MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during

25 Pages·2017·6.3 MB·English

RESEARCHARTICLE
MicroRNA and cellular targets profiling reveal
miR-217 and miR-576-3p as proviral factors
during Oropouche infection
VictorEmmanuelVianaGeddes1,AnibalSilvadeOliveira2,AmilcarTanuri1,
EuricoArruda2,MarceloRibeiro-Alves3,RenatoSantanaAguiar1*
1 DepartamentodeGene´tica,InstitutodeBiologia,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,
RiodeJaneiro,Brazil,2 DepartamentodeBiologiaCelulareMolecular,CentrodePesquisaemVirologia,
FaculdadedeMedicinadeRibeirãoPreto,UniversidadedeSãoPaulo,RibeiraoPreto,SãoPaulo,Brazil,
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3 InstitutoNacionaldeInfectologiaEvandroChagas,FIOCRUZ,RiodeJaneiro,RiodeJaneiro,Brazil
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a1111111111 *[email protected]
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Abstract
OropoucheVirusistheetiologicalagentofanarbovirusfebrilediseasethataffectsthousands
ofpeopleandiswidespreadthroughoutCentralandSouthAmericancountries.Althoughiso-
OPENACCESS
latedin1950’s,stillthereisscarceinformationregardingthevirusbiologyanditsprevalence
Citation:GeddesVEV,deOliveiraAS,TanuriA,
islikelyunderestimated.Inordertoidentifyandelucidateinteractionswithhostcellsfactors
ArrudaE,Ribeiro-AlvesM,AguiarRS(2018)
MicroRNAandcellulartargetsprofilingrevealmiR- andincreasetheunderstandingabouttheOropoucheVirusbiology,weperformedmicroRNA
217andmiR-576-3pasproviralfactorsduring (miRNA)andtargetgenesscreeninginhumanhepatocarcinomacelllineHuH-7.Cellular
Oropoucheinfection.PLoSNeglTropDis12(5):
miRNAsareshortnon-codingRNAsthatregulatesgeneexpressionpost-transcriptionally
e0006508.https://doi.org/10.1371/journal.
andplaykeyrolesinseveralstepsofviralinfections.ThelargescaleRT-qPCRbased
pntd.0006508
screeningfound13differentiallyexpressedmiRNAsinOropoucheinfectedcells.Furthervali-
Editor:PatriciaV.Aguilar,UniversityofTexas
dationconfirmedthatmiR-217andmiR-576-3pwere5.5foldup-regulatedatearlystagesof
MedicalBranch,UNITEDSTATES
virusinfection(6hourspost-infection).Usingbioinformaticsandpathwayenrichmentanaly-
Received:September22,2017
sis,wepredictedthecellulartargetsgenesformiR-217andmiR-576-3p.Differentialexpres-
Accepted:May8,2018
sionanalysisofRNAfrom95selectedtargetsrevealedgenesinvolvedininnateimmunity
Published:May29,2018 modulation,viralreleaseandneurologicaldisorderoutcomes.Furtheranalysisrevealedthe
Copyright:©2018Geddesetal.Thisisanopen geneofdecappingprotein2(DCP2),apreviousknownrestrictionfactorforbunyaviruses
accessarticledistributedunderthetermsofthe transcription,asamiR-217candidatetargetthatisprogressivelydown-regulatedduringOro-
CreativeCommonsAttributionLicense,which
poucheinfection.Ouranalysisalsoshowedthatactivatorsgenesinvolvedininnateimmune
permitsunrestricteduse,distribution,and
responsethroughIFN-βpathway,asSTING(StimulatorofInterferonGenes)andTRAF3
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. (TNF-ReceptorAssociatedFactor3),weredown-regulatedastheinfectionprogress.Inhibi-
tionofmiR-217ormiR-576-3prestrictsOROVreplication,decreasingviralRNA(upto8.3
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation fold)andvirustiter(3fold).Finally,weshowedthatvirusescapeIFN-βmediatedimmune
files. responseincreasingthelevelsofcellularmiR-576-3presultinginadecreasingofitspartners
Funding:Thisworkwassupportedbyfunding STINGandTRAF3.Weconcludedstatingthatthepresentstudy,thefirstforaPeribunyaviri-
fromFundac¸ãodeAmparoàPesquisadoEstado daemember,givesinsightsinitsprospectivepathwaysthatcouldhelptounderstandvirus
doRiodeJaneiro-FAPERJ(www.faperj.br)under
biology,interactionswithhostcellsandpathogenesis,suggestingthatthevirusescapesthe
thegrantnumber:E-26/010.001562/2014,Ref.:
210.022/2014.RSAandATwererecipientofthat antiviralcellularpathwaysincreasingtheexpressionofcognatesmiRNAs.
funding.VEVGwasrecipientofafellowship
grantedbyCoordenac¸ãodeAperfeic¸oamentode
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006508 May29,2018 1/25
miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
PessoaldeN´ıvelSuperior-CAPES(www.capes.
gov.br)underthegrantnumber:1545985.The
Authorsummary
fundershadnoroleinstudydesign,datacollection
andanalysis,decisiontopublish,orpreparationof OropoucheViruscausestypicalarboviralfebrileillnessandiswidelydistributedintropi-
themanuscript.
calregionofAmericas,mainlyAmazonregion,associatedwithcasesofencephalitis.
