Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and ...

22 Pages·2016·2.66 MB·English

RESEARCHARTICLE
Small RNA Profiling in Dengue Virus 2-
Aedes
Infected Mosquito Cells Reveals Viral
piRNAs and Novel Host miRNAs
PascalMiesen1,AlasdairIvens2,AmyH.Buck2,RonaldP.vanRij1*
1 DepartmentofMedicalMicrobiology,RadboudUniversityMedicalCenter,RadboudInstituteforMolecular
LifeSciences,Nijmegen,TheNetherlands,2 CentreforImmunity,Infection&Evolution,Universityof
Edinburgh,Edinburgh,UnitedKingdom
*[email protected]
Abstract
InAedesmosquitoes,infectionswitharthropod-borneviruses(arboviruses)triggerormodu-
latetheexpressionofvariousclassesofviralandhost-derivedsmallRNAs,includingsmall
OPENACCESS
interferingRNAs(siRNAs),PIWIinteractingRNAs(piRNAs),andmicroRNAs(miRNAs).
Citation:MiesenP,IvensA,BuckAH,vanRijRP
ViralsiRNAsareatthecoreoftheantiviralRNAinterferencemachinery,oneofthekey
(2016)SmallRNAProfilinginDengueVirus2-
InfectedAedesMosquitoCellsRevealsViralpiRNAs pathwaysthatlimitvirusreplicationininvertebrates.BesidessiRNAs,Aedesmosquitoes
andNovelHostmiRNAs.PLoSNeglTropDis10(2): andcellsderivedfromtheseinsectsproducearbovirus-derivedpiRNAs,thebeststudied
e0004452.doi:10.1371/journal.pntd.0004452
examplesbeingvirusesfromtheTogaviridaeorBunyaviridaefamilies.HostmiRNAsmodu-
Editor:GregoryDEbel,ColoradoStateUniversity, latetheexpressionofalargenumberofgenesandtheirlevelsmaychangeinresponseto
UNITEDSTATES
viralinfections.Inaddition,someviruses,mostlywithaDNAgenome,expresstheirown
Received:October13,2015 miRNAstoregulatehostandviralgeneexpression.Here,weperformacomprehensive
Accepted:January21,2016 analysisofbothviralandhost-derivedsmallRNAsinAedesaegyptiAag2cellsinfected
withdenguevirus2(DENV),amemberoftheFlaviviridaefamily.Aag2cellsarecompetent
Published:February25,2016
inproducingallthreetypesofsmallRNAsandprovideapowerfultooltoexplorethecross-
Copyright:©2016Miesenetal.Thisisanopen
talkbetweenarboviralinfectionandthedistinctRNAsilencingpathways.Interestingly,
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits besidesthewell-characterizedDENV-derivedsiRNAs,aspecificpopulationofviralpiRNAs
unrestricteduse,distribution,andreproductioninany wasidentifiedininfectedAag2cells.KnockdownofPiwi5,Ago3and,toalesserextent,
medium,providedtheoriginalauthorandsourceare
Piwi6resultsinreductionofvpiRNAlevels,providingthefirstgeneticevidencethatAedes
credited.
PIWIproteinsproduceDENV-derivedsmallRNAs.Incontrast,wedonotfindconvincing
DataAvailabilityStatement:SmallRNAsequences
evidencefortheproductionofvirus-derivedmiRNAs.NeitherdowefindthathostmiRNA
havebeensubmittedtoNCBISequenceRead
expressionisstronglychangeduponDENV2infection.Finally,ourdeep-sequencinganaly-
ArchiveunderaccessionnumbersSRX1309506to
SRX1309511.Allotherrelevantdataarewithinthe sesdetect30novelAedesmiRNAs,complementingtherepertoireofregulatorysmall
paperanditsSupportingInformationfiles. RNAsinthisimportantvectorspecies.
Funding:ThisworkisfinanciallysupportedbyaPhD
fellowshipfromRadboudUniversityMedicalCenter
(www.radboudumc.nl)toPM,aStrategicawardfrom
theWellcomeTrusttotheCentreforInfection
AuthorSummary
ImmunityandEvolution(grantno.095831),Welcome
Trust(grantno.WT097394A1A)toAHB,anECHO MosquitoesoftheAedesfamilytransmitmanyimportantviruses,includingdenguevirus,
projectgrantfromtheNetherlandsOrganizationfor
betweentheirvertebratehosts.Inthemosquito,thegrowthofthesevirusesislimitedby
ScientificResearch(NWO,grantno.711.013.001)to
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 1/22
SmallRNAsinDENV2-InfectedMosquitoCells
RPvR,andEuropeanResearchCouncilConsolidator
GrantundertheEuropeanUnion’sSeventh theantiviralRNAinterferencepathway.Keytothispathwayisaclassofsmallnon-coding
FrameworkProgramme(ERC,grantno.615680)to RNAsknownassmallinterferingRNAs(siRNAs).Inaddition,tworelatedbutdistinct
RPvR.Thefundershadnoroleinstudydesign,data
smallRNApathwaysknownasthemicroRNA(miRNA)andthePIWI-interactingRNA
collectionandanalysis,decisiontopublish,or
(piRNA)pathwayareimplicatedinregulatingvirusreplicationinmosquitoes.Thus,since
preparationofthemanuscript.
