Nucleobases and corresponding nucleosides display potent antiviral activities against dengue

18 Pages·2017·3.21 MB·English

RESEARCHARTICLE
Nucleobases and corresponding nucleosides
display potent antiviral activities against
dengue virus possibly through viral lethal
mutagenesis
LiQiu,StevenE.Patterson,LaurentF.Bonnac*,RobertJ.Geraghty*
CenterforDrugDesign,AcademicHealthCenter,UniversityofMinnesota,Minneapolis,Minnesota,United
a1111111111 StatesofAmerica
a1111111111
a1111111111 *[email protected](LFB);[email protected](RJG)
a1111111111
a1111111111
Abstract
Denguevirusaffectsmillionsofpeopleworldwideeachyear.Todate,thereisnodrugfor
thetreatmentofdengue-associateddisease.Nucleosidesareeffectiveantiviralsandwork
OPENACCESS
byinhibitingtheaccuratereplicationoftheviralgenome.Nucleobasesofferacheaperalter-
Citation:QiuL,PattersonSE,BonnacLF,Geraghty
nativetonucleosidesforbroadantiviralapplications.Metabolicactivationofnucleobases
RJ(2018)Nucleobasesandcorresponding
involvescondensationwith5-phosphoribosyl-1-pyrophosphatetogivethecorresponding
nucleosidesdisplaypotentantiviralactivities
againstdengueviruspossiblythroughvirallethal nucleoside-5’-monophosphate.Thiscouldprovideanalternativetophosphorylationofa
mutagenesis.PLoSNeglTropDis12(4): nucleoside,astepthatisoftenratelimitingandinefficientinactivationofnucleosides.We
e0006421.https://doi.org/10.1371/journal.
evaluatedmorethan30nucleobasesandcorrespondingnucleosidesfortheirantiviralactiv-
pntd.0006421
ityagainstdenguevirus.Fivenucleobasesandtwonucleosideswerefoundtoinducepotent
Editor:AdlyM.M.Abd-Alla,InternationalAtomic
antiviraleffectsnotpreviouslydescribed.Ourstudiesfurtherrevealedthatnucleobases
EnergyAgency,AUSTRIA
wereusuallymoreactivewithabettertissueculturetherapeuticindexthantheircorrespond-
Received:October26,2017
ingnucleosides.Thedevelopmentofvirallethalmutagenesis,anantiviralapproachthat
Accepted:March31,2018 takesintoaccountthequasispeciesbehaviorofRNAviruses,representsanexcitingpros-
Published:April19,2018 pectnotyetstudiedinthecontextofdenguereplication.Passageofthevirusinthepres-
enceofthenucleobase3a(T-1105)andcorrespondingnucleoside3b(T-1106),favipiravir
Copyright:©2018Qiuetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative derivatives,inducedanincreaseinapparentmutations,indicatinglethalmutagenesisasa
CommonsAttributionLicense,whichpermits possibleantiviralmechanism.Amoreconcertedandwidespreadscreeningofnucleobase
unrestricteduse,distribution,andreproductionin
librariesisaverypromisingapproachtoidentifydenguevirusinhibitorsincludingthosethat
anymedium,providedtheoriginalauthorand
mayactasviralmutagens.
sourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Authorsummary
Funding:ThisworkwasfundedbytheCenterfor
DrugDesign(LQ,SEP,LFB,RJG).Thefundershad
Denguevirusisaworld-widepublichealthmenaceestimatedtoinfecthundredsofmil-
noroleinthestudydesign,datacollectionand
lionsofpeopleperyear.Vaccinestopreventdenguevirusinfectionhavehadlimitedsuc-
analysis,decisiontopublish,orpreparationofthe
cessdueinparttotherequirementtoeliciteffectiveimmuneresponsesagainstthefour
manuscript.
dengueserotypes.Thereisanurgentunmetneedforanti-denguevirustherapies.Nucleo-
Competinginterests:TheAuthorshavedeclared
sidesareeffectiveantiviralsmallmoleculeswhichusuallyworkbyinhibitingtheaccurate
nocompetinginterestsexist.
