Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

23 Pages·2017·7.16 MB·English

RESEARCHARTICLE
Inhibition of Japanese encephalitis virus
infection by the host zinc-finger antiviral
protein
Hsin-PingChiu1,2,HanChiu1,2,Chao-FuYang2,Yi-LingLee2,Feng-LanChiu3,Hung-
ChihKuo3,Ren-JyeLin4*,Yi-LingLin1,2,5*
1 GraduateInstituteofMicrobiology,NationalTaiwanUniversity,Taipei,Taiwan,2 InstituteofBiomedical
Sciences,AcademiaSinica,Taipei,Taiwan,3 InstituteofCellularandOrganismicBiology,AcademiaSinica,
Taipei,Taiwan,4 NationalMosquito-BorneDiseasesControlResearchCenter,NationalHealthResearch
Institute,Taipei,Taiwan,5 GenomicsResearchCenter,AcademiaSinica,Taipei,Taiwan
a1111111111
a1111111111 *[email protected](RJL);[email protected](YLL)
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a1111111111 Abstract
CCCH-typezinc-fingerantiviralprotein(ZAP)isahostfactorthatrestrictstheinfectionof
manyvirusesmainlythroughRNAdegradation,translationinhibitionandinnateimmune
OPENACCESS responses.Sofar,onlyoneflavivirus,yellowfevervirus,hasbeenreportedtobeZAP-resis-
tant.Here,weinvestigatedtheantiviralpotentialofhumanZAP(isoformZAP-LandZAP-S)
Citation:ChiuH-P,ChiuH,YangC-F,LeeY-L,Chiu
F-L,KuoH-C,etal.(2018)InhibitionofJapanese againstthreeflaviviruses,Japaneseencephalitisvirus(JEV),denguevirus(DENV)and
encephalitisvirusinfectionbythehostzinc-finger Zikavirus(ZIKV).InfectionofJEVbutnotDENVorZIKVwasblockedbyZAPoverexpres-
antiviralprotein.PLoSPathog14(7):e1007166.
sion,anddepletionofendogenousZAPenhancedJEVreplication.ZAPhamperedJEV
https://doi.org/10.1371/journal.ppat.1007166
translationandtargetedviralRNAfor30-50RNAexosome-mediateddegradation.Thezinc-
Editor:JohnW.Schoggins,UniversityofTexas fingermotifsofZAPwereessentialforRNAtargetingandanti-JEVactivity.JEV30-UTR,
SouthwesternMedicalCenteratDallas,UNITED
especiallyintheregionwithdumbbellstructuresandhighcontentofCGdinucleotide,was
STATES
mappedtobindZAPandconfersensitivitytoZAP.Insummary,weidentifiedJEVasthe
Received:March19,2018
firstZAP-sensitiveflavivirus.ZAPmayactasanintrinsicantiviralfactorthroughspecific
Accepted:June19,2018
RNAbindingtofightagainstJEVinfection.
Published:July17,2018
Copyright:©2018Chiuetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which Authorsummary
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal Inadditiontoinnateandadaptiveimmunities,manycellularproteinsalsoexertantiviral
authorandsourcearecredited. activityagainstviralinvasion.Humanzinc-fingerantiviralprotein(ZAP)isacellular
DataAvailabilityStatement:Allrelevantdataare restrictionfactoragainstmanyvirusesbutitsrolewithregardtotheflavivirusfamilyis
withinthepaperanditsSupportingInformation largelyunknown.WetestedtheantiviralpotentialofZAPagainstthreeflavivirusesand
files. foundthatJapaneseencephalitisvirus(JEV)wasZAP-sensitive,whiledenguevirusand
Funding:ThisworkwassupportedbyAcademia ZikaviruswereZAP-resistant.ZAPspecificallytargetsJEVviralRNAandinducestrans-
Sinica(106-2101-01-11-01&107-2101-01-18-03) lationrepressionandRNAdegradation.OurfindingshighlighttheZAP-mediatedanti-
andtheMinistryofScienceandTechnology, JEVmechanismsandextendtheantiviralspectrumofZAPtoincludeamemberofthe
Taiwan(MOST106-0210-01-15-02&107-0210-
Flavivirusgenus.
01-19-01).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1007166 July17,2018 1/23
JEVinfectioninhibitedbyZAP
Competinginterests:Theauthorshavedeclared
Introduction
thatnocompetinginterestsexist.
