TNFα Impairs Rhabdoviral Clearance by Inhibiting the Host Autophagic Antiviral Response

20 Pages·2016·6.04 MB·English

RESEARCHARTICLE
α
TNF Impairs Rhabdoviral Clearance by
Inhibiting the Host Autophagic Antiviral
Response
RaquelEspín-Palazón1☯¤a,AliciaMartínez-López2☯,FranciscoJ.Roca1¤b,AzucenaLópez-
Muñoz1,SylwiaD.Tyrkalska1,SergioCandel1,DianaGarcía-Moreno1,AlbertoFalco2,
JoséMeseguer1,AmparoEstepa2†‡,VictorianoMulero1‡*
1 DepartamentodeBiologíaCelulareHistología.FacultaddeBiología.UniversidaddeMurcia,IMIB-
Arrixaca,Murcia,Spain,2 InstitutodeBiologíaCelularyMolecular,UniversidadMiguelHernández,Elche,
Spain
a11111 †Deceased.
☯Theseauthorscontributedequallytothiswork.
¤a Currentaddress:DepartmentofCellularandMolecularMedicine,UniversityofCaliforniaatSanDiego,
LaJolla,California,UnitedStatesofAmerica
¤b Currentaddress:DepartmentofMedicine,MRC-LaboratoryofMolecularBiology,Universityof
Cambridge,Cambridge,UnitedKingdom
‡Thesetwoauthorshavecosupervisedthisstudy.
*[email protected]
OPENACCESS
Citation:Espín-PalazónR,Martínez-LópezA,Roca
Abstract
FJ,López-MuñozA,TyrkalskaSD,CandelS,etal.
(2016)TNFαImpairsRhabdoviralClearanceby
InhibitingtheHostAutophagicAntiviralResponse. TNFαisapleiotropicpro-inflammatorycytokinewithakeyroleintheactivationofthe
PLoSPathog12(6):e1005699.doi:10.1371/journal.
immunesystemtofightviralinfections.Despiteitsantiviralrole,afewvirusesmightutilize
ppat.1005699
thehostproducedTNFαtotheirbenefit.Somerecentreportshaveshownthatanti-TNFα
Editor:SaraCherry,UniversityofPennsylvania
therapiescouldbeutilizedtotreatcertainviralinfections.However,theunderlyingmecha-
SchoolofMedicine,UNITEDSTATES
nismsbywhichTNFαcanfavorvirusreplicationhavenotbeenidentified.Here,arhabdo-
Received:May18,2015
viralinfectionmodelinzebrafishallowedustoidentifythemechanismofactionbywhich
Accepted:May20,2016 Tnfahasadeleteriousroleforthehosttocombatcertainviralinfections.Ourresultsdemon-
Published:June28,2016 stratethatTnfasignalsthroughitsreceptorTnfr2toenhanceviralreplication.Mechanisti-
cally,Tnfadoesnotaffectviraladhesionanddeliveryfromendosomestothecytosol.In
Copyright:©2016Espín-Palazónetal.Thisisan
openaccessarticledistributedunderthetermsofthe addition,thehostinterferonresponsewasalsounaffectedbyTnfalevels.However,Tnfa
CreativeCommonsAttributionLicense,whichpermits blocksthehostautophagicresponse,whichisrequiredforviralclearance.Thismechanism
unrestricteduse,distribution,andreproductioninany
ofactionprovidesnewtherapeutictargetsforthetreatmentofSVCV-infectedfish,and
medium,providedtheoriginalauthorandsourceare
advancesourunderstandingofthepreviouslyenigmaticdeleteriousroleofTNFαincertain
credited.
viralinfections.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:Thisstudywasfundedby:MINECO
BIO2011-23400(VM),MINECOCSD2007-00002
AuthorSummary
(VM),MINECOBIO2014-52655-R(VM),MINECO
AGL2014-51773-C3-1-R(AE),andFondosEuropeos Tumornecrosisfactoralpha(TNFα)isoneofthemainpro-inflammatorycytokinespro-
deDesarrolloRegional(FEDER)(VMAE).The
ducedinresponsetoabroadtypeofinfections[1].AlthoughTNFαhasacrucialrolein
fundershadnoroleinstudydesign,datacollection
andanalysis,decisiontopublish,orpreparationof protectingthehostorganismfrompathogens,itsderegulationcanpromotesusceptibility
themanuscript.