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Nucleobasesandnucleosidesasanti-denguevirusmutagens
replicationoftheviralgenome.Typically,nucleosidesmustbeconvertedwithinthecellto
theirtriphosphateformtoinhibitvirusreplication,thusinefficientphosphorylationoften
leadstosuboptimalactivity.Wescreenedasmalllibraryofnucleobasesthatrequirean
activationpathwaydifferentfromnucleosidestoachievethesameactiveform.Weidenti-
fiedsomeknownandpreviouslyundescribeddenguevirusnucleobaseinhibitorsand
theircorrespondingnucleosides.Ourinvestigationofthemechanismofactionofone
nucleobaseanditscorrespondingnucleosidefoundevidenceforenhancedmutagenesisof
thedenguevirusgenomeinthepresenceofthecompoundsincellculture.Awidescreen-
ingofnucleobaseslibrariesisapromisingstrategytodiscoverdenguevirusinhibitors
includingpotentialviralmutagens.
Introduction
Denguevirus(DENV)isaworldwidehealththreat,withhundredsofmillionsofpeople
infectedyearlyinmorethan100countries[1].TherearefourknownDENVserotypesanda
firstinfectionwithoneserotypefollowedbyasecondinfectionwithanotherserotypemay
resultinseveredisease[2,3].Fortheseandotherissues,vaccinesdesignedforapan-serotype
protection,includingthecommercialdenguevaccineapprovedandusedinafewcountries,
haveyieldedmixedresults[4,5].Safetyandpartialefficacyconcernsinadditiontocost,stor-
ageanddeliveryissuesmayhinderimplementationofvaccinesinmanycountries.
TherearecurrentlynoapproveddrugstotreatDENVinfection.Thusfar,classicalantiviral
approaches(e.g.NS5polymeraseinhibitors,entryinhibitors,proteaseinhibitors,etc.)haveyet
toprovidetreatmentsforDENVinfectionandthereforetheinvestigationofnewantiviral
strategiesiswarranted[6–8].Onesuchstrategytoexploreislethalmutagenesis[9].Theidea
ofvirallethalmutagenesisistoexploitthenaturaltendencyofRNAvirusestomutateinorder
tofavortheaccumulationofdeleteriousmutationsinthenewlyformedviruses,eventually
leadingtoviralextinction(forreviewsee[10]).DENVandotherRNAvirusesdisplayahigh
mutationrate(10−4to10−6mutationsperbppergeneration)[11,12]asanevolutionarychar-
acteristicallowingthesevirusestoescapehostimmunedefensemechanismsandadaptrapidly
tonewstressconditions[13,14].Anerror-proneviralpolymerasecombinedwithahighrepli-
cationrateareconsideredtobethemainsourcesofmutations.Itisthiscriticalsourceofviral
adaptability(e.g.thevirushighmutationrate)thatmakesRNAvirusesatargetofchoicefor
antivirallethalmutagenesisstrategies[15–17].RNAvirusesmaintainadelicatebalance
betweentheirneedtoadaptandtheirneedtopreservealevelofgeneticintegrityputatriskby
deleteriousmutations[18].Modifyingthisfragileequilibriumbyincreasingtheviralmutation
ratewithmutagenshasbeenproposedasanantiviralstrategy[15].Thewell-knownantiviral
nucleosidedrugribavirininduceslethalmutagenesisfordifferentviruses[19–22].
Thediscoveryofnewnucleosidesasantiviralmutagenshasbeenimpairedbyseveralhur-
dlesincludingthetoxicityofthepotentialdrugsaswellassyntheticchallenges.Inaddition,
potentiallymutagenicnucleosideanaloguesfrequentlysufferfrompoormetabolicconversion
totheactivetriphosphateformrequiredbytheviralpolymerase.Thefirstphosphorylationof
thenucleosideanalogueisoftentheratelimitingsteptoobtaintheactivemutagenicnucleo-
sidetriphosphateusedbytheviralpolymerase[23,24].Inordertoovercomethepotentialfirst
phosphorylationdifficulty,weproposetousenucleobases(purineorpyrimidinebasewithout
theriboseorphosphatemoietiesofanucleoside).Enzymemediatedcondensationofnucleo-
baseswith5-phosphoribosyl-1-pyrophosphatetogivethecorrespondingnucleoside-5’-mono-
phosphatecanprovideanalternativepathway.Thus,foranucleosidewherethefirst
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Nucleobasesandnucleosidesasanti-denguevirusmutagens
phosphorylationisinefficientthenusingitscorrespondingnucleobasecouldallowmetabolic
conversiontothecorrespondingnucleosidetriphosphate,therebyprovidingamoreefficient
metabolicconversiontothetriphosphate.Studiesonnucleobaseshavebeenlimited[25],with
thenotableexceptionsof5-fluorouracilandT-705(favipiravir)[26–32].However,inthecon-
textoftargetingvirusesthataffectdevelopingcountries,mutagenicnucleobasespresentkey
advantagesovermutagenicnucleosides.Inadditiontotheirdifferentmetabolicactivation
pathways,nucleobaseanaloguesareconsiderablycheaper,morediverseandcommercially
availableinhighernumberscomparedtocorrespondingnucleosideanalogues.Thechemical
synthesisofanucleobaseisfasterandsimplerthanthesynthesisofthecorrespondingnucleo-
side.Similarlytonucleosides,nucleobasespossesstheirowncellulartransporters[33,34].