Competinginterests:Theauthorshavedeclared 500,000peopleareestimatedtobeinfectedwithOropoucheworldwideandsomestatesin
thatnocompetinginterestsexist. BrazildetectedhighernumberofcasesamongotherarbovirusessuchasDengueandChi-
kungunya.Asmuchasclimatechange,humanmigrationandvectorandhostavailability
mightincreasetheriskofvirustransmission.Despiteitsestimatedhighprevalencein
CentralandSouthAmericapopulations,theliteratureconcerningthemainaspectsof
viralbiologyremainscarceandbegantobeinvestigatedonlyinthelasttwodecades.
Nonetheless,littleisknownaboutvirus-hostcellinteractionsandpathogenesis.Virus
infectionregulatescellularpathwayseitherpromotingitsreplicationorescapingfrom
immuneresponsethroughmicroRNAs.KnowingwhichmicroRNAsandtargetgenesare
modulatedininfectioncouldgiveusnewinsightstounderstandmultipleaspectsofinfec-
tion.Here,wedepictedcandidatemiRNAs,genesandpathwaysaffectedbyOropouche
Virusinfectioninhepatocytecells.Wehopethisworkserveasguidelineforprospective
studiesinordertoassessthecomplexityregardingtheorthobunyavirusesinfections.
Introduction
OropoucheVirus(OROV)istheetiologicalagentofOropouchefever,anarthropod-borne
viraldiseasecharacterizedbysymptomslikefever,headache,myalgia,arthralgia,malaise,pho-
tophobia,nausea,vomiting,dizziness,skinrash,andinfewcasesencephalitisandmeningitis
[1–7].ItwasfirstdescribedinTrinidadin1955[8]andthefirstBrazilianstrainwasisolated
fromadeadpale-throatedthree-toedsloth(Bradypustridactylus)nearahighwayconstruction
campsiteinBele´m,Para´state,northernBrazil[9].Itisestimatethatmorethan500,000people
wereinfectedinatleast30outbreaksinSouthandCentralAmericabetween1961and2009[8,
10,11,12],placingOropouchefeverasoneofthemostprevalentarboviraldiseaseinsome
statesofBrazil,afterDengue,ChikungunyaandYellowFever.However,theviruspathogenesis
isstillobscure,andOropouchefeverisstillconsideredaneglecteddisease.Duringurbanout-
breaks,thevirusismainlytransmittedbyitsmajortransmissionvector,themidgeCulicoides
paraensis[3,9,13].Otherinsectspecies,likemosquitoesofthegenusAedesandCulex,might
alsobepotentialvectors[9].OROVisclassifiedintheorderBunyavirales,Peribunyaviridae
family,Orthobunyavirusgenus,asBunyamweraVirus,LaCrosseVirusandtherecentlydis-
coveredSchmallenbergVirus[14].TheorderBunyaviralesisthelargestvirusorder,contain-
ingseveralvirusesimplicatedintheetiologyofrelevanthumandiseases,suchasLaCrosse
Virus(LACV)andOropoucheVirus(Orthobunyavirus),RiftValleyFeverVirus(RVFV)
(Phlebovirus),Crimean-CongoFeverVirus(CCFV)(Orthonairovirus)andtherodent-borne
HantaanVirus(HTNV),AndesVirus(ANDV)andSinNombreVirus(SNV)(Orthohanta-
virus).OROVhasatri-segmentednegativestrandRNAgenomewithasmallsegment(S)that
encodesthenucleocapsidproteinNandanon-structuralproteinNSs;amedium(M)segment
thatencodestheglycoproteinsGcandGnandanothernon-structuralprotein,NSm,anda
large(L)segmentthatencodestheviralRNA-dependentRNApolymerase(RdRP)[15].
DespiteitsrelevanceasahumanpathogenanditshighprevalenceinSouthAmerica,littleis
knownaboutOROVreplicativecycle,pathogenesisandvirus-hostinteractions.Arecent
studydemonstratedthattheOROVentryinHeLacellsisdependentonclathrin-coatedpits
[16].AnotherreportshowedtherelevanceofMAVS,IRF-3andIRF-7,componentsofthe
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006508 May29,2018 2/25
miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
innateimmuneresponse,inrestrictingOROVinfectioninknockoutmicemodelsandnon-
myeloidcells[17].Despitethat,theviruspathogenesisandthecellularpathwaysregulatedby
OROVinfectionarenotknownindetail.Geneexpressionandpost-transcriptionalregulation
ismediatedbyshortnon-codingRNAs(microRNAs,miRNAsormiR)thatplaysimportant
rolesduringvirusreplication.