PLOSPathogens|DOI:10.1371/journal.ppat.1005699 June28,2016 1/20
TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. topathogensbyimpairingpathogenclearanceand,ultimately,promotingmaintenanceof
infectionanddeath.Inaddition,somevirusesmightutilizethehostproducedTNFαto
theirbenefit.Thus,anti-TNFαtherapiescouldbeutilizedtotreatcertainviralinfections.
However,theunderlyingmechanismsbywhichTNFαcanfavorcertainvirusreplication
havenotbeenidentified.Here,wehaveusedaviralinfectionmodelinzebrafishtoidentify
themechanismofactionbywhichTNFαhasadeleteriousroleforthehosttocombatcer-
tainviralinfections.OurresultsdemonstratethatTnfadoesnotaffectviralabilitytoinfect
hostcellsortoantagonizethemainhostantiviralpathway,namelytheinterferonpathway.
However,Tnfaimpairsviralclearancebyblockingthehostautophagyresponse,whichis
usuallyusedbyhostcellstodegradeunnecessaryordysfunctionalcellularcomponents,
andthatwefoundtobecriticaltoeliminateintracellularviralparticles.Thismechanism
ofactionprovidesnewtherapeutictargetsforthetreatmentofSVCV-infectedfishin
aquacultureandprobablytootherviralinfectionaffectingcattleindustryandhuman.
Introduction
Tumornecrosisfactoralpha(TNFα)isoneofthemainpro-inflammatorycytokinesproduced
inresponsetoabroadtypeofbacterial,viralandfungalinfections[1].TNFαhasacrucialrole
inactivatingandorchestratingtheimmuneresponseinordertoprotectthehostorganism
frompathogens.TNFαderegulationcanpromotesusceptibilitytopathogensbyimpairing
pathogenclearanceand,ultimately,promotingmaintenanceofinfectionanddeath.Whenspe-
cificallytalkingaboutviralpathogenesis,TNFαhasbeenshowntoinhibitthereplicationof
certainvirusessuchashepatitisBvirus(HBV)andthevaricellazostervirus(VZV)[2].In
addition,anti-TNFtherapiestotreatautoimmunediseasesexacerbatetheinfectionproduced
byvirussuchasherpessimplexvirus(HSV),Epstein-Barrvirus(EBV),cytomegalovirus
(CMV)andhumanpapillomavirus(HPV)[3].Itisnotsurprisingthatduetothekeyroleof
TNFαinthehostprotectiontoviralinfections,someviruseshavedevelopeddifferentwaysto
interferewiththeTNFαpathway[4].Incontrast,itseemsthatafewvirusesmightutilizethe
hostproducedTNFαtotheirbenefit.Interestingly,humanimmunodeficiencyvirus1(HIV-1)
infectioninducesTNFαexpression.TheseincreasedTNFαlevelsinserumcorrelatesto
increasedviralreplication[5].Inaccordancetothat,TNFαinhibitorsareabletoimpairHIV-1
replication[6],andanti-TNFαtreatmentshavebeenproposedtocombatHIV-1infectionin
combinationwithothertherapies[7][5].Similarly,neutralizationofTNFαdecreasesvirus
productioninCMV-infectedmacrophages[8].TheabilityofTNFαtofavorvirusreplication
hasalsobeendemonstratedfornon-mammalianviruses,suchasthespringviremiaofcarp
virus[9],afishrhabdovirusinfectingcyprinids[10,11].Moreover,intraperitoneallySVCV-
infectedadultfish,inwhichrecombinantTNFαwasadministratedsimultaneously,hasshown
ahighermortalityratethanfishinjectedwiththevirusalone.Themechanismexplaininghow
TNFαfacilitatesviralinfectionanditsdeleteriouseffectsinthehosthasnotyetbeenproposed.
