Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient

31 Pages·2016·2.5 MB·English

RESEARCHARTICLE
Endoplasmic Reticulum Stress Induced
Synthesis of a Novel Viral Factor Mediates
Efficient Replication of Genotype-1 Hepatitis
E Virus
VidyaP.Nair1,SaumyaAnang1☯,ChandruSubramani1☯,AbhilashaMadhvi1,
KarishmaBakshi1,AkritiSrivastava1, Shalimar2,BaibaswataNayak2,RanjithKumarCT1,
MilanSurjit1*
1 VirologyLaboratory,VaccineandInfectiousDiseaseResearchCentre,TranslationalHealthScienceand
TechnologyInstitute,NCRBiotechScienceCluster,Faridabad,Haryana,India,2 Departmentof
Gastroenterology,AllIndiaInstituteofMedicalSciences,GautamNagar,AnsariNagarEast,NewDelhi,
Delhi,India
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS Abstract
Citation:NairVP,AnangS,SubramaniC,MadhviA,
BakshiK,SrivastavaA,etal.(2016)Endoplasmic
HepatitisEvirus(HEV)causesacutehepatitisinmanypartsoftheworldincludingAsia,
ReticulumStressInducedSynthesisofaNovelViral
AfricaandLatinAmerica.Thoughself-limitinginnormalindividuals,itresultsin~30%mor-
FactorMediatesEfficientReplicationofGenotype-1
HepatitisEVirus.PLoSPathog12(4):e1005521. talityininfectedpregnantwomen.Ithasalsobeenreportedtocauseacuteandchronichep-
doi:10.1371/journal.ppat.1005521 atitisinorgantransplantpatients.Ofthesevenviralgenotypes,genotype-1virusinfects
Editor:Xiang-JinMeng,VirginiaPolytechnicInstitute humansandisamajorpublichealthconcerninSouthAsiancountries.Sporadiccasesofgeno-
andStateUniversity,UNITEDSTATES type-3and4infectioninhumanandanimalssuchaspigs,deer,mongeesehavebeenreported
Received:February9,2016 primarilyfromindustrializedcountries.Genotype-5,6and7virusesareknowntoinfectanimals
suchaswildboarandcamel,respectively.Genotype-3and4viruseshavebeensuccessfully
Accepted:March2,2016
propagatedinthelaboratoryinmammaliancellculture.However,genotype-1virusreplicates
Published:April1,2016
poorlyinmammaliancellcultureandnootherefficientmodelexiststostudyitslifecycle.Here,
Copyright:©2016Nairetal.Thisisanopenaccess
wereportthatendoplasmicreticulum(ER)stresspromotesgenotype-1HEVreplicationby
articledistributedunderthetermsoftheCreative
inducingcap-independent,internalinitiationmediatedtranslationofanovelviralprotein
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany (namedORF4).Importantly,ORF4expressionandstimulatoryeffectofERstressinducerson
medium,providedtheoriginalauthorandsourceare viralreplicationisspecifictogenotype-1.ORF4proteinsequenceismostlyconservedamong
credited.
genotype-1HEVisolatesandORF4specificantibodiesweredetectedingenotype-1HEV
DataAvailabilityStatement:Mostofthedataare patientserum.ORF4interactedwithmultipleviralandhostproteinsandassembledaprotein
presentedinthemanuscript.Sequenceshavebeen
complexconsistingofviralhelicase,RNAdependentRNApolymerase(RdRp),X,host
depositedtoGenbankwithIDs:KU168733-
eEF1α1(eukaryoticelongationfactor1isoform-1)andtubulinβ.InassociationwitheEF1α1,
KU168737.
ORF4stimulatedviralRdRpactivity.Furthermore,humanhepatomacellsthatstablyexpress
Funding:RamalingaswamyFellowshiptoMSis
ORF4orengineeredproteasomeresistantORF4mutantgenomepermittedenhancedviral
gratefullyacknowledged.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto replication.ThesefindingsrevealapositiveroleofERstressinpromotinggenotype-1HEVrep-
publish,orpreparationofthemanuscript. licationandpavethewaytowardsdevelopmentofanefficientmodelofthevirus.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexists.