Zinc-fingerCCCH-typecontaining,antiviral1(ZC3HAV1)alsoknownaszinc-fingerantivi-
ralprotein(ZAP)wasfirstdiscoveredinratsasahostantiviralproteinagainstMoloney
murineleukemiavirus[1].Later,multipleRNAandDNAviruses,likeretroviruses,filoviruses,
alphaviruses,andhepatitisBviruswereshowntodisplaysensitivitytoZAP[2–6].However,
ZAPdoesnotinduceauniversalantiviralstate,sincevirusessuchasherpessimplexvirus1
(HSV-1),vesicularstomatitisvirusandyellowfevervirus(YFV)areresistanttoZAP[4].Fur-
thermore,viruseswithinthesamefamilymayexhibitdifferentsensitivitytoZAP.Forexample,
inthefamilyPicornaviridae,ZAPinhibitedcoxsackievirusB3butnotpoliovirusinfection
[4,7].
Inhumans,ZAPcontainstwomajorisoforms(ZAP-LandZAP-S),whichdifferintheC-
terminalregionthroughalternativesplicing[8].IntheN-terminusofZAP,therearefour
CCCH-typezinc-fingermotifs,whicharerequiredforRNAbindingandantiviralproperty
[9].ZAPexhibitsantiviralactivitygenerallyviaposttranscriptionalRNAregulation,suchas
mRNAdecayandtranslationinhibition.ZAPrecruitstheRNAprocessingexosomecomplex
andpoly(A)-specificribonuclease(PARN)todegradethetargetRNAfromthe30-end[6,10].
ZAPalsointeractswithp72RNAhelicasetorecruitdecappingenzymesDCP1/DCP2and
exoribonucleaseXRN1todegradethetargetRNAfromthe50-end[6,11].Besidestargeting
RNAaccumulation,ZAPcanblocktranslationofincomingviralRNAofSindbisvirus(SINV)
[4]probablybyinterruptingtheinteractionbetweentranslationalinitiationfactorseIF4Aand
eIF4G[12].Moreover,ZAP-Scanassociatewithretinoicacid-induciblegeneI(RIG-I),akey
sensortorecognizeviralRNA,topromotetheinnateimmuneresponseandcontributetoits
antiviralpotential[13].
FlavivirusesincludenumerousimportanthumanpathogenssuchasYFV,WestNilevirus
(WNV),Zikavirus(ZIKV),denguevirus(DENV)andJapaneseencephalitisvirus(JEV),caus-
ingendemicorpandemicoutbreaksintropicalandsubtropicalareas[14].Flaviviralvirions
areenvelopedandcontainasingle-stranded,positive-senseRNAgenomewith50-capbutnot
30-poly(A)tail.FlavivirusRNAencodesasingleopenreadingframe(ORF)flankedby50-and
30-untranslatedregions(UTRs),whichcontainRNAsecondarystructuresrequiredforviral
translationandtranscription[15,16].YFVwasresistanttotheantiviralactivityofratZAP[4],
butthesusceptibilityofotherflavivirusestoZAPisnotclear.Inthisstudy,wedeterminedthe
antiviralactivityofZAPagainstJEV,DENV,andZIKVandfounddifferentviralresponsesto
ZAP.WefurtherdemonstratedtheantiviralmechanismofZAPagainstJEVinfection.
Results
OverexpressionofZAPinhibitsJEVinfection
ToevaluatetheantiviralpotentialofhumanZAPagainstmembersofflavivirus,weestablished
A549cellsoverexpressingZAP-LandZAP-Sbylentiviraltransduction.Cellswithorwithout
ZAPoverexpressionwereinfectedwithJEV,DENVorZIKVandanalyzedforviralreplica-
tion.AscomparedwithcontrolEGFPcells,JEVinfectionmeasuredbyviralpropagation,viral
NS3proteinexpression,andviralRNAreplicationwerelowerincellsectopicallyoverexpres-
singZAP-LandZAP-S(Fig1A–1C).TheinhibitoryeffectofZAP-L/SagainstJEVinfection
wasalsonotedintheinducedpluripotentstemcells(iPSCs)-derivedhumanneuralprogenitor
cells(hNPCs),humanmicrogliaHMC3cellline,aswellashumanneuroblastomaBE(2)Cand
SK-N-SHcelllines(S1Fig).Nevertheless,nosignificantantiviraleffectofZAPwasobserved
afterhighandlowmultiplicityofinfection(MOI)ofDENVandZIKV(Fig1D–1F,S2andS3
Figs).Thus,differentflavivirusesexhibitdifferentsensitivitytotheantiviralactivityofZAP.