SincezebrafishisacyprinidsusceptibletoSVCVinfection,andTNFαcanexacerbate
SVCVinfection,wechosethisamenableinfectionmodeltoinvestigatehowavirusmightuti-
lizehostproducedTNFαtotheirbenefit.Tothatend,weanalyzedtheroleofzebrafishTNFα
(Tnfa)ini)thekeystepsofSVCVpathogenesis:virusadhesion,fusion,andreplication;andii)
intheantiviralhostresponse,suchasinterferonproductionandautophagy.Theresults
showedthatTnfasignalingthroughitsreceptorTnfr2inhibitsautophagy,leadingtoimpaired
viralclearanceinSVCV-infectedcells.Thismechanismofactionprovidesnewtherapeutic
PLOSPathogens|DOI:10.1371/journal.ppat.1005699 June28,2016 2/20
TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
targetsforthetreatmentofSVCV-infectedfish,andadvancesourunderstandingoftheprevi-
ouslyenigmaticdeleteriousroleofTNFαincertainviralinfections.
Results
TnfaincreasessusceptibilityofzebrafishtoSVCVinfection
Asinmostinfections,Tnfaisup-regulatedinresponsetoSVCVinfection[12].Unexpectedly,
thisup-regulationratherthanhelptocontroltheinfection,hasadeleteriousroleinadultzeb-
rafish[9].Tofurtherstudythisphenomena,wefirstinvestigatedwhetherornotTnfawasalso
abletoenhanceSVCVreplicationbothinvivoandinvitro.Forthat,wepre-incubatedthezeb-
rafishembryonicfibroblastcellline,ZF4,whichexpressesbothTnfrs[9],withzebrafish
recombinantTnfaorinterferon1(Ifn1,alsoknownasIfnphi1)for4hoursand,subsequently,
thetreatedcellswereinfectedwithSVCV.At24hourspost-infection(hpi),viralreplication,
measuredasthepresenceoftranscriptofthenucleoproteinthatformstheSVCVcapside(N
protein),wasevaluatedbyRT-qPCR(Fig1A).Nproteintranscriptssignificantlyincreasedin
Tnfa-treatedcellsandsignificantlydecreasedinIfn1-treatedcells(Fig1B),suggestingthat
TnfaenhancesandIfn1decreasesviralreplicationinvitro.
BothinsufficientandexcessTnfahavebeenshowntopromotesusceptibilitytomycobacte-
rialinfection[13].WethenaskedwhetherendogenousratherthanexogenousTnfawasbenefi-
cialordetrimentaltothehostduringSVCVinfection.Thepercentageofanimalsthatsurvived
at7dayspost-infection(dpi)wassignificantlyhigherinTnfa-depletedlarvaewhencompared
tocontrols(Tnfaexpressinglarvae)(55%versus30%,respectively)(Fig1Cand1DandS1A
andS1BFig).ThesurvivalpercentageofcontrolandTnfa-depleteduninfectedlarvaewas
100%inbothcases.Inaccordancetotheseresults,qPCRanalysisofembryosharvestedat
48hpishowedthatthehighestlevelsofviralreplication(measuredastheamountofSVCVN
proteinmRNAininfectedanimaltissues)(Fig1E),andvirusparticles(measuredasthe
amountofnegativesenseRNAencodingSVCVGglycoproteinininfectedanimaltissues)(Fig
1F),werefoundincontrollarvae.Theseresultswerefurtherconfirmedinlarvaforcedto
expressTnfaRNA,whichshoweddrasticincreasedsusceptibilitytoSVCV(Fig1GandS1C
andS1EFig).Together,theseresultsindicatethatTnfaenhancesSVCVreplicationandpatho-
genesisinvivo.
TheTnfa/Tnfr2axismediatesincreasedSVCVreplicationinzebrafish
TNFαexertsitsactivitythroughthebindingandactivationoftworeceptors,TNFR1and
TNFR2(Tumornecrosisfactorreceptor1and2,respectively)[14].Tnfreceptorsareexpressed
earlyduringzebrafishdevelopment[15],andtheybothhaveimportantrolesfortheclearance
ofviralinfections[16].TofurtherdissectthecontributionofTnfasignalinginSVCVpatho-
genesis,weperformedloss-of-functionexperimentsforbothTnfareceptorsusingspecificanti-
sensemorpholinos(MOs)[15]inSVCV-infectedembryos(Fig2AandS1F–S1IFig).