PLOSPathogens|DOI:10.1371/journal.ppat.1005521 April1,2016 1/31
EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
AuthorSummary
HepatitisEvirus(HEV)isoneofthemostcommoncausesofacuteandsporadicviral
hepatitis.ItisasmallpositivestrandRNAvirus,whichistransmittedthroughthefeco-
oralroute.Owingtolackofsanitationandunavailibilityofsafedrinkingwater,popula-
tionsofdevelopingandresourcestarvedcountriesarepronetowardsHEVinfection.
RecentreportsalsoindicateHEVinducedacuteandchronichepatitisinorgantransplant
patients.AnotherpeculiarcharacteristicofHEVisattributedtoitsabilitytocausehigh
mortality(~30%)ininfectedpregnantwomen.Evenafter30yearsofdiscoveryofthe
virus,littleinformationexistsregardingvirallifecycleandreplicationmachinery.HEVis
dividedintosevengenotypes.Genotype-3and4virusesinfecthumansandafewanimals
(suchaspigs,deer,mongeese)andhavebeenreportedfromindustrializedcountries.
Genotype-3and4viruseshavebeensuccessfullypropagatedinthelaboratoryinmamma-
liancellculture.However,genotype-1virus,whichisknowntoinfecthumanandisa
majorpublichealthconcerninsouthAsiancountries,replicatespoorlyinmammaliancell
cultureandnootherefficientmodelexiststoinvestigatethevirallifecycle.Withthegoal
ofdevelopinganefficientlaboratorymodelofgenotype-1HEV,weattemptedtoidentifya
permissivecellularconditionthatwouldallowefficientviralreplicationinhumanhepa-
tomacells.Here,wereportthatendoplasmicreticulumstressinducingagentspromote
genotype-1HEVreplicationbyinitiatingcap-independent,internaltranslationmediated
synthesisofanovelviralfactor,whichwehavenamedORF4.Furtherinvestigations
revealedthatORF4isexpressedonlyingenotype-1anditactsbyinteractingwithmultiple
viralandhostproteinsandcooperateswithhosteEF1α1(eukaryoticelongationfactor1
isoform1)tocontroltheactivityofviralRNAdependentRNApolymerase.Moreover,a
proteasomeresistantORF4mutantsignificantlyenhancedviralreplication.Thus,our
studyidentifiesanoptimalconditionrequiredforefficientreplicationofgenotype-1HEV
anddissectsoutthemolecularmechanismgoverningthat.Datapresentedherewillbe
instrumentalindevelopinganefficientmodelofthevirus.
Introduction
HepatitisEisafeco-orallytransmittedpositivestrandRNAvirusthatcausesacuteandspo-
radichepatitisinhumanandotheranimals[1].Itisalsoemergingtobeamajorcauseofinfec-
tioninorgantransplantpatientsworldwide[2].Thoughself-limitinginnormalindividuals,a
peculiarcharacteristicofHEVisattributedtoitsabilitytocausehighmortality(~30%)in
infectedpregnantwomen[3].Theviralgenomeconsistsofa7.2kb5’-cappedand3’-polyade-
nylatedRNA,whichencodesthreeknownopenreadingframes(ORF);ORF1codesfornon-
structuralproteins,ORF2codesforthemajorcapsidproteinandORF3codesforanaccessory
proteinthatassociateswithmultiplehostproteinsandissupposedtomodulatehostsignaling
pathways[1].ORF3alsointeractswithhosttumorsusceptibilitygene101(TSG101)andplays
anessentialroleinvirusrelease[4,5].ORF2hasbeenobservedtobindtotheviralgenomic
RNA[6],induceendoplasmicreticulum(ER)stress[7,8]andinhibitNFκBactivity[9]in
humanhepatomacells,suggestingapossibleregulatoryroleoftheviralcapsidprotein.Seven