smallRNAsmaycriticallyinfluencethetransmissionofdenguevirus,wesetouttoanalyze
CompetingInterests:Theauthorshavedeclared
thepopulationsofviralandmosquitosmallRNAsthatareproducedininfectedAedes
thatnocompetinginterestsexist.
mosquitocells.Wefoundthatbesidesthewell-knownviralsiRNAs,denguevirus-derived
piRNAswereproducedinthesecellsandweidentifiedthePIWIproteinsthatthesesmall
RNAsrelyon.Inaddition,wefoundthatviralmiRNAswerenotexpressedfromtheden-
guevirusgenomeandthatthelevelsofmosquitomiRNAswerebarelychangedupon
infection.Finally,ourdataallowedfortheidentificationofnovelAedesmiRNAs,comple-
mentingtherepertoireoftheseimportantregulatoryRNAsinvectormosquitoes.
Introduction
Aedesmosquitoesareessentialvectorsforthetransmissionofimportantarthropod-borne
viruses(arboviruses),includingdenguevirus(DENV),yellowfevervirus,andchikungunya
virus[1].Whileseveralofthesearboviralinfectionscausediseaseinhumans,virusreplication
generallydoesnotleadtoseverepathologyinvectormosquitoes.Infectedmosquitoesthus
serveasapersistentreservoirforarbovirusesinthewildandtheymaytransmitthesevirusesto
vertebratehoststhroughouttheirentirelives[2].
Afteringestioninamosquito’sbloodmeal,arbovirusesneedtoovercomeanumberofana-
tomicalandimmunologicalbarrierstoreachsufficientlyhightitresinthesaliva.Onlythencan
transmissiontoanaivevertebratehostefficientlyoccur.Oneofthemostimportantimmune
responsestoarboviralinfectionisantiviralRNAinterference(RNAi)[3–5].Thispathwayis
triggeredbythepresenceofdoublestrandedRNA(dsRNA),whichisproducedduringtherep-
licationofRNAandDNAviruses[6,7].ThedsRNAisrecognizedandcleavedbytheRNase-
IIIenzymeDicer-2(Dcr2)into21nucleotide(nt)smallinterferingRNAduplexes(viral
siRNA;vsiRNA)[8,9].OneofthesiRNAstrandsisincorporatedinArgonaute-2(Ago2),the
coreproteinoftheRNAinducedsilencingcomplex(RISC)[10].ThesiRNA-loadedRISCcom-
plexisguidedtocomplementaryviralRNAmoleculesandcleavesthesetargetRNAsusingthe
endonuclease(slicer)activityofAgo2[11].
MicroRNAs(miRNAs)areadistinctclassofsmallRNAsthatareproducedfromgenome-
encodedstemloop-containingtranscriptsknownasprimarymiRNA(pri-miRNAs).During
thecanonicalmiRNAbiogenesispathway,thestemloopstructures,knownasprecursor
miRNA(pre-miRNA),arereleasedfromthepri-miRNAbythemicroprocessorcomplexwith
atitscoretheRNase-IIIenzymeDrosha.Aftertranslocationintothecytoplasm,pre-miRNAs
arecleavedbyDicer-1(Dcr1)toproduceasmallRNAduplexcomprisedofthetwomature
miRNAstrands.Usually,oneofthesestrandsisthenpreferentiallyincorporatedintotheArgo-
naute-1(Ago1)containingmiRNA-inducedsilencingcomplex(miRISC),whereastheother
(cid:1)
strand(thepassengerormiRNA strand)isusuallydiscarded[12].LoadedmiRISCcomplexes
areabletobindtospecifictargetsiteswithinmRNAs.ThismiRNA-mRNAinteractionisiniti-
atedbynucleotidetwotosevenofthemiRNA,theso-calledseedsequence[13].Stablebinding
ofmiRISCtoanmRNAtarget,generallycausesdown-regulationofgeneexpressionviatrans-
lationalinhibitionandmRNAdestabilization[14].Importantly,infectingvirusescandirectly
orindirectly,asaconsequenceoftheimmuneresponse,reshapethehostmiRNAexpression
landscape.Whilequiteanumberofstudieshavereportedonthismatterinmammalian
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 2/22
SmallRNAsinDENV2-InfectedMosquitoCells
systems[15],littleisknownaboutvirus-inducedchangesinmiRNAlevelsinmosquitovectors.
InAedesmosquitoes,miRNAlevelsormodificationshavebeenreportedtobechangedupon
infectionswithDENV,WestNilevirus,andchikungunyavirus[16–20].Formostofthesedif-
ferentiallyexpressedmiRNAs,thebiologicalrelevanceaswellasthetargetedmRNAsstill
awaitexperimentalvalidation.
BesidesmodulationofhostmiRNAs,someDNAandretrovirusesencodetheirownmiR-
NAstoregulateviralandhostmRNAs[21].TheexpressionofmiRNAsfromcytoplasmic
RNAviruseshasbeencontroversial.However,functionalintroductionofartificialmiRNAs
intothegenomesofSindbisvirus(SINV)andtick-borneencephalitisvirusprovidesevidence
thatmiRNAproductionfromcytoplasmicRNAvirusesmayinprinciplebepossible[22,23].