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JEVinfectioninhibitedbyZAP
Fig1.InhibitionofJEVbutnotDENVinfectionbyhumanZAPisoforms.A549cellstransducedwithlentivirusesexpressingEGFP
(A549-EGFP),V5-taggedZAP-L(A549-ZAP-L)andV5-taggedZAP-S(A549-ZAP-S)wereinfectedwithJEVorDENV(MOI=5).Culture
supernatants,celllysatesandtotalRNAwereharvestedattheindicatedtimepoints.(AandD)Viraltitrationdeterminedbyplaqueassay.
Representativedatafromthreeindependentexperimentsareshownasmean±SD(n=3).Statisticalsignificancewasanalyzedbytwo-wayANOVA.
(cid:3)(cid:3)(cid:3)P(cid:20)0.001;NS,notsignificant.(BandE)ProteinlevelsofviralNS3,V5-taggedZAP,EGFPandactinwereanalyzedbywesternblot.(CandF)
RNAlevelsofviralgenomeandactinwereperformedbyRT-PCR.hpi,hourspost-infection.
https://doi.org/10.1371/journal.ppat.1007166.g001
VirusesmaydownregulatetheexpressionofZAPand/orblockitsfunctionstoevadethe
ZAP-mediatedantiviralaction[17–20].TounderstandwhyDENVwasresistanttoZAP,we
detectedtheendogenousproteinlevelsofZAPsinJEVorDENV-infectedcellsandnoreduc-
tionofZAPswasnoted(S4Fig).TheantiviralactivityofZAPagainstSINVwasalsonot
obstructedbyDENVinfection(S5Fig),implyingthatDENVdidnotantagonizetheantiviral
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1007166 July17,2018 3/23
JEVinfectioninhibitedbyZAP
activityofZAP.Othermechanisms,besidesaffectingZAPexpressionandfunction,needtobe
considered.
DownregulationofZAPenhancesJEVreplication
ToinvestigatetheantiviralpotentialofendogenousZAP,A549-shZAP-L/Scellsdeprivedof
ZAPexpressionwereestablishedbytransductionwithlentiviralvectorexpressingshRNAtar-
getingbothZAP-LandZAP-S(Fig2A).AscomparedtothecontrolshLacZcells,shZAP-L/S
cellssupportedsignificantlyhigherlevelsofJEVinfectionasindicatedbya2.87-and3.08-fold
increaseinviralRNAandviralprogenyproduction,respectively(Fig2Band2C).Further-
more,knockdownofZAPinhumanneuronalBE(2)CcellsenhancedJEVreplication,sup-
portingtheanti-JEVroleofendogenousZAPinneuronalcells(S6Fig).SinceJEVreplication
occursatthesurfaceoftheendoplasmicreticulum(ER)membraneinthecytoplasm[21],and
ZAPpredominantlylocalizestothecytoplasm[22],weexaminedwhetherZAPco-localized
withJEVviralRNA.A549cellswithorwithoutJEVinfectionwereprocessedforimmunofluo-
rescenceanalysiswithanti-dsRNAantibodyforviralreplicationcomplex[23,24]andanti-
ZAPantibodyforendogenousZAPprotein.Co-localizationofdsRNAandZAPwasseenat
theviralreplicationsitesinJEV-infectedcells(Fig2Dand2E).Furthermore,weapplied
immunoprecipitation/RT-PCRassayusingantibodyagainstendogenousZAPtoevaluatethe
interactionofZAPwithJEVviralRNA.ZAP-LandZAP-Sprecipitatedbyanti-ZAPantibody
alsopulleddownJEVRNA(Fig2F),indicatingassociationbetweenZAPproteinsandJEV
RNA.