genotypesofHEVhavebeenreported;genotype-1(g-1),genotype-2(g-2)exclusivelyinfect
humanwhereasgenotype-3(g-3),genotype-4(g-4)infecthuman,pig,deer,mongeeseandrab-
bit.Infectionbygenotypes5–7havenotbeenreportedinhuman.Genotype-5(g-5),genotype-
6(g-6)infectswildboarandgenotype-7(g-7)isknowntoinfectcamel[10,11].Littleisknown
aboutthelifecycleofHEVowingtolackofahandyanimalorcellculturemodel.Amongthe
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
variousgenotypes,invitrosynthesizedgenomeofg-3andg-4HEVreplicateswellinmamma-
liancellculture[12].Attemptsatachievinghighreplicationefficiencyofg-1HEVinmamma-
liancellculturehavenotbeensuccessful[13,14].Interestingly,inoneoftheg-3HEVinfected
patients,humanribosomalS17codingsequencewasfoundtobeinsertedintheORF1region,
whichconferredgrowthadvantagetothevirus[15,16].Molecularmechanismsunderlyingthe
aboveobservationremaintobeexplored.Moreover,suchaninsertionappearstobeaveryrare
occurrence.
EfficienttranslationandreplicationaretwocrucialeventsinthelifeofanRNAvirus,tight
controlofwhichisessentialforsurvivalofbothvirusanditshost.Theseeventsareunderstrict
surveillancebythehostdefencemachineryasinnateantiviralmeasures,mostcommonbeing
inductionofERstressandunfoldedproteinresponse,inactivationofeukaryotictranslation
initiationfactor2αandshutdownofcap-dependenttranslation[17].Degradationofviraldou-
blestrandedRNAbyinnateimmuneeffectors[18]andautophagy[19]alsoservesashost
defencemechanismsagainstmanyviruses.Virusesontheotherhand,employcleverstrategies
toexploittheadversitiesimposedbythehost.Innateimmuneresponseiscounteredbyvarious
strategiessuchasinhibitionoftypeIinterferonproduction[20],manipulationofpatternrec-
ognitionreceptorsignaling[21],IRF3(interferonregulatoryfactor3)inhibition[22,23]and
autophagyinhibition[24].Translationalrestrictionsareovercomebyribosomeshunting,rein-
itiation,stimulationofeIF4Fcomplexassembly,inhibitionofelF2αphosphorylation[25],
internalribosomeentrysite(IRES)mediatedtranslation[26,27]andexecutionofbothcap-
dependentandcap-independentmodesoftranslation[28,29]dependingonthecellularstate.
ThesecondstepintheRNAviruslifecyclepertainstogenomereplication.Mostviruses
encoderegulatoryproteins,RNAand/ormiRNAthatexploithostmachineriestoaugment
viralreplication.HepatitisC,DengueandPoliovirusesactivateautophagy[30,31,32].Hepati-
tisBvirusinhibitsproteasomeactivity[33],whichleadstoincreasedviralreplication.Hence,
dependinguponthehostcellularcondition,eachvirusseemstohaveevolvedsuitablesurvival
strategiesthatpermitsitsoptimalgrowth.
Sinceg-1HEVdoesnotreplicateefficientlyinmammaliancellculture,wewondered
whetheranyparticularcellularconditionmightenhanceviralreplication.Screeningofvarious
compoundsknowntoaltercellularconditionrevealedaroleofERstressinducingcompounds,
thapsigarginandtunicamycininenhancingg-1HEVreplication.Furtherstudiesledtothe
identificationofanovelviralproteinsynthesizedfromanoverlappingreadingframewithin
ORF1,whichwasnamedopenreadingframe4(ORF4).TheroleofORF4inviralreplication
wasexplored.