Tnfr2-depletedlarvaeweredistinctlymoreresistanttoSVCVinfectioncomparedtotheircon-
trolsiblings(60%versus30%,respectively)(Fig2B),whileTnfr1-depletedlarvaeshoweda
slightly,butstatisticallysignificant,reducedsurvivalcomparetotheircontrolsiblings(Fig2B).
Thisresultwassupportedbyincreased,ordecreased,SVCVreplicationinTnfr1-and
Tnfr2-depletedlarvae,respectively(Fig2C).Accordingly,thepresenceofviralgenomeswas
alsohigherinTnfr1-depletedlarvaeandlowerinTnfr2-depletedlarvaeat48hpi(Fig2D).In
addition,larvaforcedtoexpressaRNAencodingadominantnegative(DN)formofTnfr2,
whichislackingtheentireintracellularsignalingdomainandextinguishesTnfr2signalingby
trimerizationwithendogenousTnfr2[15],showedincreasedresistancetoSVCV(Fig2Eand
PLOSPathogens|DOI:10.1371/journal.ppat.1005699 June28,2016 3/20
TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
Fig1.TnfaenhancesSVCVinfectioninzebrafish.(A)Workflowoftheexperimentaldesignfollowedin(B).
RecombinantzebrafishTnfaorinterferon1(Ifn1)wereaddedtoZF4cellsgrowinginmonolayerat80%confluenceand
incubatedfor4hours.Subsequently,themediumwaswashedoutandfreshmediumcontainingSVCVwasadded.After
24hoursofincubationwiththevirus,thecellswereharvestedforqPCRanalysis.(B)NproteinmRNAexpressionlevels
assessedbyqPCRrelativetothehousekeepinggenerps11andmultipliedby105.Barsrepresentmean±S.E.M.of
indicatedgeneexpressionfromonerepresentativeexperiment.(C)Workflowoftheexperimentaldesignfollowedin
(D-G).Std(Control)orTnfamos(D-F)orantisenseorTnfaRNAs(G)wereinjectedinzebrafishembryosatone-cell-stage
ofdevelopment.At3dpf,theselarvaewereimmerseinRPMIcontaininginactivatedSVCV(control)orintactSVCVfor
subsequentlyanalysisofsurvival(D,G)orqPCRanalysisat48hourspost-infection(hpi)(E,F).Percentageofsurvivalof
Tnfa-depleted(D)andoverexpressing(G)zebrafishlarvaeexposedto109TCID /mlSVCV.(E,F)ThemRNAlevelsof
50
thegenecodingfortheSVCVNproteinasanestimationoftheviralreplication(E),andtheRNA-levelsofGprotein(F)
weredeterminedintheinfectedlarvaebyqPCRin10pooledlarvaeat48hpi(5dpf).Thegeneexpressionwas
normalizedagainstrps11andmultipliedby105forNprotein.Barsrepresentmean±S.E.M.oftriplicatereadingsfrom
pooledlarvaeandthedataarerepresentativeoftwoindependentexperiments.***p<0.001.ND,notdetected.
