Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

28 Pages·2017·7.78 MB·English

RESEARCHARTICLE
Digoxin reveals a functional connection
between HIV-1 integration preference and T-
cell activation
AlexanderZhyvoloup1☯,AnatMelamed2☯,IanAnderson1,DelphinePlanas3,Chen-
HsuinLee1,JanosKriston-Vizi4,RobinKetteler4,AndyMerritt5,Jean-PierreRouty6,
PetronelaAncuta3,CharlesR.M.Bangham2,AribertoFassati1*
1 DivisionofInfection&Immunity,UniversityCollegeLondon,London,UnitedKingdom,2 Departmentof
Medicine,ImperialCollege,St.Mary’sCampus,London,UnitedKingdom,3 DepartmentofMicrobiology,
a1111111111
InfectiologyandImmunology,FacultyofMedicine,UniversityofMontrealandtheResearchCentreofthe
a1111111111
CHUM,Montreal,Que´bec,Canada,4 MRCLaboratoryforMolecularCellBiology,UniversityCollege
a1111111111
London,London,UnitedKingdom,5 CentreforTherapeuticsDiscovery,MRCTechnology,MillHill,London,
a1111111111 UnitedKingdom,6 McGillUniversityHealthCentre,Glensite,Montreal,Que´bec,Canada
a1111111111
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Citation:ZhyvoloupA,MelamedA,AndersonI,
HIV-1integratesmorefrequentlyintotranscribedgenes,howeverthebiologicalsignificance
PlanasD,LeeC-H,Kriston-ViziJ,etal.(2017)
Digoxinrevealsafunctionalconnectionbetween ofHIV-1integrationtargetinghasremainedelusive.Usingaselectivehigh-throughput
HIV-1integrationpreferenceandT-cellactivation. chemicalscreen,wediscoveredthatthecardiacglycosidedigoxininhibitswild-typeHIV-1
PLoSPathog13(7):e1006460.https://doi.org/
infectionmorepotentlythanHIV-1bearingasinglepointmutation(N74D)inthecapsidpro-
10.1371/journal.ppat.1006460
tein.Weconfirmedthatdigoxinrepressedviralgeneexpressionbytargetingthecellular
Editor:RonaldSwanstrom,UniversityofNorth Na+/K+ATPase,butthisdidnotexplainitsselectivity.ParallelRNAseqandintegrationmap-
CarolinaatChapelHill,UNITEDSTATES
pingininfectedcellsdemonstratedthatdigoxininhibitedexpressionofgenesinvolvedinT-
Received:April26,2017
cellactivationandcellmetabolism.Analysisof>400,000uniqueintegrationsitesshowed
Accepted:June8,2017 thatWTvirusintegratedmorefrequentlythanN74Dmutantwithinorneargenessusceptible
Published:July20,2017 torepressionbydigoxinandinvolvedinT-cellactivationandcellmetabolism.Twomain
genenetworksdown-regulatedbythedrugwereCD40LandCD38.BlockingCD40Lby
Copyright:©2017Zhyvoloupetal.Thisisanopen
accessarticledistributedunderthetermsofthe neutralizingantibodiesselectivelyinhibitedWTvirusinfection,phenocopyingdigoxin.Thus
CreativeCommonsAttributionLicense,which theselectivityofdigoxindependsonacombinationofintegrationtargetingandrepression
permitsunrestricteduse,distribution,and
ofspecificgenenetworks.ThedrugunmaskedafunctionalconnectionbetweenHIV-1inte-
reproductioninanymedium,providedtheoriginal
grationandT-cellactivation.OurresultssuggestthatHIV-1evolvedintegrationsiteselec-
authorandsourcearecredited.
tiontocoupleitsearlygeneexpressionwiththestatusoftargetCD4+T-cells,whichmay
DataAvailabilityStatement:Allrelevantdataare
affectlatencyandviralreactivation.
withinthepaperanditsSupportingInformation
files.