ZAPinteractswithJEVRNAandexertsantiviralactivitythroughitszinc-
fingermotifs
Tounderstandtheanti-JEVmechanismofZAP,welookedatwhetherRNAbindingis
requiredbygeneratingconstructsexpressingthezinc-finger(ZF)domainsdeletedZAP-Land
ZAP-S(ZAP-L/S-del4ZFs)(Fig3A).Wefirsttestedthebindingabilityofwildtype(WT)and
ZF-deletedZAPwithJEVviralRNAbyimmunoprecipitation/RT-PCRassay.TheV5-tagged
WTZAP-LandZAP-Sprecipitatedbyanti-V5antibodyalsopulleddownJEVRNA,which
wasnotseeninZF-deletedZAPandEGFPcontrol(Fig3B).Furthermore,theanti-JEVactivity
ofZAPwasgreatlyreducedintheZF-deletedmutantsasshownbyviralNS3proteinexpres-
sion,viralRNAreplication,andviralprogenyproduction(Fig3C–3E).Thus,thezinc-finger
motifsofZAPwereessentialforRNAtargetingandantiviralactivityagainstJEVinfection.To
explorewhyDENVwasresistanttotheantiviralactionofZAP,wedeterminedwhetherZAP
associatedwithDENVviralRNA.Interestingly,ZAPdidnotbringdownDENVRNAwhileit
readilypulleddownthecellularTRAILR4mRNA(S7Fig)knowntobindwithZAP[25],sug-
gestingthatRNA-bindingabilitymaydictatetheantiviralpotentialofZAP.
ZAPinterfereswithviraltranslationandpreventsaccumulationofJEV
RNAbydestabilizingviralRNA
Afterenteringthecells,JEVRNAundergoesafirstroundoftranslationtoproducenonstruc-
turalproteinsrequiredforviralRNAreplication.Toassesswhetherviraltranslationwas
affectedbyZAP,wedetectedviralproteinexpressioninZAP-andEGFP-overexpressingcells
atearlytimepointsofJEVinfection.LowerJEVNS3proteinexpressionwasnotedincells
withZAPoverexpressionstartingfrom2hourspost-infection(hpi)(Fig4Aand4B).Wealso
monitoredtheviralRNAlevelsincellswithorwithoutZAPoverexpressionbyRT-qPCR.No
significantdifferencewasfoundat2hpi,butZAPsignificantlyreducedJEVRNAsince3hpi
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JEVinfectioninhibitedbyZAP
Fig2.AntiviralpotentialofendogenousZAPagainstJEVinfection.(A)Westernblotanalysisfortheexpressionof
ZAPinA549cellstransducedwithlentivirusescarryingshRNAtargetingLacZandZAP-L/S.(BandC)A549-shLacZ
and-shZAP-L/ScellswereinfectedwithJEV(MOI=5)for24h.TotalRNAandculturesupernatantswereharvested
forRT-qPCR(B)andviraltitration(C),respectively.RelativeJEVRNAlevelnormalizedbyGAPDHwasdetermined
usingRT-qPCR.Viraltiterwasmeasuredusingplaqueassay.Representativedatafromtwoindependentexperiments
shownasmean±SD(n=3)wereanalyzedbytwo-tailedStudent’sttest.(cid:3)(cid:3)P(cid:20)0.01.(D)Confocalmicroscopyofmock
andJEV(MOI=1)infectedA549cellsat16hpistainedwithanti-dsRNAandanti-ZAPantibodies.Cellnucleiwere
counterstainedwithDAPI.Scalebar=20μm.(E)Co-localizationofdsRNAwithZAPwasestimatedbyPearson’s
correlationcoefficient(PCC).Mean±SDwascalculatedfrom30cellseachgroupandthestatisticalsignificancewas
analyzedbytwo-tailedStudent’sttest.(cid:3)(cid:3)(cid:3)P(cid:20)0.001.(F)CelllysatesfrommockandJEV(MOI=1)infectedA549cells
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1007166 July17,2018 5/23
JEVinfectioninhibitedbyZAP
after16hofinfectionwereincubatedwithanti-ZC3HAV1(ZAP)antibodyorcontrolIgG.Westernblotanalysisofthe
immunoprecipitatedZAPisoforms(upperpanel).TheZAP-bindingviralRNApulleddownbyantibodieswas
amplifiedbyRT-PCRwithJEV30-UTRspecificprimers(middlepanel).RT-PCRofinputJEVviralRNA(lower
panel).