doi:10.1371/journal.ppat.1005699.g001
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TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
Fig2.Tnfr2mediatestheTnfa-triggeredsusceptibilityofzebrafishtoSVCV.(A)Workflowoftheexperimentaldesignfollowed
in(B-E).Std(Control),Tnfr1orTnfr2mos(B-D)orantisenseorDN-Tnfr2RNAs(E)wereinjectedinzebrafishembryosatone-cell-
stageofdevelopment.At3dpf,theselarvaewereimmerseinRPMIcontaininginactivatedSVCV(control)orintactSVCVfor
subsequentlyanalysisofsurvival(B,E)orqPCRanalysisat48hourspost-infection(hpi)(C,D).PercentageofsurvivalofTnfr-
depleted(B)andDN-Tnfr2overexpressing(E)zebrafishlarvaeexposedto109TCID /mlSVCV.(C,D)ThemRNAlevelsofthe
50
genecodingfortheSVCVNproteinasanestimationoftheviralreplication(C),andthetheRNA-levelsofGprotein(D)were
determinedintheinfectedlarvaebyqPCRin10pooledlarvaeat48hpi(5dpf).Thegeneexpressionwasnormalizedagainstrps11
andmultipliedby105forNprotein.Barsrepresentsmean±S.E.M.oftriplicatereadingsfrompooledlarvaeandthedataare
representativeoftwoindependentexperiments.*p<0.1;***p<0.001.
doi:10.1371/journal.ppat.1005699.g002
S1DandS1EFig).Overall,theseresultssuggestthatTnfafacilitatesSVCVreplicationthrough
Tnfr2signaling.
Tnfadoesnotaffectviraladhesionorviraldeliveryfromendosomestothecytosol. To
betterunderstandthemechanismbywhichTnfaenhancesviralreplication,weinvestigated
theroleofTnfaintwokeystepsofSVCVpathogenesis,viruscellbindingandthesubsequent
membranefusionthatallowsvirusreleasefromtheendosometothecytosol.Tointerrogateif
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TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
Fig3.SVCVadhesionorsyncytiaproductionisnotaffectedbyTnfa.(A)Workflowoftheexperimentaldesign
followedin(B).Briefly,recombinantzebrafishTnfawasaddedtoZF4cellsgrowinginmonolayerat80%ofconfluence
andincubatedfor4hours.Subsequently,themediumwaswashedoutandnewmediumcontainingSVCV±Tnfa(Tnfa
combination)wasaddedfor30minutesat4°Ctoallowvirusadhesionbutnotvirusinternalizationandreplication.After
30minutes,thecellswerewashedandharvestedforqPCRanalysisoftheGproteingeneforquantitationofthe
adheredvirus.(B)qPCRanalysisoftheGproteinencodinggene.Barsrepresentmean±S.E.M.oftriplicatereadings
fromonesampleandthedataarerepresentativeoftwoindependentexperiments.*p<0.1.ns,nonsignificant.Tnfa
comb.,TnfaaddedincombinationtotheSVCV.(C)Workflowrepresentingtheexperimentaldesignfor(D)and(E).ZF4
wereincubatedwithSVCVfor24hours.Themediumcontainingviruswaswashedoutandnewmediumcontaining
Tnfawasaddedfor45minutes.ThefusionprocesswastriggeredbydecreasingthepHto6for30minutes,andthe
nucleiinthesyncytiawerequantitated(D,E).Redlinesdenotesyncytia.
doi:10.1371/journal.ppat.1005699.g003
Tnfacouldbefacilitatingthevirusbindingtothecell,weinfectedZF4cellsfor30minutesat
4°Ctoallowthevirusadhesionbutnotitsendocytosisandreplication.TnfawasaddedtoZF4
cellsbeforeorsimultaneouslytoSVCV(Fig3A).Thenumberofviralparticlesadheredto
Tnfa-treatedcells(inbothconditions,pre-orsimultaneouslyadded)wasslightlylowerthanin
thenon-treatedcells,assessedbyqPCRofnegativesenseRNAencodingSVCVGglycoprotein
(Fig3B).ThisresultsuggeststhatTnfadoesnotfacilitatetheSVCVcellbinding.
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TNFαImpairsRhabdoviralClearancebyInhibitingAutophagy
Afterbindingtothecellmembrane,SVCVentersthecellbyreceptor-mediatedendocytosis.