Results
Increasedg-1HEVreplicationbyEndoplasmicReticulumstress
inducingagents
InordertoidentifytheinfluenceofaparticularcellularconditiononHEVreplication,Huh7
cellsweretransfectedwithwildtypecappedgenomicRNA(WTHEV).6daysposttransfec-
tion,viralreplicationwasmeasuredbymonitoringthelevelofsenseandantisenseRNAand
estimatingthepercentageofcellsexpressingviralhelicaseandORF2.Notethathelicasesyn-
thesisreflectsORF1translationfromgenomicRNAwhereasORF2issynthesizedfromthe
subgenomicRNA(generatedafterreplication).Areplicationdeficientmutantgenome(GAA
HEV),inwhich“DD”aminoacidsofRdRp(a.a.1551,1552inORF1)werealteredto“AA”;
wasusedtoensurespecificityofbothassays.Quantitativereal-timePCR(QRT-PCR)ofsense
strandRNAlevelinGAAHEVtransfectedsamplesreflectedthelevelofinputRNA(quantity
oftransfectedRNA,Fig1A).SensestrandRNAlevelwasapproximatelyfourfoldhigherin
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
WTHEVRNAtransfectedsample(comparedtoGAAHEV),reflectingreplicationmediated
increase.Asexpected,noantisenseRNAwasdetectedinGAAHEVtransfectedsamplesbut
wasdetectableatbasallevelsinWTHEVexpressingsamples.UpontreatmentwithknownER
stressinducers;thapsigargin(TG)andtunicamycin(TUN),senseandantisenseRNAlevels
werefurtherincreasedby2–3fold(Fig1A).Similarlytreatedsampleswereanalyzedbyimmu-
nofluorescenceassaytomeasurethepercentageofhelicaseandORF2positivecells(Fig1B).
Representativeimagesareshown(S1Fig).GAAHEVtransfectedsamplecontained2%heli-
casepositiveandnoORF2positivecells,inagreementwithQRT-PCRdata,confirmingspeci-
ficityoftheassays(Fig1B).20%helicaseand5%ORF2positivecellsweredetectedinDMSO
treatedWTRNAtransfectedcells.Thapsigarginandtunicamycintreatedsamplescontained
significantlyhigherpercentageofhelicaseandORF2positivecellsinWTHEV.Helicaseposi-
tivecellswereabsentinthapsigarginandtunicamycintreatedGAAHEVsample,rulingout
thepossibilityofincreasedORF1translationbythesecompounds.Wenexttestedwhether
thapsigarginandtunicamycinenhancedthereplicationofg-3HEVusingaGaussialuciferase
secretingrepliconofg-3HEV[16].Therewasnoincreaseinluciferaselevelupontreatmentof
therepliconexpressingcellswiththapsigarginandtunicamycin(Fig1C),suggestingthatboth
compoundshadastimulatoryeffectonlyong-1HEVreplication.
Anovelproteinisexpressedfromanoverlappingreadingframelocated
withinORF1ofg-1HEVinvitroandinvivo
AssumingthatthemechanismunderlyingtheobservedstimulatoryeffectofERstressong-1
HEVreplicationisencodedintheviralgenome,weanalysedtheg-1HEVgenome(SAR55
strain,GenbankID:AF444002.1)using“ATGpr”,asoftwaretopredictpotentialopenreading
frames[34].AllknownORFsofHEVwerepredicted.AnunknownORFof158aminoacids
withinORF1,locatedin+1readingframe(withreferencetoORF1,2835–3308basesfrom5’)
wasalsopredicted,whichwasnamedORF4(S1Table,Fig1D).AnORFcodingforatruncated
ORF1proteinwasalsopredicted.Incontrast,sequenceanalysisofotherHEVgenotypesdid
notrevealanyORFresemblingthatofORF4(S1Table).Next,weperformedabioinformatics
analysisofseveralg-1HEVgenomicsequencesavailableinpublicdatabasetofindoutwhether
openreadingframe4ispresentinallandwhethertheORF4proteinsequenceisconserved
amongthevariousisolates.Additionally,weanalyzedtheviralgenomicsequencefromfive
newcasesofg-1HEVinfection,recentlyisolatedbyusattheAllIndiaInstituteofMedicalSci-
ences,NewDelhi,India(GenbankID:KU168733-KU168737,Fig1E).Allg-1HEVgenomes
containanORFattheexpectedpositionofORF4,withasuboptimalKozaksequencestarting
eitherat2832or2834nucleotides,from5’end(Fig1D).Threedifferentpatternswereobserved
withrespecttoterminationoftheputativeORF4;in8cases,itterminatesat3311nucleotides
(158aminoacids,fulllengthORF4protein),intwocases,at3277nucleotides(147amino
acids)andinremaining9cases,itterminatesat3256nucleotides(139aminoacids,Fig1E).