MicroRNAsspanbetween19–22nucleotidesinlengthandtheirfirstdescriptionwasmade
innematodes[18,19],thoughnowtheyhavebeenidentifiedinseveralphylaofplantsandani-
mals[20],andeveninviralgenomes[21].Inmammals,theycanbegeneratedfromintronic
andexonicregionsofprotein-codinggenesorintergenicregions[22].Theycanbefoundas
singlemiRNAgenesorinclustersthatencodeslongprecursormolecules,thepri-miRNA,
rangingfromhundredtothousandnucleotidesinlength[23,24].Pri-miRNAsbeginstobe
editedinthenucleusbytheenzymeDroshaintopre-miRNAs,shorter70nucleotideslong
moleculeswithhairpinstructures[25,26].Thosepre-miRNAsareexportedfromthenucleus
intothecytoplasmbyproteinssuchasexportin5andRAN-GTP[27],andarefurtherpro-
cessedbyDicerintoa22nucleotideslongdouble-strandedRNA(commonlyreferredas
miRNA:miRNA(cid:3))[28,29].Thedouble-strandedRNAisloadedintoanArgonaute-driven
RNAinducedsilencingcomplex(RISC),whichselectsonestrandandbindstoatargetmRNA
(commonlyinthe3’-untranslatedregion,or3’-UTRregion)[30,31]bybasecomplementarity.
ThemiRNAinteractionwithitstargetmRNAinducesgenesilencingbydegradation(when
fullcomplementaritybetweenthemiRNAandthetargetsequenceoccurs)[32],ortransla-
tionalinhibition(incaseofpartialcomplementarity)[33,34].Sincetheseedsequence(the
minimalcomplementaritysitebetweenmiRNAandmRNA)isusually7–8nucleotideslong,a
singlemiRNAcouldregulatesexpressionofseveralgenes,aswellasasinglegenecouldbereg-
ulatedbymanymiRNAs[35,36].
MiRNAshavealreadybeendescribedinfluencingdiseaseprogression,pathogenicityand
replicativecycleofseveralviruses,beingeitherinhibitoryorstimulatoryoftheinfection[37,
38].Theliver-specificmiRNA-122stimulatesHCVtranslation,stabilizingandprotectingthe
5’-UTRofviralRNAsfromdegradation,leadingtoanaccumulationofthesameinthecyto-
plasm[39–42].InrestingCD4+Tlymphocytes,HIV-1viralproductionisimpairedbycellular
miRNAsthatcontributetoestablishthevirallatency[43].AnothermiRNA,miR-29a,targets
HIV-1RNAtoaccumulateinRNAprocessingbodies(P-bodies),inhibitingvirusinfection
throughtranslationsuppression[44].Evendifferentstrainsofthesameviruscanelicitdiffer-
entmiRNAregulationresponses,asdemonstratedforthehighly-pathogenicavian-derived
InfluenzaAH7N7strainandthelow-pathogenicswine-derivedInfluenzaAH1N1strain[45],
suggestingthatmiRNAsignatureprofilescouldraisecluesaboutpathogenicityvariation.
ConcerningmiRNAregulationbybunyaviruses,astudywithpathogenicandnon-patho-
genicstrainsofhantavirusesdemonstratedthevariationonmiRNAprofileamongthediffer-
entspecie-specificvirusesandcelltypes[46].AnotherstudywiththeHantavirusRespiratory
Syndrome(HPS)-causingagent,AndesVirus(ANDV),identifieddown-regulationofmiR-
126expression,amiRNAthatactsasregulatorofSPRED1[47].Increasedexpressionof
SPRED1wassuggestedtobeoneofthemechanismsthataugmentendothelialcellspermeabil-
ity,leadingtoHPS.ArecentstudywithPBMCofpatientspresentingacutehemorrhagicfever
causedbytheCrimean-CongoHemorrhagicFeverVirus(CCHFV)showedthederegulation
ofseveralmiRNAs,someofthemassociatedwithinnateimmunityandviralescapemecha-
nisms[48].TheonlystudywithphlebovirusesdescribedtheassociationbetweenmiR-142-3p
andtheendocyticvesicleproteinVAMP3,suggestingacontrolmechanismfortheintracellu-
lartraffickingofUukuniemiVirus(UUKV)[49].
DuetothescarcityofinformationregardingtheregulationofbunyavirusesbymiRNAand
theincreasingnecessityofbetterunderstandingofvirus-hostinteractionsofrelevantemerging
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
pathogens,weaimedtoevaluateandidentifythecellularmiRNAprofileandtargetgenes
inducedbyOROVinfectioninvitro.WedemonstratedthatmiRNAsmiR-217andmiR-576-
3p,differentiallyexpressedduringinfection,couldberegulatingcrucialpathways,likeinnate
immunityresponse,mainlyinupstreamproteinsofinterferon-βinductionpathway(adaptor
andkinaseproteins,aswellastranscriptionfactors),proteinshutoffandapoptosis.