Subsequently,theseearlyendosomesareacidifiedafterfusingtolysosomes.Endosomalacidifi-
cationtriggersconformationalchangesintheGproteinofrhabdovirus,releasingthevirus
genomeintothecytoplasmandallowingtheirreplication[10,17,18].Inordertoinvestigate
whetherornotthiscriticalstepinvirusreplicationwasaffectedbyTnfa,weperformedafusion
assay[19]inSVCV-infectedZF4cellspre-treatedwithTnfa,whereG-dependentcellfusionis
triggeredatpH=6(Fig3C).Theresultsshowthatthefusionprocess,evaluatedbythenumber
ofnucleiinsyncytia,inTnfa-treatedcellswasunaffectedcomparedtonon-treatedcells(Fig
3Dand3E).Takentogether,thesedataindicatethatTnfadoesnotfacilitatevirusbindingto
thecellmembraneorviralgenomereleaseintothecytoplasm.
TnfadoesnotantagonizetheantiviralroleofinterferonduringSVCV
infection
Interferonisoneofthemostpowerfulantiviralcytokine[20].Ithasbeenshownthatinterfer-
onscanactsynergisticallywithTNFαtosuppressvirusreplication[21,22].However,TNFα
andinterferoncanhaveantagonisticrolesincertaincellssuchashumanfibroblast-likesyno-
viocytes[23].Therefore,wedecidedtoinvestigateiftheenhancingroleofTNFαinSVCVrep-
licationwastheresultofimpairingtheinterferonresponseduringSVCVinfection.ZF4cells
werepre-treatedwithTnfaand/orIfn1for4hours.Subsequently,thesecellswereinfectedwith
SVCValoneorincombinationwithTnfa.After24hours,qPCRanalysiswasperformedto
detecttheexpressionofantiviralhostgenesandviralNproteintranscript(Fig4A).Theaddi-
tionofTnfabeforeorincombinationwithSVCVdidnotalterthetranscriptlevelsofthegenes
encodingmajorantiviraleffectors,suchasmyxovirus(influenza)resistanceb(Mxb),radicalS-
adenosylmethioninedomaincontaining2(Rsad2),Mxcandproteinkinasecontaining
Z-DNAbindingdomains(Pkz)comparedtountreatedcells(Fig4Band4CandS2AandS2B
Fig).Incontrast,Ifn1-treatedcellsshoweddrasticallyincreasedlevelsofthetranscriptsforthe
samehostgenes(Fig4Band4CandS2AandS2BFig).Cellswerethenpre-incubatedwith2
differentdilutionsofIfn1(1/100and1/500),aloneorincombinationwithTnfa,andsubse-
quentSVCVinfectionwasperformed.BothIfn1dilutionswereabletoincreasetheRNAlevels
ofmxb,thoughtheselevelswereunaffectedbythesimultaneousadditionofTnfa,inbothunin-
fectedandinfectedcells(Fig4D).Altogether,theseresultsindicatethatTnfadoesnotantago-
nizetheantiviralroleofIfn1duringSVCVinfection.ToverifythatIfn1wasindeedinterfering
withSVCVreplicationinZF4cells,SVCVreplicationwasquantitatedbyRT-qPCRanalysisof
theNproteintranscriptsinSVCV-infectedZF4cellspre-treatedwithTnfaandIfn1(Fig4A
and4E).WhiletheNproteinmRNAlevelswereup-regulatedinTnfa-treatedcells,theywere
down-regulatedintheIfn1,aswellasintheTnfa/Ifn1combination(Fig4E).Thisfindingsug-
geststhatIfn1hasaprotectiveroleagainstSVCVreplicationandthatTNFαdoesnotantago-
nizetheantiviralroleofIfn1duringSVCVinfection.
Tnfainhibitsautophagy
Sinceautophagyisanefficientantiviralmechanisminresponsetomanyviralinfections
includingSVCV[24,25],weaskedifTnfacouldinterferewiththeautophagy-mediatedclear-
anceofSVCVbyhostinfectedcells.ZF4cellswereincubatedwithTnfafor4hoursandautop-
hagylevelswereassessedbycellularLC3distribution.Cellsweretreatedwithautophagy
modulators,suchas3-Methyladenine(3MA)andrapamycin(Rapa)torespectivelyinhibitor
enhanceautophagy.Asexpected,autophagy(redpunctaindicatingL3Crecruitment)was
clearlydiminishedin3MA-treatedcellsand,incontrast,stronglyincreasedinRapa-treated
cells(bothinnumberandsizeoftheautophagosomes)(Fig5A).Interestingly,Tnfatreatment
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Fig4.TheantiviralroleofinterferonisnotdisruptedbyTnfaduringSVCVinfection.(A)Workflowrepresentingthe
experimentaldesignfollowedin(B-E).Briefly,TnfaorIfn1wereaddedto80%confluentZF4cellsandincubatedfor4hours.