Yet,thepresenceandbiologicalrelevanceofmiRNAsencodedinthegenomesofflaviviruses
suchasDENVisstillanissueofdebate[24–26].
Thethird,mostenigmaticclassofsmallRNAsarePIWIinteractingRNAs(piRNAs).These
areprocessedfromlongRNAprecursorsthataretranscribedfromgenomiclociknownas
piRNAclusters.InsharpcontrasttosiRNAsandmiRNAs,theirbiogenesisintomaturepiR-
NAsisDicer-independent.InDrosophila,piRNAmaturationinvolvesendonucleolyticcleav-
ageofprecursortranscriptsbytheZucchininucleaseandthethreePIWIproteinsPiwi,
Aubergine(Aub)andArgonaute-3(Ago3)[27–30].TheprimaryfunctionofthepiRNApath-
wayinthismodelorganismisthedefenceagainsttransposableelements,mainlyingerm-line
tissues.Interestingly,piRNAsofviralorigin(vpiRNA)havebeenfoundinsomatictissueof
Aedesmosquitoes,suggestingthattheycontributetotheregulationofvirusreplication[31].At
present,vpiRNAshavebeendiscovereduponinfectionwithanumberofAlphaviruses,Bunya-
virusesandFlaviviruses,includingDENV[31–39].However,withtheexceptionofSINV
(Alphavirus),theirmolecularbiogenesishasnotbeeninvestigated[37].
Here,wemakeuseofsmallRNAdeep-sequencinginthesiRNA,miRNA,andpiRNAcom-
petentAedesaegyptiAag2celllinetoinvestigatetheproductionofsmallRNAsduringDENV
infection.Wefindthatinadditiontothewell-characterizedvsiRNAs,specificvpiRNAsare
producedfromDENV,whichfortheirbiogenesisinAag2cellsrelyonPiwi5andAgo3and,to
alesserextent,onPiwi6.WedonotdetectDENV-derivedmiRNAs,orprominentchangesin
hostmiRNAlevelsuponinfection.Finally,weidentifynovelhostmiRNAsinoursmallRNA
deep-sequencinglibraries,complementingthecurrentlyannotatedmiRNArepertoireinAedes
aegyptivectormosquitoes.
MaterialsandMethods
Cellsandviruses
Aag2cellswereculturedat25°CinLeibovitzL-15medium(Gibco)supplementedwith10%heat
inactivatedfetalcalfserum(FCS;PAA),2%tryptosephosphatebrothsolution(Sigma),1xMEM
non-essentialaminoacids(Gibco),and50U/mlpenicillinand50μg/mlstreptomycin(pen/
strep;Gibco).U4.4andC6/36werekeptinthesameculturemediumat28°C.BHK-21cellswere
culturedat37°C,5%CO inDulbecco’smodifiedEaglesmedium(DMEM)supplementedwith
2
10%FCSandpen/strep.StocksofDENVserotype2(DENV2),NewGuineaC(NGC)and
16681strainsweregrownonC6/36cellsandtitredonBHK-15cellsasdetailedin[40].
InfectionofAag2cellswithDENV2
Aag2cellswereseededonedaypriortoinfectionandinfectedwithDENV2atamultiplicityof
infection(MOI)of0.5bydirectlyaddingthevirustotheculturemedium.Threedayspost
infectiontheculturemediumwasremovedandcellswereharvestedforRNAandproteinisola-
tionasdetailedbelow.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 3/22
SmallRNAsinDENV2-InfectedMosquitoCells
Westernblot
ForthedetectionoftheDENVNS1proteininsamplesusedforsmallRNAdeep-sequencing,
5%ofthecellswereharvestedin50μllysisbuffer(50mMTris-HClpH7.8;150mMNaCl;1
mMEDTA;0.5%NP-40;1xProteaseinhibitorcocktail(Roche);1mMDTT).12.5μlof5x
Laemmlibufferwasaddedtoeachsample,incubatedat95°Cfor5min,and30μlofeachsam-
plewasloadedona12.5%polyacrylamidegel.Aftergelelectrophoresis,proteinsweretrans-
ferredtoanitrocellulosemembrane(Bio-Rad)usingasemi-dryblottingsystem(Bio-Rad).
Themembranewasblockedin5%non-fatdrymilk(Bio-Rad)in0.1%Tween20inPBS
(PBS-T)for30minatroomtemperature.MouseantiDENVNS1antibodywaskindlypro-
videdbyDr.PeterMason[41].Theantibodywasaddedtothemembraneina1:1,000dilution
in5%blockingbuffer.Afteranincubationfor1.5hoursatroomtemperature,themembrane
waswashedthreetimesinPBS-T.IRdye680conjugatedgoatantimouseantibody(1:15,000
dilutioninPBS-T;Licor)wasthenaddedtothemembraneandincubatedatroomtemperature
for1.5hours.Afterthreewashingsteps,themembranewasimagedonanOdysseyinfrared
imagesystem(Licor).
dsRNAproductionandtransfectionofAag2cells
dsRNAstargetingPIWI/AGOtranscriptsorluciferaseasanegativecontrolwereproducedby
invitrotranscriptionfromT7-promoterflankedPCRproductsasdetailedin[37].Primersto
produceT7-flankedPCRproductsareindicatedinS1Table.