Inthisstudy,wedescribetheidentificationoffivenucleobasesandthreecorresponding
nucleosidesthatpossesspotentanti-DENVactivity.Thesecompoundshavenotbeenprevi-
ouslydescribedtohaveanti-DENVactivity,exceptforthenucleoside1b(ribavirin)[35,36].
Wecomparedtheantiviralactivitiesandtoxicitiesofthenucleobaseswiththeircorresponding
nucleosides.Forviruspassagedinthepresenceofanucleobase3a(T-1105)ornucleoside3b
(T-1106),wedetectedanincreaseinmutationscomparedtoviruspassagedinDMSOindicat-
ingapossiblereductioninvirustiterviaincreasedmutagenesis.Toourknowledge,ourstudy
isthefirsttofullycomparetheantiviralmechanismsandefficaciesofanucleobaseanditscor-
respondingnucleoside,highlightingthedifferences,similaritiesandpotentialadvantagesof
nucleobasesversusnucleosides.Ourstudyalsohighlightsthepotentialoflethalmutagenesis
inductionduringDENVreplicationasanalternativetoclassicalantiviralstrategies.
Methods
Compounds
5a(T-705)(CAS#259793-96-9,6-fluoro-3-hydroxypyrazine-2-carboxamide)waspurchased
fromASTATech.3a(T-1105)(CAS#55321-99-8,3-Hydroxy-2-pyrazinecarboxamide)was
purchasedfromAlfaAesar.3b(T-1106)wassynthesizedaccordingtoknownprocedures
(Preparationofnucleosideswithnon-naturalbasesasanti-viralagentsCan.Pat.Appl.(2006),
149pp.CODEN:CPXXEB;CA2600359).1a(ribavirinbase)(CAS#3641-08-5,1,2,4-Triazole-
3-carboxamide)waspurchasedfromArkPharm.1b(ribavirin)(CAS#36791-04-5)waspur-
chasedfromCarbosynth.2a(mizoribinebase)(CAS#56973-26-3,5-Hydroxy-1H-imidazole-
4-carboxamide)waspurchasedfromArkPharm.2b(mizoribine)(CAS#50924-49-7)waspur-
chasedfromCarbosynth.4a(diaminopurine)(CAS#1904-98-9,2,6-diaminopurine)waspur-
chasedfromSigma-Aldrich.4b(diaminopurineriboside)(CAS#2096-10-8,
2-Aminoadenosine)waspurchasedfromBerryandAssociates.6(mycophenolicacid)(CAS#
24280-93-1)waspurchasedfromSigma-Aldrich.
Celllinesandvirus
Thehepatocyte-derivedcellularcarcinomacelllineHuh-7[37]wasusedforDENVinfection
anddrugtreatment.TheAfricangreenmonkeykidneyVerocellline(ATCCCRL-81)was
usedtotiterDENVviaplaqueassay.ThebabyhamsterkidneycelllinecarryingaDENVsub-
genomicreplicon,BHKpD2-hRucPac-2ATG30[38](obtainedfromDr.M.Diamond,Wash-
ingtonUniversity,SchoolofMedicine),wasusedforDENVrepliconassay.Allcelllineswere
maintainedinDulbecco’smodifiedEagle’s(DME)mediumsupplementedwith10%fetal
bovineserum(FBS),100IUstreptomycin/penicillinpermland10μg/mLplasmocin(Invivo-
Gen)at37˚Cina5%CO incubator.DENVrepliconcellsweresupplementedwith3μg/mL
2
puromycin(LifeTechnologies).DENV-2stocksfromNewGuineaCstrain(ATCCVR-1584)
weregeneratedfromC6/36mosquitocellcultures(ATCCCRL-1660)growninMinimum
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EssentialMedium(MEM)supplementedwith10%FBS,1%non-essentialaminoacidsand1%
sodiumpyruvateat28˚Cwith5%CO .TheC6/36cellsonT-150flaskswereinoculatedwith
2
virusandthesupernatantharvestedaftercompletecytopathiceffects.Viralstocktiterswere
determinedbyplaqueassayonVerocells.
