NP:NPfocistainedwiththeanti-NP
antibody were visualized using gold particles. B, IPS-1-HeLa cells infected with NDV for 9h were fixed, stained with anti-FLAG antibody, and
subjected to ultra thin sectioning. The area enclosed by a red rectangle is enlarged. NP: morphologically similar structures are in A. IPS-1 was
visualizedusinggoldparticles.ThearrowheadsindicateboundariesbetweenIPS-1andNPfoci.
doi:10.1371/journal.ppat.1001012.g006
expressedon,andimplicatedinthefusion ofthemitochondrial Physical interaction between IPS-1 and MFN1
inner membrane [20]. Three independent siRNA targeting ToexplorethemolecularmechanismofhowIPS-1isregulated
OPA1,down-regulatedOPA1expression(Fig.9C)andpartially by MFN1, co-immunoprecipitation was performed using cells
(up to 50%) blocked NDV-induced signaling (Fig. 9D). How- stably expressing IPS-1. FLAG-IPS-1 was precipitated by anti-
ever, the knockdown of dynamin-related protein 1 (DRP1) FLAG and the associated proteins were analyzed by immuno-
(Fig.9E),whichregulatesmitochondrialfission[21]resultingin blotting(Fig.10).BothMFN1andMFN2constitutivelyassociated
elongated mitochondria, did not have a significant effect with IPS-1 in the cells, but an unrelated mitochondrial outer
(Fig. 9F). To explore the site where MFN1 is active, we membrane protein, BCL-XL, did not associate with IPS-1.
temporarily overexpressed the dominant active RIG-I (RIG-I Furthermore, OPA1 and DRP1 did not co-immunoprecipitate
CARD) [17] or IPS-1 in wt and Mfn-knockout MEFs. Unlike with FLAG-IPS-1. These data suggest that IPS-1 selectively
the signal generated by the overexpression of IPS-1, the signal associates withMFN1and MFN2.
generated by overexpression of the RIG-I tandem caspase
recruitment domain (CARD) clearly required MFN1. MFN1
however, is dispensable if IPS-1 is overexpressed (Fig. 9G). Knockdown of MFN1 inhibits the redistribution of IPS-1
Again, MFN2 exhibited little influence on the signaling induced by viral infection
triggered by either stimulus. These results indicate that Next,weexaminedwhateffecttheknockdownofMFN1would
MFN1, but not MFN2, is essential for signal transduction have on the virus-induced redistribution of IPS-1. Three
mediated by RIG-I and IPS-1. independent siRNA efficiently knocked down MFN1 expression
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RedistributionofIPS-1
Figure7.AdominantnegativemutantofRIG-IfailstoinduceIPS-1redistribution.A,IPS-1-HeLacellsstablyexpressingwild-typehuman
RIG-I(RIG-IWT)ormutantRIG-I(RIG-IK270A)weremock-treatedorinfectedwithNDVfor12handexpressionofIFNB1mRNAwasanalyzedbyqRT-
PCR.OpenandfilledbarsindicateRNAsamplesfrommock-treatedandNDV-infectedcells,respectively.Datarepresentmeans6s.d.(n=3).B,IPS-1-
HeLacellsexpressingRIG-IWTorRIG-IK270AwereinfectedwithNDVfor12handstainedwithanti-RIG-Iantibodyandanti-NPantibody.RIG-I
stainingisdiffuseinuninfectedcellshoweverinfectionbyNDVproducedRIG-Ifoci.SomeRIG-Ifociareco-localizedwithNDVNPfoci.C,IPS-1-HeLa
cellsexpressingRIG-IWTorRIG-IK270AwereinfectedwithNDVfor12handIPS-1redistributionwasexamined.IPS-1andNPwerestainedwithanti-
IPS-1antibodyandanti-NPantibody,respectively.Theareaenclosedbytheredrectangleisenlargedattheright.AlthoughtheredistributedIPS-1
surroundsNPfociinRIG-IWTcells,K270AmutationofRIG-IfailedtoinducetheredistributionofIPS-1,butnottheformationofNPfoci.
doi:10.1371/journal.ppat.1001012.g007
(Fig. 11A) resulting in a strong inhibition of the NDV-induced IFN gene activation. This correlates with prior observations that
IFN-b gene expression in HeLa cells and IPS-1-HeLa cells although IFN production is inhibited by LGP2 overexpression,
(Fig. 11B and 11C). This once again suggests that IPS-1-HeLa viral yield does not increase [22]. When control siRNA-treated
cellstendtobehavelikenormalcells.UponNDVinfection,IPS-I cells were infected with NDV, a redistribution of IPS-1 was
displayedaspeckledstainingpatternincontrolcells,butnotinthe observed (69.3615.7% of cells positive for NP). In MFN1-
MFN1-knockdown cells (Fig. 11D). Though the intensity of NP knockdown cells, although NDV infection resulted in the
stainingdidnotincrease,MFN1-knockdownsignificantlyinhibited formation of NP foci, IPS-1 redistribution did not occur
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RedistributionofIPS-1
Figure8.MFN1isinvolvedinantiviralsignaling.A,SchematicrepresentationoftheMFN1domain.B,L929cellsweretransfectedwithavirus-
responsive reporter gene (p-125 Luc) and either an empty vector (Empty), an expression vector for MFN1, or an expression vector for MFN2 as
indicated.48hafterthetransfection,cellsweremock-treatedorinfectedwithNDV.Luciferaseactivitywasdeterminedat12hafterinfection.Cand
D,L929cellsweretransfectedwithavirus-responsivereportergene(p-125Luc)andeitheranemptyvector(Empty)oranexpressionvectorforMFN1
oritsmutant(MFN1T109A)asindicated.At48haftertransfection,cellsweremock-treated,infectedwithNDV,ortransfectedwith59OH-RNAor
59ppp-RNA.Luciferaseactivitywasdeterminedat12h(C)or9h(D)afterinduction.E,L929cellsweretransfectedwithavirus-responsivereporter
gene(p-125Luc)andcombinationsoftheindicatedvectors.48hafterthetransfection,cellsweremock-treatedorinfectedwithNDV.Luciferase
activitywasdetermined12hafterinfection.
doi:10.1371/journal.ppat.1001012.g008
(4.561.3%ofcellspositiveforNP).Asimilareffectwasobserved studyexhibitedverylowbasalexpressionofIFNgenes,whichled
when MFN1-knockdown cells were infected with SeV (Fig. 12). us to speculate that IPS-I inhibitory protein(s) is up regulated in
These results strongly suggest that MFN1 is critical to the theseclones.WealsoexaminedtheexpressionlevelofNLRX1,an
redistribution of IPS-1 triggered by RIG-I mediated sensing of IPS-1 inhibitor [23], and noted no change in its expression level
viral RNA. (notshown).Similarly,levelsofMFN1andMFN2didnotchange
intheIPS-1-HeLa clones. (Fig.10,input).
Discussion WeobservedthattheIPS-1leveldidnotchangeforupto12h
in virus-infected cells and no specific modification of IPS-1 was
RIG-Imediatedantiviralsignalingisacriticalantiviralresponse identified up to that point. We therefore hypothesize that the
whichisinitiatedwhentheRIG-IsensorrecognizesviralRNA.A activationstatusofIPS-1isdeterminedbyitslocalizationpattern.
signalisrelayedtoIPS-1,amitochondrialregulatorwhichdelivers We speculate that the mechanism of mitochondrial fusion is
thesignaldownstream.Interestingly,theIPS-1-HeLaclonesinthis mediated by MFN1, and that IPS-1 translocates from some
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