Methods
Celllines,virusandinfection
CelllinesVero(ATCC,CCL-81),U87-MG(ATCC,HBT-14)andHeLa(ATCC,CCL-2)were
maintainedinDMEM(Gibco)supplementedwith10%v/vFetalBovineSerum(FBS)(Gibco)
and1%v/vofpenicillin-streptomycin(10.000U/ml-10.000μg/ml)(Gibco)at37˚Cand5%
CO .HuH-7cellsweremaintainedinDMEMwithoutsodiumpyruvate(Gibco)supplemented
2
with10%v/vHyCloneserum(GELifeSciences),1%v/vantibiotics,1%200mML-Glutamine
(Gibco)and1%v/vnon-essentialaminoacids(Gibco)at37˚Cand5%CO .Jurkat(ATCC,
2
TIB-152)andTHP-1(ATCC,TIB-202)weremaintainedinRPMI-1640medium(Gibco)sup-
plementedwith10%v/vFBS,1%v/vantibioticsand1%v/vsodiumbicarbonate(Gibco)at
37˚Cand5%CO .OROVstrainBeAn19991wasoriginallyobtainedfromtheEvandroChagas
2
InstituteandpropagatedbyserialpassagesinVerocellsbyroutinemethodsusingDMEM.
TheOROVstockusedinthepresentexperimentswaspropagatedinHeLacellsandtitratedto
2x106PFU/ml.InfectionswereperformedatMOI1during1hat37˚Cand5%CO in
2
mediumwithoutFBS,underbiosafetylevel3conditionsataBSL-3laboratoryatUniversidade
FederaldoRiodeJaneiro.
Virustitration
VirustitrationwasperformedbyplaqueassayinVerocellsplatedat3x105cells/wellin12
wellplates1daypriortoinfection.After1hincubationwiththevirus,cellswerereplenished
byDMEMsupplementedwith1%v/vFBS,1%v/vantibioticsand1%v/vcarboxymethylcellu-
lose(CMC)(Sigma-Aldrich),andincubatedat37˚Cand5%CO during4days.Cellswere
2
fixedwith4%formaldehydefor20minatroomtemperature,washedinPhosphateBuffered
Saline(PBS)(Gibco)andstainedwith20%v/vethanol-violetcrystalsolutionfor15min.
THP-1PMAtreatment
Inordertoinducemonocyte-to-macrophagedifferentiation,THP-1cellswerestimulatedwith
100nMphorbol12-myristate13-acetate(PMA)(Sigma-Aldrich)instandardRPMImedium
for24hor3daysfollowedby5daysincubationatRPMImediumwithoutPMA.FreshRPMI
mediumwasprovidedtocellsaftertreatmentandbeforeinfections.THP-1derivedmacro-
phagescellswereinfectedasdescribedabove,at24hor8dayspostPMAtreatment.
Flowcytometryandimmunofluorescencestaining
Cells(105cells/sample)werefixedwith4%paraformaldehydefor20minandpermeabilizedin
1%v/vTritonX-100PBSsolution.Blockingwasperformedin5%v/vDonkeySerum(Sigma-
Aldrich)PBSsolutionfor1hat37˚C.OROVinfectedanduninfectedcellswereincubatedwith
mousepolyclonalanti-OROVantibodyat1:300dilutioninblockingsolutionat37˚Cfor30
min.CellswerethenwashedthriceinPBSandincubatedwith2μg/mlDonkeyanti-mouse
AlexaFluor488secondaryantibody(ThermoFisherScientific)at37˚Cfor30min.Afterincuba-
tionwiththesecondaryantibody,cellswerewashedandresuspendedinPBS.Flowcytometry
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
wasperformedinAccuriC6cytometer(BDBiosciences).Atleast10,000gatedeventswere
countedperexperimentalreplicaatFITCchannel.
Cellviabilityassay
HuH-7wereplatedat2x104cells/welldensityon96-wellplateandincubatedat37˚Cand5%
CO for12h.Afterthat,cellswereinfectedasdescribedabove.Cellviabilitywasevaluatedby
2
CellTiter-Blue(Promega)accordingtomanufacturer’sinstructions.Thefluorescencewasmea-
suredatSpectraMaxParadigmMulti-ModeDetectionPlatform(MolecularDevices).
RNAisolationandqualityassessment
TotalcellularRNAformicroarrayandtargetmRNART-qPCRanalysiswasisolatedusingMir-
Vana kit(ThermoFisherScientific)accordingtomanufacturer’sinstructions.RNAquantification
andqualitywasassessedby2100BioanalyzerusingRNA6000Nanokit(AgilentTechnologies).
OnlysampleswithaRNAIntegrityNumber(RIN)(cid:21)9.0wereusedformicroarray.Extraction
ofRNAformiRNAvalidationwithspecificprimerswasperformedusingPureLinkRNAMini
Kit(ThermoFisherScientific)andquantificationandintegritywereassessedinNanoVueSpec-
trophotometer(GELifeSciences).AllRNAsweretreatedwithDNase(TURBODNA-freeKit,
ThermoFisherScientific)beforeRT-qPCRexperimentstoavoidDNAcontamination.