Cellviabilityassay
Thesensitivityofthecelllinestothecompoundswasexaminedusingthe3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)-basedtetra-
zoliumreductionCellTiter96AqueousNon-Radioactivecellproliferationassay(Promega
G5430).Thecompoundswereinitiallytestedat10and50μMfinalconcentrations.Eachplate
alsocontainedDMSOalone,mediumalone,andaninhibitorycompound,6.DENVreplicon
orHuh-7cellswereplatedatadensityof1,500or8×103cells,respectively,perwellin96-well
platescontaining100μlofculturemediumovernight.Compoundswereaddedtotriplicate
wellsinculturemediumandincubatedforanadditional72h.MTSreagentwasthenaddedto
eachwellandincubatedat37˚Cinahumidified5%CO atmosphere.Theplateswerereadat
2
varioustimepointsatawavelengthof490nmusingaMolecularDevicesM5eplatereader.
Meanvaluesoftriplicatewellsweredeterminedandcomparedtothemeanvalueforthewells
thatreceivedDMSOalone.Forcompoundsselectedfordose-responseexperiments,theCC
50
wasdeterminedbycomparingcellviabilityforeightserialdilutionsofthecompoundand
DMSOtreatedcellsusingGraphPadPrismsoftware.TheCC valuewasdefinedasthecom-
50
poundconcentrationresultingina50%reductionreadoutcomparedwiththeDMSO.
DENVrepliconassay
CompoundswereevaluatedforantiviralpropertiesusingBHKcellscontainingaDENV-2
viralreplicon.1.5×103replicon-containingcellsperwellwereplatedinwhiteopaque96-well
platesintheabsenceofantibioticselectionandthenextday,compoundsdissolvedinDMSO
wereaddedtotriplicatewellsinculturemedium.Thecompoundswereinitiallytestedat10
and50μMfinalconcentrationsandeachplatealsocontainedDMSOalone,mediumalone,
and6.Threedayslater,mediumwasreplacedwitha1:1000dilutionofViVi-RenLiveCell
Substrate(Promega)inDMEminusphenolredand10%FBS.Luminescencewasmeasured
withaMolecularDevicesM5eplatereader.Meanvaluesoftriplicatewellsweredetermined
andcomparedtothemeanvalueforthewellsthatreceivedDMSOalone.Forcompounds
selectedfordoseresponseexperiments,theconcentrationofcompoundthatreducedlucifer-
aseactivityby50%wasdefinedasthe50%effectiveconcentration(EC ).TheEC wasdeter-
50 50
minedbycomparingluciferaseactivityforeightserialdilutionsofthecompoundandDMSO
treatedcellsusingGraphPadPrismsoftware.
Titerreductionassay
Huh-7cellswereseededin12-wellplatesatadensityof4×105cellsperwellin1mLculture
medium.Thenextday,cellswerewashedandinoculatedwithDENVatamultiplicityofinfec-
tion(m.o.i.)of0.2in500μlinfectionmedium(MEMcontaining2%FBSand10mMHEPES).
Theinoculumwasremovedafter1h,cellswerewashedwithPBSandthenincubatedin1mL
MEM,2%FBS,1%pen/streppluscompoundfor72h.Viralsupernatantswereclarifiedby
centrifugationfor5minat1500×gandaliquotedandstoredat-80˚C.Viraltitersweredeter-
minedusingaplaqueassayonVerocells.Briefly,confluentVerocellmonolayersin24-well
plateswereincubatedat37˚Cfor1hwithduplicate300μlsamplesof10-foldserialdilutions
ofviralsupernatants.Thecellswerethenwashedtoremoveunboundviralparticlesandover-
laidwith500ulMEMcontaining1.3%methylcellulose,5%FBSand10mMHEPES.After5
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daysofincubationat37˚Cand5%CO ,cellswerewashedwithPBS,fixed,andstainedusing
2
1%Giemsa.Infectiousvirustiter(pfu/mL)wasdeterminedusingthefollowingformula:num-
berofplaques×dilutionfactor×(1/inoculationvolume).Theviraltiterwaspresentedasthe
meanofduplicatesamplesfromadilutionyieldingapproximately20–50plaquesperwell.