ClustalWanalysisoftheputativeORF4proteinsequenceoftheseisolatesrevealed~80%con-
servationofaminoacids(Fig1E).
ToverifyORF4and/orΔORF1expression,aninvitrotranscription-translationassaywas
performedusingaTNTkit.Twobandscorrespondingtounprocessedandprobablypartially
(cid:1)(cid:1)
processedORF1protein( )weredetected(Fig2A,topandmiddle).Twobandscorrespond-
(cid:1)
ingto~20kDaand~40kDa( )werealsoobserved(Fig2A,top).Nosuchbandsweredetected
inmock.MutatingtheinitiatormethioninecodonofORF1toLysine(ATG-AAAsubstitution,
26ATGmutORF1)resultedindisappearanceofORF1specificbandswithoutaffecting20kDa
and40kDabands.Similarly,blockingORF1translationinitiationbyinsertingawellcharacter-
isedstemloopformingsequence[35]upstreamoftheinitiatorcodonofORF1(SLinsORF1)
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
Fig1.Tunicamycinandthapsigarginpromoteg-1HEVreplication.(A)QRT-PCRofwildtype(WTHEV)andreplicationdeficient(GAAHEV)HEVin
Huh7cellstransfectedwithinvitrosynthesizedgenome.TG:thapsigargin,TUN:tunicamycin.Valuesaremean±SEM.(B)QuantitationofviralORF1
(Helicase)andORF2expressioninHuh7cells,transfectedwithwildtype(WTHEV)orreplicationdeficient(GAAHEV)HEVgenomicRNAandtreatedwith
theindicatedcompounds.Tenrandomfieldsofapproximately30cellsineachfieldwerecountedforhelicase,ORF2,DAPI(nuclearstain)fluorescenceand
percentage±SEMcalculated.(C)MeasurementofsecretedGaussialuciferaseactivityintheculturemediumofHuh7cellsexpressinginvitrotranscribed
HEVgenotype-3repliconRNAandtreatedasindicated.Valuesaremean±SEM.(D)HEVgenomeorganisation.Numbersindicatenucleotidepositionfrom
5’-end.Mutatedbasesareinbold.(E)ClustalWalignmentofORF4proteinsequenceofindicatedg-1HEVisolates.
doi:10.1371/journal.ppat.1005521.g001
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
Fig2.TunicamycinandthapsigargininduceORF4expression.(A)Top:AutoradiogramshowingTNTofindicatedplasmids.Mock:emptyvector,“**”:
ORF1protein,“*”:unknownproteins.Middle:samplesfromtopresolvedby7%SDS-PAGE,followedbyautoradiography.Bottom:samplesfromtop
resolvedby15%SDS-PAGEandwesternusinganti-ORF4.(B)ImmunofluorescenceofORF4inHuh-7cellstransfectedwithindicatedinvitrosynthesized
RNA.Scale:20μm.Shownaremergedimagesofnuclei(blue)andORF4(green).“!”:positivestaining,“►”:unstained.(C)ImmunofluorescenceofHelicase
andORF4inHuh-7cellstransfectedwithinvitrosynthesizedwildtypeHEVg-1(WTHEV)org-3(WTg-3HEV)genomicRNA.Scale:20μm.Shownare
mergedimagesofnuclei(blue)andHelicaseorORF4(green).“!”:positivestaining.
doi:10.1371/journal.ppat.1005521.g002
abolishedthebandscorrespondingtoORF1withoutimpacting20kDaand40kDabands(Fig
2A).Correlating“ATGpr”predictionwithabovedatasuggestedthat20kDabandmaycorre-
spondtotranslationproductofORF4.40kDabandcouldbeadenaturationresistantdimeric
formofORF4oranunrelatedprotein.