FordsRNAtransfection,7.5x105Aag2cellswereseededinonewellofa24-wellplate.For
eachcondition,threewellswereplated.Thefollowingday,transfectionmixescontaining
300μlnon-supplementedL-15medium,450ngdsRNAand1.8μlX-tremeGENEHP(Roche)
werepreparedaccordingtothemanufacturer’srecommendations.100μlofthemixwasadded
dropwisetoonewell.After2–3hoursthemediumwasreplacedwithfullysupplementedL-15
medium.48hourslater,thetransfectionwasrepeatedtoenhanceknockdownefficiencies.
Whereindicated,thecellswereinfectedwithDENV2whichwasaddedtotheL-15medium
usedtoreplacethetransfectionmediumasspecifiedabove.
RNAisolation
Aag2cellswerelysedinIsol-RNALysisreagent(5PRIME)asdescribedinthemanufacturer’s
instructions.Briefly,200μlofchloroformwasaddedto1mlofLysisreagentandmixedwell.
Aftercentrifugation,theaqueousphasewascollectedandtotalRNAwaspurifiedusingisopro-
panolprecipitation.RNAwasquantifiedonaNanodropspectrophotometerandRNAintegrity
wascheckedbyethidiumbromidestainingofribosomalRNAbandsafteragarosegel
electrophoresis.
DNaseItreatment,reversetranscriptionand(quantitative)PCR
ForRT-(q)PCR,1μgoftotalRNAwasDNaseI(Ambion)treatedaccordingtothemanufactur-
er’sinstructions.TheRNAwassubsequentlyreversetranscribedina20μlreactionusingthe
Taqmanreversetranscriptionkit(AppliedBiosystems).ComplementaryDNA(cDNA)was
diluted5–10timesbeforePCRamplification.EndpointPCRwasperformedusingThermoper-
fectDNAPolymerase.QuantitativePCR(qPCR)analysiswasperformedusingtheGoTaq
qPCRSYBRmastermix(Promega)onaLightCycler480instrument(Roche).Therelative
changesingeneexpressionwerecalculatedusingtheΔΔCtmethod[42]usinglysosomalaspar-
ticprotease(LAP)asaninternalnormalizationcontrol.SequencesofthePCRprimersareindi-
catedinS1Table.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 4/22
SmallRNAsinDENV2-InfectedMosquitoCells
β-elimination
Sodiumperiodate(NaIO )oxidationandβ-eliminationoftotalRNAwasperformedas
4
describedpreviously[43].TotalRNA(10μgin47.5μlnuclease-freewater)wasmixedwith
12.5μl200mMNaIO and40μl5xboratebuffer.Asacontrol,RNAwastreatedwithwater
4
insteadofNaIO .Thereactionwasincubatedatroomtemperaturefor30minand10μlglyc-
4
erolwasaddedtothereaction.Thereactionwasincubatedforanother10minbefore10μlof
500mMsodiumhydroxide(NaOH)wasaddedtoinduceβ-elimination.Thereactionwas
incubatedat45°Cfor90min.Afterthesetreatments,totalRNAwaspurifiedbyethanolprecip-
itationinthepresenceof300mMNaCland5μgofglycogen.Electrophoreticmobilityof
AedesaegyptimiR-2940-3pandDENV2piRNAswasthenanalyzedbysmallRNAnorthern
blottingasdetailedbelow.
SmallRNAnorthernblotting
SmallRNAnorthernblotwasperformedasdescribedin[44].Briefly,totalRNAwassize-sepa-
ratedon0.5xTBE,7MUrea,15%Polyacrylamidegels,transferredtoHybondNXnylonmem-
branes(Amersham),andcross-linkedusing1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC;Sigma).IndividualsmallRNAsweredetectedwithDNAoligonucleotidesthatwere5’
end-labelledwith[32P]γ-adenosine-triphosphate(PerkingElmer)usingT4Polynucleotide
kinase(Roche).Hybridizationtotheoligo-probeswasperformedovernightat42°CinUltra-
hybOligohybridizationbuffer(Ambion).Membraneswerethenwashedthreetimesat42°Cin
0.1%SDSwithdecreasingconcentrationsofSSC(2x,1x,0.1x).MembraneswereexposedtoX-
rayfilms(Carestream)orPhosphorimagerscreens(BioRad).SequencesofDNAoligonucleo-
tideprobesareindicatedinS1Table.Quantificationofnorthernblotpanelswasperformed
usingImageJsoftware.Bandsweredefinedusingtherectangularselectiontoolandthepixel
density(areaunderthecurve)wasmeasuredandnormalizedtouninfecteddsLucsamples.