ViralRNAextraction,RT-PCRamplification,quantitativePCR,detection
ofviralgenomemutations
ForqPCRdeterminationofviralgenomecopynumber,viralRNAwasisolatedfrom140μLof
drug-treatedorDMSO-treatedinfectedculturesupernatantusingQIAampViralRNAmini
kit(Qiagen),followingmanufacturer’sprotocol.ViralRNAwasquantifiedusingtheTaqMan
RNA-to-C 1-StepqPCRKit(AppliedBiosystems).PrimersusedforqPCRwere5’-CAT-
T
GATGGGAAAAAGAGAGAAGAAGCT-3’(forward)and5’-GGCTCTGCTGCCTTTTGC-3’
(reverse)amplifyingaregionnumbering8928–8988inthegenome(numberingstartingfrom
thebeginningofgenome,accessionnumberKM204118).TheqPCRFAMprobesequenceis
5’-TTGCCGAACTCCCC-3’.Serial10-folddilutionsofplasmidcontainingtheNS5geneof
DENVwereusedtogenerateastandardcurveforthequantificationofviralRNAgenome
copynumberbasedoncyclethreshold(C )values.ThelimitofdetectionforNS5plasmid
T
dilutionswas30copies(S1Fig).One-wayANOVAwasperformedtodeterminestatisticalsig-
nificanceofmeangenomecopynumbersamongtreatmentsateachviruspassageandTukey’s
honestlysignificantdifference(HSD)usedtodeterminestatisticalsignificant(p<0.05)
betweenDMSOand3aor3bateachpassage.StatisticalanalysisviaGraphPadPrism5soft-
ware.ToobtainsequencedatafromviralRNAisolatedateachpassage,cDNAwasgenerated
viaM-MLVreversetranscriptaseandrandomhexamers(NewEnglandBiolabInc.)permanu-
facturer’sinstructions.Anapproximately1600-basefragmentcoveringmembraneprotein
(prM)andenvelopproteingene(E)ofDENV-2viralgenomewasamplifiedusingPfuUltraII
FusionHSDNApolymerase(Agilent)withprimersprMEfor(5’-AACTCAGAATTCTTCC
ATTTAACCACACGTAAC-3’)andprMErev(5’-AACTCAGAATTCTCCTTTCTTAAACC
AGTTGAG-3’).PCRproductswerepurifiedusingQiaquickPCRpurificationkit(Qiagen)
andthendigestedwithEcoRIandligatedintopcDNA3.1forsequencing.Sequenceforapprox-
imately40–50individualclonespersamplewasobtainedfromtheUniversityofMinnesota
GenomicsCenter.Sequenceswerealignedovera980-baseregionthathadadequatequality
sequencingreadsforallclones.Onlymutationspresentinboththeforwardandreversereads
ofaclonewerecounted.Allincidence-baseddeterminantsofmutationfrequency(Mfmin,
Mfmax,Mfe)werecalculatedasdescribed[39].Atwo-tailedMann-WhitneyUtest(Graph-
PadPrism5software)wasusedtodetermineiftherewerestatisticallysignificantdifferences
forthemeannumberofmutationsperclonebetweenDMSO-treatedandeachdrug-treated
viruspassage.
ConstructionofDENV-2NS5plasmid
TheDENV-2viralRNAwasisolatedfrom140μLoflabstockDENV-2usingQIAampViral
RNAminikit(Qiagen).cDNAscorrespondingtoviralRNAsweregeneratedwithrandom
hexamers(NewEnglandBiolabInc.).A2600-basefragmentofNS5cDNAwasamplified
usingPfuUltraIIFusionHSDNApolymerase(Agilent)withprimersNS5for(5’-GGCCAGT
GCCAAGCTTGAACTGGCAACATAGGAAGAACGC-3’)andNS5Rev(5’-CCGGGGAT
CCTCTAGACCACAGGACTCCTGCCTCTT-3’).PCRproductwerepurifiedusingQiaquick
PCRpurificationkitandinsertedintoaXbaIandHindIIIdigestedpUC18vectorusingIn-
FusionHDCloningKit(Clontech).Apositiveclonewasidentifiedandthenucleicacid
sequenceofNS5confirmedbysequencingattheUniversityofMinnesotaGenomicsCenter.