MiRNAscreening
InordertoevaluatetheexpressionprofileofmiRNAs,anarrayusingTaqmanchemistrywas
performedasfollows:12hafterinfection,sixindependentreplicasofmock-infectedorOro-
poucheinfected(4x106cells/replicaatMOI1)HuH-7cellsweretrypsinized(Trypsin0.25%,
Gibco)andthetotalcellularRNAwasextractedandquantifiedasdescribedabove.cDNAwas
generatedusingTaqManMicroRNAReverseTranscriptionKit(ThermoFisherScientific)with
100ngofRNApersampleaccordingtomanufacturer’sinstructions.ThecDNAwaspreampli-
fiedusingMegaplexPreAmpPrimers(ThermoFisherScientific)andTaqmanPreAmpMaster
Mix(ThermoFisherScientific)asinstructedbymanufacturer.TheqPCRreactionwasperformed
usingTaqmanOpenArrayHumanMicroRNAPanels(ThermoFisherScientific),TaqmanOpen-
ArrayReal-TimeMasterMixandtheOpenArrayAccufillsystemOpenArrayreal-timerobotics
(ThermoFisherScientific).Thisplatformisabletoquantify754humaninventoriedmiRNAs.
MiRNAarraystatisticalanalysis
Rstatisticallanguage[50]wasusedforbackgroundcorrectionanddataexploratoryanalysis(Rn
intensitycumulativecurveandHighResolutionMelting—HRMgraphs)foreachRT-qPCR
reaction.Forrelativeexpressionquantification,afourparameterssigmoidalcurveadjustment
wasdoneusingtheqpcRfunctionsinRlanguage[51].Quantificationcycle(Cq)wasdetermined
astherelativecycletosecondderivativemaximumpointofadjustedsigmoidalcurve(cpD2).
Theamplificationefficiencywasdeterminedattheexponentialamplificationregion,atthe
meanpointbetweenrelativecyclestothefirstderivativemaximumpointandsecondderivative
maximumpointofadjustedsigmoidalcurve[expR=cpD2-(cpD1-cpD2)],andcalculatedasthe
ratiobetweentheexpRcorrespondingcyclefluorescenceandthepriorcyclefluorescence.For
eachmiRNA,theamplificationefficiencywasdeterminedasthemeanofefficienciescalculated
forthecorrespondingmiRNA.Endogenoussmall-nucleolarRNAsRNU44,RNU48andU6
RNAwerecandidatesfornormalizationcontrolsselectedbythegeNormmethod[52].Asan
alternativenormalizationmethod,thenormalizationfactorwascalculatedbythegeometric
meanofallmiRNAexpressedineachsample[53].Fornormalizedexpressioncomparison
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
betweentwosamplegroups,weperformedanon-parametricT-testwith1,000permutations
[54].Forthreeormoregroupscomparisonweusedaone-waynon-parametricANOVAwith
unrestrictedpermutation(n=1,000)followedbyanon-parametricpairwiseT-testmeancom-
parisonwithpermutation(n=1,000)followedbyBonferronicorrection[54].Resultswere
presentedasmean±S.E.M(standarderrormean).Two-tailedp-valuesinsamplegroups’com-
parisonlowerorequalto0.01,0.05or0.1wereconsideredashighlysignificant,significantand
suggestive,respectively.TherelationshipbetweensampleprofileswasinvestigatedbyBayesian
InfiniteMixturesModelclusteranalysis[55]andrepresentedby2Dheatmapwithdendrograms
(bi-cluster).Forthepurposeofdisplayintheheatmap,k-nearestneighborsmethod(k=5)was
performedtopredictthemissingvaluesinuninfectedcellsformiR-217,miR-26a-2-3pand
miR-92a-5p.Afterimputationofthemissingvalues,ascaled(Z-score)normalizationwasper-
formed(subtractedmiRNAmeandividedbymiRNAstandarddeviation).
ValidationofmiRNAthroughprimerdesignedRT-qPCR
ReversetranscriptionwasperformedusingmiRNA1st-StrandcDNASynthesisKit(Agilent
Technologies) andqPCRreactionsweremadewithHigh-SpecificitymiRNAQPCRCoreKit
(AgilentTechnologies) andforwardspecificprimersforeachmiRNAinvestigated.HumanU6
RNAforwardprimer(AgilentTechnologies) wasusedasnormalizationcontrol.Alltheexperi-
mentsweredoneinfourindependentreplicasforeachtimepointandsamplegroup.The
qPCRreactionwasperformedin7500Real-TimePCRSystem(AppliedBiosystems).The
cyclingparametersweresetforstandardSYBRGreenmethodaccordingtomanufacturer’s
instructionsasfollow:95˚C–10minand95˚C–10sec,60˚C–15sec,72˚C–20secfor40
cycles.ThemiRNAforwardprimerssequencesaredepictedinS1Table.Statisticalanalysis
wasperformedusingnon-parametricalMann-Whitneytests.
Targetgenesbioinformaticsprediction
WeonlyconsideraputativetargetfordifferentiallyexpressedmiRNA(miRNA:mRNAinter-
action)theonespredictedinatleast3outof6publicdatabasesasfollows:TargetScan, (avail-
ableathttp://www.targetscan.org/index.html)miRTarget2 (availableathttp://mirdb.org/
miRDB/),PicTar (availableathttps://pictar.mdc-berlin.de/),miRBase(availableathttp://www.
mirbase.org/),TarBase (availableathttp://carolina.imis.athena-innovation.gr/diana_tools/
web/index.php?r=tarbasev8%2Findex)andmiRandaV3.3a.Interactionnetworktreewas
designedusingCytoscapev3.2.1software(CytoscapeConsortium).