https://doi.org/10.1371/journal.ppat.1007166.g002
(Fig4C).TofurtheraddresswhetherthedecreaseofviralproteinatearlytimepointofJEV
infectionisthroughrepressingtranslationand/orreducingviralRNA,weusedreplication-
deadJEVrepliconRNA(Fig4D)totransfect293T/17cellswithorwithoutZAPoverexpres-
sion(Fig4E).Thecelllysateswereseparatedintotwoportionsformeasurementsofluciferase
activityandRNAlevel.ReductionofluciferaseactivitywasnotedincellswithZAPoverex-
pressionsince1hpost-transfectionwhentheRNAlevelwasnotyetaffected(Fig4F),suggest-
ingrepressionofviraltranslationbyZAP.ToclarifywhetherZAPinfluencedJEVviralRNA
stability,wealsousedreplication-deadJEVrepliconRNAforlongertimepoints.Adecreasein
repliconRNAwasnotedat3hand9hpost-transfectionincellswithZAPoverexpressionas
comparedtothosewithEGFPcontrol(Fig4G),suggestingthatZAPpromotedJEVviralRNA
decay.Thus,ZAPinhibitedJEVtranslationandimpairedviralRNAstability.
JEVsuppressionbyZAPisdependentonthe30-50 RNAdecaypathway
ZAPmodulatestargetRNAbyrecruitingcellular50-30XRN1-dependentand30-50RNAexo-
some-dependentRNAdecaymachineries[6,10].ToevaluatewhethercellularRNAdecay
machineriesparticipatedintheanti-JEVactivityofZAP,wedepletedtheexpressionofXRN1
orEXOSC5inZAP-L,ZAP-SandEGFP-overexpressingcellsbyusingashRNA-targeting
approach.ComparedwiththeshLacZcontrol,knockdownofXRN1didnotaffecttheanti-
JEVactivityofZAP(Fig5A–5C),whereastheanti-JEVeffectofZAPwashamperedbyknock-
downofEXOSC5(Fig5D–5F).Thedatasuggestthat30-50RNAdegradationbytheexosome
complexisinvolvedintheantiviralmechanismofZAPagainstJEVinfection.
ZAPenhancesinnateimmuneresponsesthroughtheRIG-Isignaling
pathway
ToaddresswhetherZAPboostedinnateimmuneresponsesduringJEVinfection,wemea-
suredthemRNAlevelsoftypeIIFNandproinflammatorycytokinesinA549cellswithor
withoutZAPoverexpression.AscomparedwiththeEGFPcontrol,ZAP-LandZAP-Sslightly
increasedthebasallevelofIFN-β,TNF-αandIL-6(Fig6A–6C),aswellaschemokines
CXCL10andCCL5(S8Fig)inuninfectedcellsaspreviouslyreported[13],andfurther
increasedtheexpressionofthesecytokines/chemokinesinJEV-infectedA549cells(Fig6A–
6C,S8Fig).WefurtherdeterminedtheupstreamRLRsinvolvedinthesensingeventsby
knockingdowntheexpressionofRIG-IorMDA5bylentiviraltransduction(Fig6D).Com-
paredwiththeshLacZcontrol,knockdownofRIG-I,butnotMDA-5,reducedtheinduction
ofinterferon-stimulatedgenes(ISGs),e.g.,IFIT1,IFIT3,andISG15thatwasseeninZAPover-
expressingcells(Fig6Eand6F),indicatingthatZAP-enhancedinnateimmuneresponses
mainlywentthroughtheRIG-IsignalingpathwayinJEV-infectedcells.Interestingly,knock-
downofMDA5evenelevatedtheinductionofISGs(Fig6F),similarlytoapreviousreport
showinghigherIFN-βproductioninMDA5-/-butnotRIG-/-mouseembryonicfibroblasts
infectedwithJEV[26].Thus,depriveofMDA5inJEV-infectedcellsmighttriggersome
compensationalIFN-relatedsignalingeventsthroughunclearmechanism.Moreover,theanti-
JEVeffectofZAPmeasuredbyviralNS3proteinexpressionwasstillnotedincellsdeprivedof
RIG-IwithdecreasedISGsexpression(Fig6E)aswellasinthetypeIIFN-deficientVerocells