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Virusandcompoundpassage
ViruswaspassagedonHuh-7cellssupplementedwith200μM3a,500μM3borDMSO
(0.5%).Huh-7cellswereseededin12-wellplatesandinoculatedwithDENV-2asdescribed
forTiterreductionexperimentsaboveexceptanm.o.i.of0.01wasused.After3daysofcom-
poundtreatment,50μLoftheharvestedsupernatantwasusedtoinoculatefreshHuh-7cells
inthecontinuedpresenceofcompound.Thevirustiterintheharvestedsupernatantwas
determinedbyplatingten-foldserialdilutionsontosinglewellsofa24-wellplate.Thewells
werewashedandoverlaidasdescribedintheTiterreductionassayabove.Ifnoplaqueswere
obtainedinanyoftheharvestedsupernatantdilutions,theundilutedsupernatantwasused.If
plaqueswerenotdetectable,thevirustiterwasconsideredtobeatthelimitofdetection,1pla-
que(3.3pfu/mL).Supernatanttiterwasdeterminedfromasinglewellwheretherewere
approximately20–50plaqueswhenpossible.Thisexperimentwasrepeatedthreetimesatthe
compoundconcentrationslisted.
Results
Antiviralnucleosideidentificationcanbehinderedbydifficultiesinchemicalsynthesisand
poorconversionofthenucleosidetotheactivetriphosphateform.Tocircumventtheseissues,
weproposetousenucleobasesinourinitialscreenforantiviralagentsbecauseoftheirdiffer-
entactivationpathwaytotheactivenucleotide(Fig1),theirlowcostandreadycommercial
availability.Phosphoribosyltransferasesofthecellularnucleotidesalvagepathwaydirectly
convertsomenucleobasestothecorrespondingnucleosidemonophosphateandthereforethe
correspondingnucleosideanalogueneednotbeanefficientsubstrateforanucleosidekinase
(Fig1)[40,41].Inthatregard,3aandanalogue5a(Fig2)aresubstratesofhumanphosphori-
bosyltransferasesandareconvertedinonesteptothecorrespondingnucleosidemonophos-
phate[41].
OurstrategytoidentifynucleobaseandnucleosideDENVinhibitorswastoscreenfor
activityandtoxicityofselectedcompoundsat10μMand50μMusingaluciferase-reporting
DENVrepliconcellline,BHKpD2-hRucPac-2ATG30[38].Compoundsthatdemonstrated
inhibitoryactivityagainsttherepliconcelllinewereusedindose-responseanalysistoassign
EC andCC values.Thenucleobasesweregenerallymoreactivewithahighertissueculture
50 50
therapeuticindex(CC /EC )thantheircorrespondingnucleosides(Fig2).TheEC values
50 50 50
oftheactivenucleobasesrangefrom2.4to110μM,comparabletotheEC valuesoftheactive
50
nucleosidesthatrangefrom1.3to113μM(Fig2).Nucleobase3ais5timesmoreactivethan
nucleobase5a(favipiravir).TheCC valuesofthenucleobases1a,3aand5awerebeyond
50
665μM(Fig2).Remarkably,nucleobase1adidnotshowcytotoxicityat1000μMcomparedto
1bnucleoside(Fig2)wheretheCC was20μM.2anucleobasedisplayedaclearantiviral
50
effectat2.4μM.RepresentativeEC curvesfor2apluscorrespondingnucleoside2bandfor
50
3apluscorrespondingnucleoside3bareshowninFig3.Inactivenucleobasesatinitialscreen-
ingarelistedinS1Tableinthesupplementarymaterial.
Wefocusedontheantiviralmechanismofnucleobase3aandcorrespondingnucleoside3b
forthefollowingreasons:1)amongthe5activenucleobases,5awasalreadyknowntoinduce
virallethalmutagenesisbutitscorrespondingnucleosidewasnotavailableandunabletobe
synthesizedforourstudy;2)1aand2aarelikelytopossessacomplexmechanismofaction
duetotheirprobableinhibitoryeffectofinosinemonophosphatedehydrogenase(IMPDH)
afterconversiontothenucleotideform;3)4bwastootoxictoperformafullstudycomparing
thenucleobasetothenucleoside(Fig2).Therefore,wechose3a,knowntobesubstrateof
humanphosphoribosyltransferase[41],anditscorrespondingnucleoside3btobethebest
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Nucleobasesandnucleosidesasanti-denguevirusmutagens
Fig1.Activationpathwaysofantiviralnucleobasesandnucleosides.
https://doi.org/10.1371/journal.pntd.0006421.g001
candidatesforantiviralmechanismofactionstudiesandtocomparetheeffectsofnucleobase
andcorrespondingnucleoside.