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
Inagreementwiththeaboveproposition,TNTofORF4codingsequenceproduced20and
40kDabands(Fig2A,pSGIORF4).Apeptidebasedrabbitpolyclonalantibodywasgenerated
againsttheputativeORF4proteininordertoidentifytheunknownbands.Functionalityand
specificityoftheantibodywasvalidated(S2AandS2BFig)andaliquotsofTNTsampleswere
westernblottedusingthisantibody.Onlythe20kDabandwasdetectablebyORF4antibodyin
WTORF1,26ATGmutORF1,SLinsORF1andpSGIORF4(Fig2A,bottom).
TwosuboptimalKozaksequencescontaininginitiationcodonsarepresentintheORF4
codingregion(Fig1D).BothweremutatedtoLysine(ATG-AAA)inHEVORF1construct
(ORF4ATGDMORF1),followedbyTNTtoconfirmtheidentityof20and40kDabands.
Bothbandswereabsentintheautoradiogramandwestern,withoutaffectingORF1level(Fig
2A).Asexpected,inhibitingbothORF1andORF4translationinitiationbystemloopinsertion
andATG-AAAsubstitution,respectively,resultedindisappearanceofallbands(SLinsORF1
DMORF4).
AnimmunofluorescenceassaywasconductedusingORF4antibodytodetectitsexpression
inWTg-1HEVgenometransfectedHuh7cells.ORF4signalwassignificantlyhigherintunica-
mycinandthapsigargintreatedcellscomparedtotheDMSOcontrol(Fig2B).Specificityofthe
signalwascontrolledbyusingtunicamycintreatedDMHEV(mutantg-1HEVgenome,in
whichORF4initiationcodonsaremutatedtoLysine)transfectedcells,whichfailedtoshow
ORF4signal.Tunicamycintreated26ATGmutHEVorGAAHEVRNAtransfectedcellsalso
expressedORF4,clearlyrulingoutanyinfluenceofORF1translationorreplicationonORF4
production,respectively(Fig2B).
InordertoconfirmthatnoORF4likeproteinisexpressedingenotype-3HEV(g-3HEV),
invitrotranscribedgenomeofaluciferaserepliconofg-3HEV(pSKHEVp6luc)orWTg-1
HEVwastransfectedintoHuh7cells,followedbythapsigargintreatmentandsubsequent
immunofluorescencestainingusinganti-ORF4oranti-Helicaseantibodies.Helicaseexpression
wasdetectableinbothsampleswhereasORF4expressionwasdetectableonlyinthecaseofg-1
HEV(Fig2C).
Next,weanalysedORF4expressioninthefiveg-1HEVinfectedpatients,inwhichORF4
sequencewasconserved(KU168733-KU168737,Fig1E).ORF4expressionwasassessedindi-
rectlybymonitoringthelevelofanti-ORF4antibody,ifany.PurifiedGST-ORF4proteinwas
readilydetectedbyserumfromall5patients(KU168733-KU168737)whereasserumfromtwo
healthy(CS1-CS2)individualswerenegative(Fig3A).