PreparationofsmallRNAlibraries
SmallRNAlibrarieswerepreparedasdescribedpreviously[37,45].Briefly,three25cm2flasks
ofAag2cellswereinfectedinparallelwithDENV2NGC.Threeadditionalflaskswereleft
uninfected.TotalRNAwasthenisolatedfromthesesixflasksasspecifiedaboveand30μgof
totalRNAwassizeseparatedona15%Polyacrylamide,7Murea,0.5xTBEgel.Subsequently,
thesmallRNAsinthesizerangefrom18ntto33ntwereexcisedfromgelusingradioactively-
labelledRNAoligos,loadedintheadjacentlanesofthegel,asrulers.Thegelwascrushedand
thesmallRNAswereelutedin300mMsodiumacetateovernightat4°Cunderconstantrota-
tion.TheRNAwasrecoveredfromtheelutionbufferusingethanolprecipitationandelutedin
10μlofnuclease-freewater.5μlofthesamplewasdirectlyusedasinputforsmallRNAdeep-
sequencinglibrarypreparationusingtheTruSeqsmallRNAlibrarypreparationkit(Illumina)
followingthemanufacturer’srecommendations.TheRNAwasligatedto3’and5’adapters,
reversetranscribed,andPCRamplified.ThesmallRNAlibrarieswerethensize-purifiedfrom
1xTBE,6%polyacrylamidegelusingovernightelutionin300mMsodiumacetatefollowedby
ethanolprecipitation.TheindividualsmallRNAlibrarieswerepooledandsequencedonasin-
glesequencinglaneonaHighSeq2500byBaseclear(Leiden,TheNetherlands).
ViralsmallRNAprofiling
FASTQsequencereadsweregeneratedusingtheCasavapipeline(v.1.8.3)andinitialquality
analysiswasperformedusingtheIlluminaChastityfilterandanin-housefilteringprotocolby
Baseclear.SubsequentqualityassessmentwasbasedontheFASTQCqualitycontroltool
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 5/22
SmallRNAsinDENV2-InfectedMosquitoCells
(v.1.10.0).TheindividualsmallRNAsequencinglibrarieswereseparatedbasedontheTruSeq
indices(no.1to6)thatwereintroducedduringPCRamplification.Theindividuallibraries
weresubsequentlyanalyzedusingtheGalaxybioinformaticstoolshed[46,47].Fortheanalysis
ofviralsmallRNAs,readsweremappedtotheDENV2NGCgenome(GenBankaccession:
KM204118.1)usingBowtie(v.1.1.2)[48].Sizeprofileswereobtainedfromallreadsthatalign
tothisreferencesequencewithamaximumofonemismatch.Thegenomedistributionof21nt
siRNAs,22–24ntsmallRNAs,or25–30ntpiRNAswasobtainedbyplottingthenumberof5’
endsofthesereadsateachpositionofthegenome.ForthepileupplotsofUTR-derived
miRNA-likesmallRNAs,the22–24ntsmallRNAswereselectedfromtheinitialFASTQfiles
andmappedtotheDENV-NGCgenome.FromtheresultingSAMfilesthereadsmappingto
the(+)strandofthevirusgenomewereselectedandusedasinputforthe‘Generatepileup
fromBAMdataset’tool(v.1.1.2).ThevaluesatthenucleotidepositionsoftheDENV25’UTR
(1–96)and3’UTR(10273–10723)wereselectedfordisplay.
Re-analysisofthedatapublishedbyHessetal.[36]wasperformedonthedatasetwiththe
accessionnumberSRR921363.TheSOLiD-formatteddatasetwasgroomedtofittherequire-
mentsformanipulationinGalaxy.Afteradapterclipping,readsweremappedtotheDENV2--
JAM1409genome(GenBankaccession:M20558)usingBowtie2[49].Sizeandgenomeprofiles
wereobtainedasdescribedabove.Unlessspecifieddifferently,allsmallRNAreadcountswere
normalizedagainstthesizeofthecorrespondingsequencinglibraryandareexpressedas‘%of
thelibrary’(i.e.readsperhundred).
miRNAanalysisandprediction
AnalysisofmiRNAexpressionlevelswasperformedusingthemiRDeep2tool.Rawdatawere
assessedforqualityusingFASTQC.Subsequently,adapterswereremovedfromtherawreads,
andthereadswerequalitytrimmedusingcutadaptsoftware(http://dx.doi.org/10.14806/ej.17.1.
200)withparameters-O6-m17-n5-q20.Withineachlibrary,theresultingreadswerecol-
lapsedtogenerateanon-redundantsetofFASTAsequences,subsequentlyprocessedtothefor-
matrequiredformiRNApredictionwithmiRDeep2software[50].Collapsedreadslongerthan
17nucleotideswerealignedtotheAedesgenome(assemblyAaegL3,downloadedfromvector-
base)andtheDENV2NGCgenomeusingthemapper.plcomponentofmiRDeep2(parameters:
-o20-l19-r100-c).Theresultingoutputswereparsedtoremovealignmentsthatwerenotfull
lengthandperfectmatch(FLPM).miRDeep2predictionsweregeneratedfromtheFLPM
alignedsequences,withmiRBasev21ArthropodamaturemiRNAandpre-miRNAsequencesas
templates[51].The‘miRNAs_expressed’outputfrommiRDeep2,whichcomprisestalliesfor
eachknownmiRNAineachsample,wasfurtherprocessedintheR/Bioconductorenvironment.