Subsequently,themediumwasremovedandfreshmediumcontainingSVCV±Tnfawasaddedduring24hoursforthe
followingqPCRanalysisfrommRNAextractedfromthecell.(B,C)mRNAlevelsofthegenesencodingfortheantiviralgenes
mxb(B)anfrsad2(C)ofSVCV-infectedZF4determinedbyqPCR.Geneexpressionisnormalizedagainstrps11and
multipliedby10formxband102forrsad2.Barsrepresentmean±S.E.M.oftriplicatereadingsfromonesampleandthedata
arerepresentativeoftwoindependentexperiments.(D)qPCRanalysisofmxbexpressionlevelsinnoninfectedorSVCV-
infectedcellspreviouslytreatedwithTnfaortwodifferentdilutionsofIfn1(1/100or1/500).Thegeneexpressionisnormalized
againstrps11andmultipliedby103.Barsrepresentmean±S.E.M.oftriplicatereadingsfromonesampleandthedataare
representativeoftwoindependentexperiments.(E)qPCRanalysisoftheNproteinexpressionlevelsinSVCV-infectedZF4
cellspreviouslytreatedwithTnfaorIfn1.Thegeneexpressionisnormalizedagainstrps11andmultipliedby105.Bars
representmean±S.E.M.oftriplicatereadingsfromonesampleandthedataarerepresentativeoftwoindependent
experiments.***p<0.001.ns,nonsignificant.
doi:10.1371/journal.ppat.1005699.g004
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Fig5.Tnfainhibitsautophagy.(A)ZF4cellsweretreatedwith0.1μgofTnfaorinactivatedTnfa(CTnfa),1μMofRapa,
10mMof3-methyladenine(3MA)orremainuntreated.After4hourscellswerefixedandthenincubatedwithanantibody
anti-LC3.Cellswerefinallystainedwithafluorophore-conjugatedsecondaryantibody(redfluorescence,LC3)andDAPI
(blue,cellnuclei).ThenumberofcellswithLC3puncta(n=10)wasdetermined(leftpanel).Barsrepresentmean±S.E.
M.Imagesarerepresentativeoftheresultsobtainedin3independentexperiments(rightpanel).(B-D)Wholecelllysates
wereobtainedfromcellstreatedwithTnfa,CTnfa,Rapaor3MA(B)orpre-treatedwithRapafor4hoursandthenTnfaor
CTnfawasaddedforanother4hours(C,D).LC3-IandLC3-IIbandswerevisualizedbyWBusingananti-LC3antibody
andtheproteincontentofthestainedbandsestimatedbydensitometry.Thedensitometryvalueswereusedtocalculate
LC3-II/LC3-Iratios.Actinbandsweredetectedasaproteinloadinternalcontrolusingananti-actinantibody.Dataare
shownasthemean±S.E.M.of3independentexperiments.*p<0.05.**p<0.01.
doi:10.1371/journal.ppat.1005699.g005
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diminishedautophagosomeformationsuggestingthatTnfainhibitsautophagy(Fig5A).In
contrast,cellstreatedwithheat-inactivatedTnfa(controlTnfa,CTnfa)didnotaffectautop-
hagy(Fig5A).