Genesetenrichmentanalysis(GSEA)
Ontologyenrichmentanalysis[56]wasperformedforthepredictedtargetsofthedifferentially
expressedmiR-217andmiR-576-3p.Theontologieswereenrichedmainlytobiologicalpro-
cesses,molecularfunction,cellularcomponents,andgeneinteraction/regulationpathways.
Onlygenespredictedinatleast3outof6databaseswereconsideredcandidatetargets.Gene
EntrezidforthepredictontologieswereusedintheGeneOntologyDatabase(GO,availableat
http://www.geneontology.org/),KEGG(availableathttp://www.genome.jp/kegg/)andREAC-
TOME(availableathttp://www.reactome.org/PathwayBrowser)forthispurpose.Onlygenes
overrepresentedinhypergeometrictestswithp-value(cid:20)0.001wereconsidered.
MicroRNAinhibitorstransfection
HuH-7cellswereseeded(105cells/replica)intriplicateinto24wellsplateovernight.Negative
controlinhibitor,miR-217inhibitorandmiR-576-3pinhibitor(Integrated DNATechnologies)
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
weretransfectedatafinalconcentrationof75nMusing2μlofLipofectamine2000(Thermo
FisherScientific)perreplica.GreenfluorescentshortRNAsiGLO(Dharmacon,GELifeSci-
ences)wasusedtoassesstransfectionefficiencyandestablishthemiRNAinhibitorconcentra-
tionforinhibitionexperiments.3hpost-transfection,cellswereinfectedwithOROVatMOI
1andRNAwereextractedformiRNAquantification(6hpost-infection)ortargetgeneand
OROVRNAquantification(18hpost-infection)byRT-qPCR.OROVsegmentSRNAwas
quantifiedusingprimersandprobe[57]withTaqman2xUniversalPCRMasterMix(Thermo
FisherScientific)andnormalizedbyGAPDHusingPrimeTimeprimersandprobemix(Inte-
gratedDNATechnologies).
ValidationoftargetmRNAthroughRT-qPCR
Cellswereseeded(106cells/sample)andinfectedwithOROVatMOI1.TheRNAwas
extractedat12hpostinfectionandreversetranscriptionwasperformedusingHigh-Capacity
cDNAReverseTranscriptionKit(ThermoFisherScientific)and1μgofRNA.Quantitative
PCRwasdoneinsixreplicaperconditionusing50ng/wellofcDNAonCustomTaqman
ArrayFastplates(96well)(ThermoFisherScientific)usingspecificprimersandprobesand
TaqmanFastUniversalPCRMasterMix(ThermoFisherScientific)accordingtomanufactur-
er’sinstructionson7500FastReal-TimePCRSystem(ThermoFisherScientific).Statistical
analysiswasdoneasdescribedformicroarrayusingendogenous18S,GAPDH,HPRT1and
GUSBasnormalizationgenes.FortargetkineticsSYBRGreenPCRMasterMix(AppliedBio-
systems)andpre-designedPrimeTimeprimers(Integrated DNATechnologies) wereused
accordingtomanufacturer’sinstruction(forprimerssequencesseeS1Table).
Results
OROVinfectshepatocytesandbloodcelllinesinvitro
InordertoexpandtheknowledgeontherangeofOROV-permissivecells,bloodandhepato-
cytecelllineswereusedtoevaluateinvitroinfection(Fig1A).TCD4+lymphocytes(Jurkat),
monocytes(THP-1)andhepatocytes(HuH-7)celllineageswereinfectedwithOROVatMOI1
and,at12hpostinfection,infectivitywasassessedbyimmunofluorescenceusingspecificanti-
bodiesagainstOROVproteinsandvirus-positivecellswerecountedbyflowcytometry.Atindi-
catedtimepoints,21%ofJurkatcellswereinfected,whileTHP-1presentednosusceptibilityto
theOROVinfection.THP-1cellscanbeinducedtodifferentiateintomacrophagebyPMA
treatment,becomingpermissivetosomeviralinfections,asdescribedelsewhere[58–61].In
ordertoassessifTHP-1cellsdifferentiatedintomacrophageswerepermissivetoOROVinfec-
tion,THP-1cellsweretreatedwithPMAfor24horfor3days,followedbyincubationin
mediumwithoutPMAfor5moredays.DifferentiationofTHP-1intomacrophage-likepheno-
typewasaccompaniedbymicroscopyandattachment.At12hpostinfection,31%and50%of
THP-1treatedwithPMAfor24hor8days,respectively,wereinfectedwithOROV,suggesting
anincreasingpermissivenesstoOROVinfectionasthecellsshiftfrommonocytetomacro-
phage-likephenotypes(Fig1A).AtthesameMOI,HuH-7cellsshowedtobemorepermissive
toOROVinfection,presenting90%infectedcellsat12hpost-infection(Fig1A).Basedonthis
resultwithHuH-7cells,andconsideringpreviousdemonstrationsthattheliverisanimportant
replicationsiteduringexperimentalOROVinfectioninhamster[62,63]andmouse[64],we
chosethehepatocytecelllineHuH-7asourinvitromodelforfurtherexperiments.