Briefly,miRNAreadcountswerenormalizedtothenumberofAedes-specificgenomereads
withineachsamplegroup,usingthelowestnumberofreadsaligningasthebaseline.Subse-
quently,thecountswereconvertedtoabundanceswithineachsample,convertedtolog2equiva-
lentcounts,andallsamplesquantilenormalizedpriortolinearmodelfittingwiththelimma
package[52].MiRNApredictionsfromthemiRDeep2outputweremanuallycuratedusingthe
followingcriteria:i)high-confidencemiRNApredictionshavereadsmappingtobothapre-
dictedmatureandstarsequence,ii)thematuresequenceshaveahomogenous5’end(80%of
(cid:1)
readsstartatsameposition),andiii)miRNA-miRNA duplexshouldresembleaDicerproduct
onagenomically-encodedhairpin,havingatwont(+/-1nt)overhangatthe3’end.miRNA
predictionssupportedby>1000readsaswellasmiRNApredictionswithaseedmatchto
knowninsectmiRNAswerealsokept.Toberetained,thesepredictionsrequiredahomogenous
5’end,butdidnotrequirethepresenceofreadsmappingtotheexpectedstarstrand.miRNA
namesandaccessionnumberswereassignedbythemiRBaserepository.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 6/22
SmallRNAsinDENV2-InfectedMosquitoCells
AlldeepsequencinglibrarieshavebeensubmittedtoNCBISequenceReadArchiveunder
theaccessionnumberSRA303329.AllsourcedataareavailableinS1Dataset.
Results
DENV2-derivedsmallRNAsininfectedAag2cells
DENVisapositive(+)strandRNAvirusbelongingtotheFlavivirusgenusintheFlaviviridae
family.ItsRNAgenomeisapproximately10.7kilobasesinsizeandencodesasinglepolypep-
tidethatisprocessedbyproteolyticcleavageeventsintothreestructuralproteinsandseven
non-structuralproteins(Fig1A).SincevariousclassesofsmallRNAshavebeenimplicatedin
modulatingDENVinfectionsinitsmosquitovectors,weaimedtocharacterizetherepertoire
ofvirusandhost-derivedsmallRNAsinAedesaegyptiAag2cells.Tothisend,weprepared
threeindependentsmallRNAdeep-sequencinglibrariesfromuninfectedandDENV2(NGC
strain)infectedcells,each.Theefficiencyofthethreeinfectionswascomparableasassessedby
westernblotfortheDENV2NS1protein(Fig1B).Asexpected,DENV2-derivedviralsmall
RNAsshowedaclearpopulationof21ntvsiRNAsmappingtoboththeviralpositive(+)
Fig1.SmallRNAproductioninDENVinfectedAag2cells.(A)SchematicrepresentationoftheDENV
NGCgenome(accessionKM204118;10723bp).Structuralproteinsareindicatedingreyscale,non-
structuralproteinsaredisplayedinbluetogreenscale.(B)WesternblotagainsttheDENV2NS1proteinin
thethreeinfectedanduninfectedsamplesusedforsmallRNAlibrarypreparation.(C)Sizeprofileofsmall
RNAsmappingtotheDENV2genomewithamaximumofonemismatch.Blackbarsrepresentreads
mappingtothe(+)strandofthegenome,greybarsdepictreadsfromthe(-)strand.Thereadcountshave
beennormalizedtothesizeofthesmallRNAlibraryandthemean+/-standarderrorofthemean(SEM)is
presented(n=3).(D)Distributionof21ntvsiRNAs(leftpanel)or25–30ntsmallRNAs(rightpanel)across
theDENVgenome.Readsfromthe(+)and(-)strandsaredepictedinredandblue,respectively.Theread
countshavebeennormalizedasdescribedinC,themeanreadcountofthethreelibrariesisshown.
NumbersinredindicategenomepositionsofthevpiRNAspikes.
doi:10.1371/journal.pntd.0004452.g001
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 7/22
SmallRNAsinDENV2-InfectedMosquitoCells
strandandthenegative(-)strandinroughlyequalnumbers(Fig1C).Interestingly,besides
siRNAs,asecondpopulationofviralsmallRNAswasproducedthatresembledvpiRNAs.
Thesewere25–30ntinlengthandalmostexclusivelyderivedfromtheviral(+)strand(Fig
1C).IncontrasttovsiRNA,whichweredistributedalongtheentirelengthoftheviralgenome,
theseputativevpiRNAswereproducedonlyfromfewspecificpositions(Fig1D).Infact,85%
ofallthe25–30ntreadswerederivedfromfourindividualvpiRNAsequences,presentinthe
NS5geneatpositions9180and9985,9989and9990oftheDENV2NGCgenome.Totest
whetherthesesmallRNAprofilesreflectthosefromadultmosquitoes,were-analyzeddeep
sequencingdatafromDENV2infectedAedesaegyptimosquitoespublishedbyHessetal.[36].