Ourapproachtostudymechanismofactionandpossiblelethalmutagenesiswastopassage
virusinHuh-7cellsinthepresenceofacompoundanddeterminethecompound’seffecton
virustiter,genomecopynumberandgenomesequence.Wehadinitiallyidentifiedinhibitors
usingaDENVrepliconBHKcelllinesowewantedtoverifyinhibitoryactivityinHuh-7cells
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Nucleobasesandnucleosidesasanti-denguevirusmutagens
Fig2.Activenucleobasesandtheircorrespondingnucleosides.Dose-responseresultsforselectedcompoundsevaluatedinDENVrepliconcells.Doseswere
performedintriplicateandeachdose-responseexperimentwasperformedindependentlyatleasttwice.Meanvaluesplusstandarddeviationforresultsfromeach
compoundareshown.Mycophenolicacidservedasanon-nucleobase/nucleosidecontrolinhibitor.TTIisthetissueculturetherapeuticindex(CC /EC ).N/A=not
50 50
available.
https://doi.org/10.1371/journal.pntd.0006421.g002
usingreplicationcompetentDENV.WechoseHuh-7cellsbecausetheyareahumancellline,
theyaresusceptibletoDENVinfectionandtheyproducereadilydetectableinfectiousvirus.
TheBHKrepliconcellsareaconvenienttooltoidentifyinitialDENVinhibitors.Weused
humancellsformoredetailedstudiesforthesecompoundsthatrequireconversiontothe
activeformbycellularenzymes.Werepeatedthedose-responseexperimentsfor3aand3b
usingatiter-reductionassaywithHuh-7cellsaspreviouslydescribed[42].Thevaluesobtained
forthecompounds(Table1)wereconsistentwiththosefromtherepliconassay.Basedupon
thedatainTable1,weusednon-toxiclevelsof3a(200μM)and3b(500μM)thatweempiri-
callydetermined(S2Fig)wouldreducevirusreplicationsteadilyduringmultiplepassagesand
ultimatelyleadtoundetectablelevelsofinfectiousvirus.Theresultsforviruspassageinthe
presenceof3a(200μM),3b(500μM)orDMSO(0.5%)areshowninFig4.Compoundswere
addedtoHuh-7cellsshortlyafterinoculation.After3daysincubationincompound,the
supernatantwascollectedandafixedvolume(50μL)ofsupernatantwasusedtoinoculate
freshcells.Thelevelofinfectiousviruspresentinthesupernatantforeachpassagewasdeter-
minedbyplaqueassayandthenumberofviralgenomesbyRT-qPCR.FortheRT-qPCR,we
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Nucleobasesandnucleosidesasanti-denguevirusmutagens
Fig3.Dose-responsecurvesfornucleosidesandnucleobases.Thepercent(%)inhibitionofeachcompoundoverthe
log ofconcentrationscomparedtoDMSOaloneisshown.Doseswereperformedintriplicateandrepeatedatleast
10
twice.Averagevaluesplusstandarddeviationforeachdoseforonerepresentativeexperimentareshown.Errorbarsin
AandBmaybetoosmalltobeseen.
https://doi.org/10.1371/journal.pntd.0006421.g003
choseprimerswithintheNS5genetoincreaselikelihoodofdetectingfull-lengthgenomes.
Bothcompoundsinducedasteadydeclineininfectiousvirusproductionandsupernatant
genomecopynumberoverrepeatedpassages(Fig4).Inthepresenceof3b,infectiousvirus
Table1. Titerreductionassaydose-response.