Afterautophagyinduction,thecytosolicsolubleformofLC3(LC3-I)isconjugatedto
phosphatidylethanolaminetoformLC3-phosphatidylethanolamineconjugate(LC3-II),which
isrecruitedtoautophagosomalmembranes.Thisprocessisconservedamongvertebratesand
presentinmammals[26]andinfish[25].Therefore,LC3-II/LC3-Iratioiscommonlyusedto
quantifyautophagosomeformationbywestern-blot(WB)[27].Inordertoquantifyautophagy
formationinTnfa-treatedcells,western-blotforLC3wasperformedfromlysatesofTnfa-
treatedZF4cellsat4hourspost-treatment(Fig5B).TheLC3-II/LC3-Iratiodecreasedby
2-foldinTnfa-treatedcells,butwasunalteredinheat-inactivatedTnfa(CTnfa),indicatingthe
negativeimpactofTnfainautophagy(Fig5B).Asexpected,theLC3-II/LC3-Iratiodecreased
after3MAtreatmentandincreasedafterRapaaddition(Fig5B).TofurtherverifytheTnfa-
mediateddown-regulationofautophagy,ZF4cellswerepre-treatedwithRapafor4hoursand,
subsequently,Tnfawasaddedfor4hoursandwestern-blotforLC3wasperformedusingthe
celllysates(Fig5C).TheadditionofTnfatoRapa-treatedcellsledtoa2-foldreductioninthe
autophagyactivitycomparedtocellstreatedwithRapaalone(Fig5D).Asexpected,incubation
ofTnfaalonereducedtheautophagyactivityby2-foldcomparedtountreatedcells(Fig5D).
Alltogether,theseresultsindicatethatTnfareducesautophagyinZF4cells.Moreover,these
datasuggestaroleforTnfaasapotenteffectorinrevertingautophagyafterthisprocesshas
beeninitiated.
Tnfaimpairsautophagy-mediatedclearanceofSVCVinZF4cells
WehavepreviouslydemonstratedthatautophagyhasaprotectiveroleduringSVCVandviral
hemorrhagicsepticemiavirus(VHSV)infection[25].ToinvestigatewhetherTnfa-mediated
reductionofautophagyimpairsSVCVclearance,ZF4cellswerepre-incubatedwithTnfaprior
toSVCVinfection.AsshowninthediagramofFig6A,thevirusfociformingunits(ffu)were
firstdetectedbyimmunofluorescenceagainstNproteinalone(Fig6B),orincombinationwith
LC3(Fig6D)orP62(Fig6E).Toevaluatewhethertheseffucorrelatedwiththeinfectiveviral
particles,theSVCVpresentinthesupernatant(viralyield)wasalsoisolatedandtitratedby
plaqueformingunits(PFU)(Fig6Aand6C).TheffunumberincreasedinTnfa-treatedcells
comparedtountreatedandCTnfa-treatedcells(Fig6B).However,nodifferencesonthefoci
sizewerefoundbetweenthesetwotreatments(Fig6B).Asexpected,3MAincreasedtheffu
number,whileRapadecreasedit(Fig6B).SupernatantfromcellstreatedwithTnfacontained
2.5-timesmoreinfectiveviralparticles(4.5x105pfu/ml)thanun-treated(1,8x105pfu/ml),or
CTnfa-treatedcells(1,4x105pfu/ml)(Fig6C).Asexpected,3MA-treatmentalsoincreasedthe
SVCVpfu/ml(5,5x105),whileRapasignificantlydecreasedit(2,7x104pfu/ml)(Fig6C).Nota-
bly,althoughviralparticlescolocalizationwithLC3andP62punctawashardlyobservedin
controlcells,probablyreflectingtherapiddegradation/lossofimmunogenicityofthevirus,it
wasnicelyobservedincellstreatedwithTnfa(Fig6Dand6E).Takentogether,theseresults
demonstratethatTnfaimpairsviralclearancethroughtheinhibitionoftheautophagyresponse
ininfectedcells.
TnfainhibitsautophagyinvivoduringSVCVinfectioninzebrafishlarvae
ToanalyzetheimpactofTnfaontheregulationofthehostautophagicresponsetoSVCV
infection,weusedaGFP-LC3transgeniclinethatallowsareal-timevisualizationofautophagy
activity[28].Morpholino-dependentTnfadepletionresultedinincreasedbasalautophagyin
wholelarvae,observedatlowmagnificationasanincreasedfluorescenceduetoLC3
PLOSPathogens|DOI:10.1371/journal.ppat.1005699 June28,2016 10/20