Weanalyzedthe9dayspostinfectionsample,whichshowedthehighestnumberofviralsiR-
NAsandpiRNAs.WhereasnormalizedvsiRNAlevelswereonly2.2-foldlowerintheselibrar-
iesthaninourAag2data,vpiRNAswereaboutfortytimeslower.Yet,theviralsmallRNA
profileswerestrikinglysimilar,with21ntreadsbeingscatteredthroughouttheentireviral
genomeandpiRNA-sizedreadsbeingpredominantlyproducedfromfewpositionslocated
towardsthe3’endoftheviralgenome(S1Fig).Thesedatasuggestthatsimilarmechanisms
mightproduceviralpiRNAsinAag2cellsandadultmosquitoes.
vpiRNAproductionfromDENV2RNA
ToexcludethepossibilitythatthepiRNA-likemoleculesaresequencingartefactsandtochar-
acterizethissmallRNApopulationinmoredetail,weperformedsmallRNAnorthernblotting
forthehighly-abundantsmallRNAsstartingatDENV2genomepositions9180or9985–9990.
Indeed,smallRNAsintheexpectedsizerangecouldreadilybedetectedspecificallyinDENV2
infectedAag2cells(Fig2A).Inaddition,thesesequenceswerealsopresentinDENV2-infected
U4.4andC6/36cellsderivedfromAedesalbopictusmosquitoes,whichwehavepreviously
showntobecompetentinproducingSINV-derivedvpiRNAs[32,37](Fig2B).Thelevelsof
vpiRNAsdidnotcorrelatewiththeexpressionofviralgenomicRNA.WhereasviralRNAlev-
elswereroughlyeighteentonineteenfoldhigherinU4.4andC6/36cellsthaninAag2cells,
vpiRNAsweremostabundantinAag2cells(Fig2B).Thesedatasuggestthatthecomposition
ofhostfactorsrequiredfortheirbiogenesisismostfavourableinAag2cells.Asexpected,mam-
malianBHK-21cells,whichlackanactivepiRNApathway,didnotproducevpiRNAs(Fig2B).
ToexcludethatviralpiRNAproductionisanartefactoftheuseofthespecificDENV2strain,
weanalyzedpiRNAaccumulationinAag2cellsinfectedwitheithertheDENVNGCorDENV
16681strain.SmallRNAnorthernblottingrevealedthatinfectionwitheitherstrainresultedin
theproductionofthoseviralpiRNAsequencesthatwehadfoundbydeep-sequencing(Fig
2C).Next,weaimedtotestwhetherDENV2-derivedpiRNAsaremethylatedattheir3’end.
Tothisend,weperformedsodiumperiodateoxidationfollowedbybeta-elimination,which
revealsmodificationsofthe3’terminalnucleotideofRNAmolecules[43].Unmodifiedsmall
RNAs,suchasanimalmiRNAs,aresusceptibletothistreatmentandwillbeshortenedbyone
nucleosideresultinginincreasedelectrophoreticmobility.IncontrasttomiRNAs,piRNAsare
protectedagainstthistreatmentbymethylationofthe2’OHontheriboseofthe3’terminal
nucleotide.Indeed,beta-eliminationresultedinincreasedelectricmobilityofmiR-2940-3p.
Yet,themigrationofDENV2piRNAbandswasnotaffectedbybeta-elimination,indicating
thattheir3’terminalnucleotidesaremodified,mostlikelymethylated(Fig2D).SincepiRNA
methylationoccursafterloadingintoPIWIproteincomplexes,thesedatasuggestthattheiden-
tifiedDENV2piRNAsarematurepiRNAsassociatedwithaPIWIprotein.
ToidentifywhichPIWIproteinsarerequiredforthebiogenesisofDENV2piRNAsinAag2
cells,weindividuallyknockeddownexpressionofalltheeightAedesPIWIproteinsandana-
lyzedtheproductionofvpiRNAsbynorthernblot.Weconfirmedknockdownefficiencyof
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 8/22
SmallRNAsinDENV2-InfectedMosquitoCells
Fig2.vpiRNAproductioninAag2cells.(A)NorthernblotofhighlyabundantvpiRNAs.TwoindividualDNAoligonucleotideprobes(leftpanels),ora
combinationoftheseprobes(rightpanel)wereusedtodetectthesmallRNAs.ThecombinationofprobeswasusedinallsubsequentsmallRNAblots.(B)
Upperpanel:SmallRNAnorthernblotofvpiRNAsintheindicatedcelllinesafterinfectionwithDENV2.Lowerpanel:RT-PCRforDENVgenomicRNAinthe
samesamplesusedforthenorthernblot.NumbersontopindicateDENVgenomicRNAlevels(relativetoAag2cells)asdeterminedbyRT-qPCR(n=1).(C)
NorthernblotofDENV2piRNAsinAag2cellsinfectedwiththeDENV2NGCor16681strain,bothatanMOIof0.5.(D)NorthernblotofDENV2piRNAsand
AedesmiR-2940-3pinuninfectedorDENV2infectedAag2cells.Whereindicated,totalRNAwassubjectedtoβ-elimination.(E)RT-qPCRfortheindicated
PIWIproteinsaftergene-specificknockdown(KD)inAag2cellsnormalizedtoacontrolKD(dsLuc).Barsrepresentthemeanofthreeexperiments+/-SEM.