ExpressionofORF4isessentialforbasalandtunicamycindependent
increaseinHEVreplication
AstablecelllineofHuh7constitutivelyexpressingFlag-taggedORF4wasgenerated(ORF4--
Huh7)toexploretheroleofORF4inHEVreplication(Fig3B).WTHEVorGAAHEV
genomewastransfectedintoORF4-Huh7anditscontrol(pCDNA5-Huh7).Thelevelofsense
andantisenseRNAofWTHEVwasapproximatelytwofoldhigherinDMSOtreatedORF4--
Huh7cellscomparedtocontrol(Fig3C).Asexpected,GAAHEVmutantwasunabletorepli-
cate.TunicamycintreatmentincreasedsenseandantisenseRNAbytwofoldincontroland
fourfoldinORF4-Huh7cells.DMHEVbehavedlikeGAAHEVincontrolcellsinthepres-
enceandabsenceoftunicamycin.IncontrasttoGAAHEV,DMHEVproducedbothsense
andantisenseRNAatlevelsequivalenttoWTHEVinDMSOtreatedORF4-Huh7cellsand
remarkably,theselevelsremainedunalteredinthepresenceoftunicamycin(Fig3C).Similar
patternwasobtainedinimmunofluorescenceanalysisofhelicaseandORF2positivecells(Fig
3D).Thapsigargintoodisplayedapatternsimilartotunicamycin(Fig3D).
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
Fig3.ORF4antibodyisdetectedinHEVpatientsanditsoverexpressionenhancesviralreplication.(A)Top:PurifiedGST-ORF4stainedwith
coomassieblue(leftmost)andwesternofequalaliquotsofthesameusinghealthy(CS1,CS2)andacuteHEVinfected(KU168733-KU168737)patientsera.
Bottom:Topblotsreprobedwithanti-ORF4antibody.(B)WesternofwholecellextractfromindicatedcellsusingORF4andGAPDHantibodies.(C)
QRT-PCRofsenseandanti-senseRNAinpCDNA5-Huh7andORF4-Huh7cellstransfectedwithinvitrosynthesizedwildtype(WT)ormutantHEVgenome
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EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
andtreatedasindicated.(D)QuantitationofviralORF1(helicase)andORF2expressioninpCDNA5-Huh7andORF4-Huh7cells,transfectedwithwildtype
(WTHEV)orORF4expressiondeficientmutant(DMHEV)HEVgenomicRNAandtreatedwiththeindicatedcompounds.Tenrandomfieldsof
approximately30cellsineachfieldwerecountedforhelicase,ORF2,DAPI(nuclearstain)fluorescenceandpercentage±SEMcalculated.
doi:10.1371/journal.ppat.1005521.g003
InternalinitiationofORF4translation,drivenbyanInternalribosome
entrysite-like(IRESl)elementlocatedwithinORF1
ORF1translationiscap-dependent[1].However,ORF4couldbetranslatedintheabsenceof
cap-dependenttranslation(SLinsORF1,Fig2A).ConsideringitslocationdeepinsideORF1,
wewonderedwhetherORF4synthesiswasdrivenbyaninternaltranslationinitiationmecha-
nism.BioinformaticsanalysisofviralRNAflankingORF4regionusing“RegRNA”[36]indi-
catedthepresenceofaputativeIRES-likeelementbetween2701–2787bases(Fig1D,IRESl).
Analysisofsamesequenceusing“IRESite”[37]predictedweakhomologywithEquineRhinitis
Avirus-1IRES[38].Secondarystructureanalysisof2664–2845basesencompassingthepre-
dictedIRES-likeelementusing“mfold”[39]revealedthepresenceofthreestemloopswithin
2701–2787bases(Fig4A,sequenceincyan).Increaseinsequencelength(315bases,2619–
2933bases)didnotalterthosestemloops,indicatingtheirstability(S3Fig).Adualluciferase
reporterassaywasconductedtoevaluatethefunctionalityoftheIRES-likeelementbyplacing
itbetweenRenillaandFireflycodingsequences(Fig4B).Threeconsecutivestopcodonswere
introduceddownstreamoftheRenillacodingsequencetoensureterminationofcap-depen-
denttranslationofRenillaluciferase.315basesfromHEVgenomeencompassingtheIRES-like
element(HIRESl315)or468basesfrom3501–3968nucleotides(negativecontrolforback-
groundFireflyactivity,HEVcRNA)wereinserteddownstreamofRenilla,precedingtheFirefly
startsite(Fig4B).MeasurementoftheFireflyandRenillaluciferaseratiosrevealedasignifi-
cantlyhigherFireflyactivityinHIRESl315sample(Fig4C).ThecoreIRES-likeelement
(2701–2787bases,HIRESl87)alsodisplayedsimilaractivity(Fig4C).Next,severalmutant
constructsweregeneratedinwhichindividualstemloopsweredestroyedbyalteringafew
(cid:1)
nucleotidesatatime.ImpairingstemloopsA,B,Corbulge(A )didnotaffectFireflyactivity.