Compound EC50(μM) CC50(μM)
3a 20±11 >1000
3b 60±22 >1000
https://doi.org/10.1371/journal.pntd.0006421.t001
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006421 April19,2018 9/18
Nucleobasesandnucleosidesasanti-denguevirusmutagens
Fig4.Anti-DENVeffectsduringserialpassageofvirusinthepresenceofantiviralnucleobaseandnucleosides.ViruspassagedinHuh-7cellsin
thepresenceof3a(200μM,blue)and3b(500μM,green).Cellswereinitiallyinoculatedatanm.o.i.of0.01andculturedincompoundatthe
indicatedconcentration.Every72hoursafixedvolumeofsupernatantwasusedtoinoculatefreshcellsmaintainedincompound.A.Thetiterofvirus
inthesupernatantwasobtainedbyserialdilutionandcountingplaquesinsingledilutioncontaining20–50plaqueswhenpossible(seeMethods).
Thisexperimentwasrepeatedthreetimesatthesecompoundconcentrationswithsimilarresultsandarepresentativeexperimentisshown.The
dashedlineindicatesthelimitofdetectionof3.3pfu/mL(seeMethods).B.Genomecopyequivalentsweremonitoredforthepassagesindicated
usingRT-qPCR.Meanvaluesandstandarddeviationsofduplicatemeasurementsareshown.Allpointshavestandarddeviationsrepresentedby
errorbarshoweversomeerrorbarsmaybetoosmalltobeseen.Thisexperimentwasperformedtwicewithsimilarresults.Thedashedlineindicates
thelimitofdetectionof30copiesofNS5gene(seeMethods).C.Specificinfectivitywascalculatedaslog titerdividedbylog genomecopy
10 10
equivalentsforeachpassageandtreatment.
https://doi.org/10.1371/journal.pntd.0006421.g004
wasundetectableatpassages4and5andgenomicRNAwasundetectableatpassage5(Fig4).
For3a,noinfectiousviruswasdetectedatpassages5and6andnogenomicRNAwasdetected
atpassage6(Fig4).Themeangenomecopynumbers(Fig4B)forthethreetreatmentswere
statisticallydifferentateachpassage(p<0.001,OneWayANOVA)withstatisticallysignifi-
cantdifferencesbetween3aandDMSOandbetween3bandDMSOateachpassage(p<0.05,
Tukey’sHSD).BothcompoundsdisplayedareductionintheratioofinfectiousvirustoRNA
genomecopynumber(sometimesreferredtoasRNAspecificinfectivity)whencomparedto
DMSO(Fig4).ThisdelayinreductionofgenomicRNAcomparedtoinfectiousvirusisahall-
markofamutagenesis-basedantiviralactivity[43–46].Theseobservations,alongwiththe
knowledgethat5a(analogueof3aand3b)inducesmutagenesisininfluenzaA,norovirusand
theflavivirusWestNilevirus[27–29],motivatedustodetermineifamechanismofactionfor
3aor3bincludedanenhancedmutagenesisoftheviralgenome.
Wehypothesizedthatanincreaseinmutationsinducedbyaparticularnucleobaseornucle-
osidewouldbedetectedbyanalyzingtheviralgenomesequenceatapassagenearwherethe
titerwassignificantlyreducedorundetectable.Therefore,weamplifiedaregioncontaining
thepre-membrane(prM)andenvelope(E)genesfromviralgenomiccDNAderivedfrompas-
sage33b-treatedcellsandpassage43a-treatedcellsbecausethosepassageswerejustbefore
viraltiterwasundetectable.WeamplifiedtheprM/EregionoftheviralRNAsinsteadofthe
downstreamNS5genetoincreasethelikelihoodwewouldobtainPCRproductswhenviral
titersweregreatlyreduced.ThepresenceoftheNS5genewouldrequireanalmostcomplete
genomicRNAwhereastheprM/Eregionwasclosertothe5’endoftheviralgenomicRNAits
presencewouldnotrequireacompleteviralgenome.Theamplifiedproductswereinserted
intoacloningvectorandthenucleotidesequencewasanalyzedfortheresulting35–50inde-
pendentclones.Wesequencedatleast31,000nucleotidesforeachsetofclonessimilartoother
publishedstudiesofvirallethalmutagenesis[27,43]andcomparedthesequencestothatof
theconsensussequenceobtainedfromDMSO-treatedcellsupernatantsatpassages3and4.
Aninitialplotandanalysisofthenumberofmutationsperclonefor3atreatmentpassage4
and3bpassage3clearlyindicatetherewasasignificantdifference(p<0.0001)inthenumber
ofmutationspercloneforsequenceobtainedfromcompound-passagedviruscomparedto
PLOSNeglectedTropicalDiseases|https://doi.org/10.1371/journal.pntd.0006421 April19,2018 10/18