Statisticalsignificancewasdeterminedusingtwotailed,unpairedstudentt-test.*p<0.05;**p<0.01.(F)Upperpanel:SmallRNAnorthernblotofvpiRNAs
uponKDoftheindicatedPIWIproteins.RNAsamplesanalyzedinEwerepooledforthisblot.Lowerpanel:Quantificationoftwoindependentblotsincluding
theoneshownintheupperpanelusingImageJsoftware.Fortheotherblot,seeS1Dataset.EthidiumbromidestainingofribosomalRNAwasusedas
loadingcontrolinpanelA,B,C,andF.
doi:10.1371/journal.pntd.0004452.g002
roughly90%forthefourPIWIproteinsthataredetectablebyRT-qPCRinAag2cells(i.e.
Piwi4,Piwi5,Piwi6,Ago3;Fig2E).ExpressionlevelsofPiwi1-3andPiwi7weretoolowto
allowreliablequantification.DENV2piRNAswerealmostundetectableuponknockdownof
Piwi5andAgo3andclearlyreduceduponknockdownofPiwi6(Fig2F).Thesedataconfirm
thatthe25–30ntpopulationofDENV2-derivedsmallRNAsarebonafidepiRNAsthatrequire
hostPIWIproteinsfortheirbiogenesis.TotestwhetherknockdownofPIWIexpressionresults
inenhancedDENV2replication,weperformedRT-qPCRtocompareviralRNAlevelsinthe
differentknockdownconditions.Wefoundthatnoneoftheknockdownsresultedinasignifi-
cantchangeinviralRNAlevels(S2AFig).Yet,alsoknockdownofthewell-establishedantiviral
factorAgo2[53]onlyresultedinaminor,statisticallynot-significant,increaseofviralRNA
replicationalthoughknockdownefficiencywashigherthan90%(S2BFig).Thissuggeststhat,
inourhands,knockdownofsmallsilencingpathwaycomponentsinAag2cellsisnotsuitedto
uncoverrobustantiviralactivityagainstDENV2.
DENV2miRNA-likesmallRNAsarenotexpressedinAag2cells
ThesignificanceofviralmiRNAproductionfromDENVgenomicRNAisheavilydebated
[24–26,54].ToinvestigatewhetherviralmiRNA-likemoleculesareproducedinDENV2
infectedAag2cells,wefiltered22–24ntsmallRNAreadsthatmaptotheDENV2genome
withamaximumofonemismatch.Ingeneral,thenumberof22to24ntreadswasratherlow
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 9/22
SmallRNAsinDENV2-InfectedMosquitoCells
Fig3.DENVmiRNA-likesmallRNAsarenotproducedininfectedAag2cells.(A)Distributionof22–24ntRNAreadsacrosstheDENVgenome.Red
barsindicatethenumberof5’endsofsmallRNAsthatmaptothe(+)strandofthegenome,bluebarsrepresentthesmallRNAsmappingtothe(-)strand.
Readcountswerenormalizedtothesizeofthecorrespondinglibraryandthemeanofthethreelibrariesisplotted.(B)Pile-upof22–24ntsmallRNAs
mappingtothe(+)strandofDENV25’(leftpanel)and3’UTRs(rightpanel),respectively.Themeanofthethreelibrariesisshown.Greyshadingshighlight
theboundariesofthematureDENV2vsRNAsequencesasreportedin[24].
doi:10.1371/journal.pntd.0004452.g003
(~5%ofallDENV2mappingreads)whencomparedto21ntsiRNAs(~28%)and25-30ntpiR-
NAs(~40%).Furthermore,therewereonlyfouroutstandingpeaksthatgaverisetoasome-
whathighernumberofsmallRNAs(Fig3A).Allofthemcoincidedwiththepositionofa
vpiRNApeaks(Fig1D),suggestingthatthesesmallRNAswereby-productsofvpiRNApro-
duction.Parallelanalysisofthevirus-derivedreadsusingmiRDeep2didnotidentifyconvinc-
ingmiRNA-likecandidates:somereadsmappedtotwopredictedhairpinsequences(genome
positions:9542(+)strand;4888(-)strand),howeverthemappingpatternsshowedheterogene-
ityofthe5’startsitesanddidnotsuggestDicerprocessing(S3AFig).
Recently,eightmiRNA-likesmallRNAswerecomputationallypredictedbasedonhairpin
structuresintheDENV2genome,buttheywerenotexperimentallyvalidated[54].Wespecifi-
callylookedforsmallRNAreadsinoursequencingdatamappingintheproximityofthese
predictedviralmiRNAs,allowingamarginof3ntaroundthestartsite.Foreachofthepre-
dictedmiRNAs,weidentifiedonlyveryfew(<20)readsinthecombinedsetofDENV2-in-
fectedsmallRNAlibraries(totalof>3.7(cid:3)107readsofwhich>3.6(cid:3)105areDENVspecific).In
anotherpublication,several‘miRNA-like’RNAs(termedvsRNA-1to6)wereproposedtobe
producedfromthe5’and3’UTRsoftheDENV2RNA,basedontheanalysisofsmallRNA
sequencingdata[24].TheDENVUTRsareindeedpronetoformRNAstructuresandhairpins,
whichweresuggestedtobeprocessedbythemiRNAmachineryintospecificsmallRNAspe-
cies[24].WespecificallylookedfortheproposedvsRNAsequencesinourdatasetbutcould
onlyidentifyasmallRNApopulationthatresembledvsRNA-2locatedattheterminalhairpin
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004452 February25,2016 10/22