AmoderateandhighreductioninFireflyactivitywasseeninsamplescontainingdualmuta-
tionsofbothA,CandB,Cstemloops,respectively(Fig4C).
DualmutationsofbothBandCwereintroducedintoplasmidscontainingHEVORF1and
HEVgenome(IRESlmutORF1andIRESlmutHEV,respectively).InTNTofIRESlmut
ORF1construct,ORF4-specificbanddisappearedwithoutaffectingthatofORF1(Fig2A).No
ORF4wasdetectedincellstransfectedwithIRESlmutHEVRNAupontunicamycintreatment
(Fig2B).Expectedly,IRESlmutHEVgenomereplicationwassignificantlyreducedirrespective
oftunicamycintreatmentinpCDNA5-Huh7cells,whichcouldberestoredinORF4-Huh7
cells,thoughinatunicamycininsensitivemanner(Fig3C).
ORF4directlyandindirectlyassociateswithseveralviralproteinsand
promotestheassemblyofamultiproteincomplex
Toexplorethemechanism(s)bywhichORF4stimulatedviralreplication,weidentifiedits
interactionpartnersamongviralproteins.ORF4directlyinteractedwithhelicase,XandORF3
proteinsofg-1HEV,evidentfromYeastTwoHybrid(Y2H)assay(Table1).Xproteinofg-3
HEValsointeractedwithORF4,howeverneitherg-3helicasenorg-3ORF3interactedwith
ORF4(Table1).UsingoverlappingdeletionsofORF4,theinteractiondomainwasmappedto
54–122aminoacidsforXandORF3and1–124aminoacidsforhelicaseproteinofg-1HEV
(Table2).Coimmunoprecipitation(CoIP)ofHuh7cellstransfectedwithplasmidsencoding
ORF4andvariousg-1HEVproteinsconfirmeditsinteractionwithX,helicaseandORF3(Fig
PLOSPathogens|DOI:10.1371/journal.ppat.1005521 April1,2016 9/31
EndoplasmicReticulumStressPromotesGenotype-1HepatitisEVirusReplication
Fig4.AnIRESl(Internalribosomeentrysite-like)elementlocatedupstreamofORF4coding
sequencedrivesitstranslationindependentofORF1.(A)PredictedsecondarystructureofIRESlusing
“mfold”.CyanlettersindicatecoreIRESlsequence.A,B,C:stemloops,A*:bulge.Substitutionsthatimpair
IRESlareinbold.(B)OrganizationofDualluciferasereporterplasmid.(C)Dualluciferasereporterassay.
Valuesaremean±SEM.
doi:10.1371/journal.ppat.1005521.g004
5A,5Band5C).Interestingly,CoIPalsodemonstratedthatORF4interactedwithg-1RdRpin
Huh7cells(Fig5D).NootherviralproteinsinteractedwithORF4inCoIP(S4Fig).SinceX
andORF3interactedwiththesameregionofORF4andhelicaseappearedtointeractwitha
broaderregion/multipledomainsofORF4,wenextdeterminedwhetherXandORF3com-
petedorcooperatedwithhelicaseforbindingtoORF4.InORF4-Huh7cellscoexpressing
PLOSPathogens|DOI:10.1371/journal.ppat.1005521 April1,2016 10/31