Toassessthemostsuitableconditionstoensurethatmostcellswouldbeinfectedatindi-
catedtimepoints,HuH-7cellswereinfectedwithdifferentMOIsandtheinfectivitywasmea-
suredbyflowcytometry(Fig1B).Wereached30%ofinfectivityatMOI0.1withaplateauof
90%inhigherconcentrationsofvirus(MOIs1,5and10),withnofurtherincreaseof
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
Fig1.Oropoucheinfectioninbloodandhepatocytecelllines.Jurkat,THP-1andHuH-7cellswereinfectedwithOROVatMOI1.At12hpostinfection,infectivity
wasassessedbyflowcytometryusingpolyclonalanti-OROVantibody.(A)InfectioninTlymphocyte(Jurkat),monocyte(THP-1)andhepatocyte(HuH-7)celllines
(x-axis)wasperformedasdescribedinmaterialsandmethodssection.Errorbarsrepresentsstandarddeviation(SD)offourindependentexperiments.THP-1
NA=THP-1notactivated;THP-1(24)=activatedfor24hinPMA;THP-1(8d)=activatedfor3daysinPMAfollowedby5dayswithoutPMA.(B)OROVinfection
inHuh-7cellsatdifferentMOIs(x-axis).Infectivitywasassessedbyflowcytometry.ErrorbarsrepresentsSD.(C)RelativecellviabilityofHuH-7cellsinfectedwith
OROV(MOI1)atdifferenttimepoints.Cellviabilitywasassessedbyfluorimetricassay.Absolutevaluesofuninfectedcellsweresetas1(lefty-axis).Blackcolumns
representuninfectedcellsandgraycolumnsrepresentOROVinfectedcells(MOI1).ErrorbarsrepresentsSDforfivereplicatesoftwoindependentexperiments.Gray
linerepresentsmeanvirustiterinsupernatantmeasuredbyplaqueassayatindicatedtimepoints(righty-axis).(cid:3)(cid:3)=p(cid:20)0.01.
https://doi.org/10.1371/journal.pntd.0006508.g001
infectivitylevels(Fig1B).Toassurethatcellswerestillviableforfurtherexperiments,we
assessedthecytopathiceffectat6,12,18and24hpostinfectionwithMOIof1usingCell
Titer-Blue(Fig1C).WedidnotdetectcelldeathassociatedtotheOROVinfectionatleast18
hpostinfection.However,only44%ofcellswereviableat24hpostinfection.Wealsoquanti-
fiedthevirustitergeneratedinthosecellsbyplaqueassayandat6hpost-infection,thetiterin
thesupernatantwas6x103PFU/ml(Fig1C,grayline).Astheinfectionprogressed,apeakof
8.6x105PFU/mlcouldbedetectedat18hpostinfection,reachingaplateauwithnofurther
increaseinviraltiterat24hpostinfection(Fig1C,grayline).Basedonthesedata,wepro-
ceededusingHuH-7cellsinfectedwithMOI1insubsequentexperimentstoevaluatethe
virus-hostinteractions.
CellularmiRNAsignatureinHuH-7cellsinfectedwithOROV
MiRNAscanbeinformativeofcellulartargetsmodulatedbyvirusinfection.Inordertoiden-
tifycandidatecellularpathwaysdifferentiallyexpressedinOROVinfectedcells,weperformed
anexploratoryscreeningof754humanmiRNAsthroughprobe-basedRT-qPCR.MiRNAs
expressionwasevaluatedinfouruninfected(control)andfiveOROVinfectedbiologicalrepli-
casat12hpostinfection.WefoundthirteenmiRNAsdifferentiallyexpresseduponOROV
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
infectioninHuH-7cellswithstatisticalsignificance:twelveup-regulatedafterinfectionand
onlyonedown-regulated(miR-450b-5p)(Fig2andTable1).Thereproducibilityofeffectsin
miRNAswasindicatedbysmallvariancenotedamongbiologicalreplicas,asdepictedinthe
heatmaphierarchicaldendrogram(Fig2).
ThedifferentialexpressionofthemiRNAsinOROVinfectedcellswasclassifiedintothree
groups:up-regulated,down-regulatedandinfection-dependentmiRNAs(selectivelyexpressed
miRNAs).MiRNAsmiR-324-3p(1.73x),miR-1227(1.95x),miR-362-3p(1.85x),miR-99b-3p
(2.21x),miR-19b-1-5p(4.11x),miR-628-3p(2.77x),miR-26a-1-3p(42.47x),miR-576-3p
(2.49x)andmiR-27a-5p(108x)wereup-regulated,inOROV-infectedcellsrelativetounin-
fectedcells.MiR-450b-5pwasdown-regulated4.65timesininfectedcellscomparedtounin-
fectedcells.TheinductionofmiR-26a-2-3pandmiR-217wereinconsistentandobservedonly
inthreeoutoffiveinfectedreplicas.FromthethirteenselectedmiRNAsfromthescreening,
onlymiR-576-3pandmiR-26a-1-3psustainedsignificance(p(cid:20)0.05,p(cid:20)0.01,respectively)
afterBonferronicorrectionaccordingtothemethodusedinthisstudy.Nonetheless,some
miRNAspresentedborderlinelimitsofsignificance(p=0.0595),namely,miR-1227,miR-19b-
1-5pandmiR-450b-5p(Table1).