withectopicZAPexpression(S9Fig).Thus,ZAP-enhancedinnateimmuneresponsewasnot
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JEVinfectioninhibitedbyZAP
Fig3.Zinc-fingermotifsofZAParerequiredforJEVRNAbindinganditsantiviralactivity.(A)Schematic
representationofhumanZAPisoforms(ZAP-L,902a.a.andZAP-S,699a.a.).ThefourtandemCCCH-typezinc-
finger(ZF)motifswithintheN-terminusofZAPareshownassolidblackboxes.DeletionofthefourZFs(a.a.73–193)
areindicatedwithadashedline.(B)293T/17cellsinfectedwithJEV(MOI=5)for16hweretransfectedwithplasmids
expressingEGFP,WTorZF-deletedZAP-L-V5andZAP-S-V5foradditional24h.TheviralRNAboundwith
V5-taggedproteinswaspulleddownbyanti-V5agaroseaffinitygelandamplifiedbyRT-PCRwithJEV30-UTR
specificprimers(middlepanel).RT-PCRofinputviralRNAinJEV-infectedcells(lowerpanel).Westernblotanalysis
oftheimmunoprecipitatedZAP-LandZAP-S(WTanddel4ZFs)(upperpanel).(C-E)Theindicatedcellswere
infectedwithJEV(MOI=5)for10h.Celllysates,totalRNA,andculturesupernatantsweredeterminedforthe
indicatedproteinsbywesternblot(C),viralRNAbyRT-PCR(D),andviraltiterbyplaqueassay(E).Representative
datafromthreeindependentexperimentsshownasmean±SD(n=3)wereanalyzedbytwo-tailedStudent’sttest.(cid:3)(cid:3)(cid:3)
P(cid:20)0.001.
https://doi.org/10.1371/journal.ppat.1007166.g003
involvedintheanti-JEVeffect,probablybecauseJEVcanblocktheJAK-STATpathwayandis
somewhatresistanttotypeIIFN[27,28].
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JEVinfectioninhibitedbyZAP
Fig4.InhibitionofJEVtranslationandreplicationbyZAP.(AandC)A549-EGFP,ZAP-LandZAP-ScellsabsorbedwithJEV(MOI=10)onicefor
2hwerewashedandthenincubatedat37˚C.(A)Attheindicatedtimepoints,proteinswereharvestedforwesternblotwiththeindicatedantibodies.(B)
RelativeNS3proteinlevelsnormalizedwithactinfromtwoindependentexperimentswerequantifiedbyImageJsoftware.(C)RNAwascollectedfor
viralRNAdeterminationbyusingRT-qPCR.RelativeJEVRNAlevelwasnormalizedbythatofGAPDH.(D)IllustrationofSP6promoter-drivenRdRP-
deadJEVreplicon(E)Westernblotof293T/17cellsexpressingEGFP,ZAP-L-V5andZAP-S-V5withtheindicatedantibodies.(F)293T/17-EGFPand
-ZAP-Scellwerecotransfectedwith50-cappedRdRP-deadJEVrepliconRNAandcontrolfireflyluciferaseRNAfor1and2h.Attheindicatedtime
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1007166 July17,2018 8/23
JEVinfectioninhibitedbyZAP
points,cellswerecollectedandseparatedintotwoportionsforthemeasurementsofluciferaseactivityandRNAlevel,respectively.Therelativeluciferase
activityandRNAlevelofRenillaluciferasereporternormalizedwithtransfectioncontrolfireflyluciferaseareshownasthepercentagetothatofEGFPat
1hposttransfection.(G)50-cappedRdRP-deadJEVrepliconRNAandcontrolfireflyluciferaseRNAcotransfected293T/17-EGFPand-ZAP-Scells
wereharvestedat3and9hpost-transfectiontodeterminetherepliconRNAlevel.TherelativerepliconRNAlevelnormalizedwiththatoffirefly
luciferaseisshown.Representativedataareshownasmean±SDfrom3independentexperimentsandanalyzedbytwo-tailedStudent’sttest.(cid:3)P(cid:20)0.05;
(cid:3)(cid:3)P(cid:20)0.01;(cid:3)(cid:3)(cid:3)P(cid:20)0.001.