InordertovalidatethemiRNAsthatweresignificantlyderegulatedinthearray(miR-26a-
1-3pandmiR-576-3p)andtoverifytheexpressionofthemiRNAsonlydetectedininfected
cellsintheexpressionprofilearray(miR-217,miR-26a-2-3pandmiR-92a-1-5p),wedesigned
specificprimersforeachmiRNAandcheckeditsexpressionbyRT-qPCR(Fig3).Ourvalida-
tionexperimentsshowedthesametendencyofthemiRNAspanelwithanincreasingexpres-
sionofbothmiR-217(Fig3A)andmiR-576-3p(Fig3B)duringinfection,reachingapeakof
expressionat6hpost-infection(about5.5foldincreaseforbothmiRNAs).Thekineticsof
expressionofmiR-217suggestsanearlyinductionduringinfectioncomparedtomiR-576-3p,
sincemiR-217wasup-regulated2.26timesasearlyas3hpost-infectionwhilemiR-576-3pwas
onlyup-regulated1.44atthesamepoint.However,atlaterstagesofinfection,miR-217expres-
sionwasalreadyclosertouninfectedexpressionlevels(up-regulatedonly1.7at12hpost-
infection),whereasmiR-576-3pwasstillup-regulated2.83timesininfectedcells,indicatinga
slightlydifferentkineticsforthosemiRNAs.
Toconfirmtherobustnessofouranalysis,wefurthervalidatedtheexpressionofthree
otherlessstablestarmiRNAs:thehighlysignificantmiRNAmiR-26a-1-3pandtwomiRNAs
detectedonlyuponinfection,miR-26a-2-3pandmiR-92a-1-5p(Fig3C).ThosethreemiRNAs
wereup-regulated5.3,4.5and6.3fold,respectively,at12hpost-infectionincomparisonwith
uninfectedcells(p(cid:20)0.01).Altogether,theseresultswithspecificprimerstoeachmiRNAcor-
roboratewithourlarge-scalepaneldata,identifyingmiRNAsthataremodulatedduring
OROVinfectionshowingthesametendencywithdifferentapproaches.StarmiRNAnomen-
claturecorrespondstopassengerstrandslessfavorabletoprocessingbyRISCwithlowerlikeli-
hoodtoregulategeneexpression[65,66].AsmostofthosemiRNAsarepreviouslyannotated
asstarmiRNAs(asexampleofmiR-26a-1-3ppreviouslyannotatedasmiR-26a-1(cid:3))andsome
predictiondatabasealgorithmsuseprovedinteractionascriteriaforprediction,weonly
selectedmiR-217andmiR-576-3p,bothmaturestrandmiRNAs,forfurthertargetprediction
analysis(onedetectedonlyininfectedcellsandtheotheroneup-regulatedsignificantlyupon
infectioninthearray,respectively,andbothvalidated).
CellulartargetgenesregulatedbymiR-217andmiR-576-3p
ToinvestigatepossiblepathwaysregulatedbymiR-217andmiR-576-3pduringOROVinfec-
tion,weperformedtargetpredictionusingTargetScan,miRTarget2,PicTar,miRBase,TarBase
andmiRandadatabases.Targetgenespredictedbyatleast3outof6ofthosedatabaseswere
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miR-217andmiR-576-3pareproviralfactorsforOropouchereplication
Fig2.HeatmapofdifferentiallyexpressedmiRNAsinOROVinfectedHuH-7cells.Depictionofthe13miRNAs
differentiallyexpressedinOROVinfectedcellsrelativetouninfectedcells.LinesrepresentindividualmiRNAsand
columnsrepresentindependentreplicates(fourmockinfectedandfiveOROVinfected).Colorscalerepresents
normalizedexpressionlevelsofmiRNAsinthetwoconditionsinlog scale;reddenotesup-regulationandgreen
2
denotesdown-regulation,respectively.Dendrogramrepresentingthe1Dclusterizationofsamplesandthe2Dmap
correspondingtothelevelsofstandardizedgeneexpressionprofiles(z-score).Reddottedlinesinthedendrogram
indicateweakunions,discouragedbytheBayesianclusteringanalysis.Valuesrepresentedinthedendrogrambranches
correspondtolog-oddsoftheunionofcorrespondingbranches.Forthepurposeofdisplay,missingvaluesof
uninfectedcellsformiR-217,miR-26a-2-3pandmiR-92a-5pwerepredictedbyk-nearestneighborsmethodand
imputedafternormalizationasdescribedinMethodssection.
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