https://doi.org/10.1371/journal.ppat.1007166.g004
ZAPtargetsthe30-UTRofJEV
BothRNAsequenceandstructurehavebeenconsideredasimportantforZAPrecognition
[29,30];however,thecommonfeaturesofZAP-responsiveelements(ZRE)arestillincon-
clusive.ToelucidatethepossibleregionsofJEVgenometargetedbyZAP,weperformed
UVcrosslinkingandimmunoprecipitation(CLIP)topulldowntheRNAinteractingwith
ZAPinJEV-infectedZAP-SoverexpressingA549cells.TheenrichedRNApopulationwas
subjectedtonextgenerationsequencing(NGS)byuseoftheIonTorrentplatform.The
readcoveragesofseveralpeaks/regionswereabovetheaveragereadcoverageespecially
withintheJEV30-UTR(Fig7A).Wethusfocusedon50-and30-UTR,whichcontaincon-
servedcomplementarysequencesandextensivesecondarystructurestoregulateviral
translationandtranscription[15,16],toelucidatethepotentialZREintheJEVgenome.
Wedesignedreporterconstructscontainingthe50197nt.(50-UTRplusthefirst102nt.of
thecoregene)and/ortheentire30-UTRflankingfireflyluciferase(Fluc)inthepGL3-pro-
motervectorunderaSP6promoter(Fig7B,leftpanel).Theinvitrotranscribedreporter
RNAandcontrolRenillaluciferase(Rluc)RNAwerecotransfectedinto293T/17cells
withorwithoutZAP-Soverexpression.AscomparedwiththeEGFPcontrol,ZAPdidnot
influencetheluciferaseactivityofthereporterRNAwith50-UTR ,whileZAPsignifi-
197
cantlyreducedthosewith30-UTR(Fig7B,rightpanel),revealingthepossibleZREinthe
JEV30-UTR.InvitroRNApull-downassayalsoconfirmedthattheinteractionbetween
ZAP-SandJEV30-UTRinaZFdomaindependentmanner(Fig7C)wasstrongerthan
thatwith50-UTR (S10Fig).
197
MappingtheZREinJEV30-UTR
TofurtherevaluatethepossibleZREinthe30-UTRofJEVgenome,CLIP-seqresults
withinthethreedefineddomainsofJEV30-UTR:domainI(variableregion),domainII
(dumbbellstructure)anddomainIII(30conservedsequenceandterminalstem-loop)[31]
areshown(Fig8A).WemeasuredthebindingabilityoftheseRNAdomainswithZAPby
usingbiotinylatedRNA.Theproteinspulleddownbystreptavidinweresubjectedtowest-
ernblottingwithanti-V5antibodyforZAP-Slevel.DomainII(showinga45%binding
abilityofthefull-length)hadthestrongestinteractionwithZAPwhencomparedto
domainI(12%ofthefull-length)anddomainIII(3%ofthefull-length)oftheJEV30-
UTRRNA(Fig8B).Interestingly,domainIIcontainedhighfrequencyofCGdinucleotide
(Fig8A),whichwasrecentlyreportedtoconferZAPbindingandrecognition[32].Fur-
thermore,thebindingofZAPtodomainI+IIRNAcontainingmostoftheCGdinucleo-
tidesreached88%ofthefulllength30-UTRRNA(Fig8B).Toverifytheregionstargeted
byZAP,wegeneratedfivereporterRNAscontainingdomainI,II,III,I+II,andII+III,
respectively(Fig8C,leftpanel).SignificantreductionofluciferaseactivitybyZAP-Swas
notedinthereporterswithWT,domainII,domainI+IIanddomainII+III,indicatingthe
importanceofdomainIIinconferringthesensitivitytoZAP(Fig8C,rightpanel).Thus,
domainIIofJEV30-UTRcontainingdumbbellstructuresandhighcontentofCGdinucle-
otidemayfunctionasZREandcontributetoZAPsensitivity.
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JEVinfectioninhibitedbyZAP
Fig5.The30-50RNAexosome-mediated,butnotthe50-30XRN1-mediated,RNAdegradationisrequiredfortheanti-JEV
activityofZAP.A549cellswithshRNAtargetingLacZ,XRN1,andEXOSC5weretransducedbylentivirusesexpressingEGFP,
ZAP-L-V5,andZAP-S-V5.After10hofJEVinfection(MOI=5),cellslysateswereanalyzedbywesternblotfortheindicated
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1007166 July17,2018 10/23