Funding:ThisworkwasfundedbytheEuropean
UnionFrameworkProgram7HIVINNOV(Grant
305137)(toAF);theUKMedicalResearchCouncil Authorsummary
(toAF);theWellcomeTrust(InvestigatorAward
HIV-1integratesmorefrequentlywithintranscribedhostgenes,howeverwedonot
WT100291MAtoCRMB);theMRCLMCB
UniversityUnitcorefunding(toRKandJKV)and understandthebiologicalsignificanceofthis.Wefoundthatadrugcalleddigoxininhibits
theoperatingfundsfromtheCanadianInstitutesof wildtypeHIV-1morepotentlythananHIV-1bearingasinglepointmutationinthe
HealthResearch(CIHR,MOP-114957toPA).The
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006460 July20,2017 1/28
AfunctionallinkbetweenHIV-1integrationandT-cellactivation
fundershadnoroleinstudydesign,datacollection
andanalysis,decisiontopublish,orpreparationof capsidprotein.HereweshowthatdigoxinrepressesHIV-1geneexpressionandinparallel
themanuscript. inhibitsCD4+T-cellactivationandmetabolism.Whenweanalysedtheintegrationsites
Competinginterests:Theauthorshavedeclared ofwildtypeandmutantHIV-1,wediscoveredthatwildtypevirusintegrateswithinor
thatnocompetinginterestsexist. neargenesinvolvedinCD4+T-cellactivationandmetabolismmoreoftenthanthe
mutantvirus.Becausethesearetheverysamegenesrepressedbydigoxin,theintegration
biasofwildtypevirusmakesitmoresusceptiblethanmutantvirustosilencingbythe
drug.DigoxinunmaskedafunctionallinkbetweenHIV-1integrationandT-cellactiva-
tion,whichmayaffectHIV-1latencyandreactivation.
Introduction
HIV-1infectsimmunecellsexpressingCD4andCCR5/CXCR4,whichserveasHIV-1recep-
torandco-receptorforentry,suchashelperT-lymphocytesandmacrophages.Uponentry
andreversetranscription,thevirusmustaccessthenucleusofinfectedcellsandintegrateinto
hostchromosomes,howeverlittleisknownaboutthestepsoccurringbetweennuclearentry
andintegration.TheHIV-1capsidprotein(CA)wasshowntobeadominantdeterminantof
nuclearimport[1].SeveralstudieshavesinceindicatedthatCAisinvolvedinnuclearentry
andpost-nuclearentryevents(reviewedin[2]).CAinteractswithnucleoporinsNup153
[3,4,5,6]andNup358,viatheCypAbindingdomain[7,8,9],althoughthesignificanceofthis
interactionisunclear[10].CAalsointeractswiththemRNAprocessingfactorCPSF6and
withthenucleartransportreceptorTransportin3(TNPO3),whichwereshowntobeimpor-
tantforHIV-1pre-andpost-nuclearentrysteps[11,12,13,14,15].SmallamountsofHIV-1CA
havebeendetectedinsidethenucleibybiochemicalfractionation[15],andtheantibioticCou-
mermycin-A1wasshowntoimpairHIV-1integrationbytargetingCA[16].Theseobserva-
tionsledtotheproposalthatCAisinvolvedinpost-nuclearentrystepsleadingtoefficient
integration[15].ImagingapproacheshaveshownthatnuclearCAisassociatedwithviral
nucleicacids[17,18,19]andCoumermycin-A1wasreportedtoblockintegrationbyprevent-
ingthecompletionofvirusuncoatinginsidethenucleus[20].HIV-1preferentiallyintegrates
withinactivelytranscribedgenes[21,22].Remarkably,CAbindingtoCPSF6inthenucleusis
criticalfortargetingHIV-1integrationneartranscribedgenes,whereasbindingofhostfactor
LEDGF/p75toHIV-1integraseseemsmoreimportantforintegrationtargetingwithintran-
scribedgenes[23,24].
CertainCApointmutations,suchasN74D,areindependentofNup358,Nup153,CPSF6
andTNPO3forinfection,andshowadifferentintegrationsiteselectioncomparedtowild
typevirus[7,23,25].ThisindicatesthatasinglepointmutationinCAcanchangetheusageof
HIV-1hostfactorsduringtheearlystepsofinfection,resultinginanalteredintegrationpat-
tern.Howeverthefunctionalsignificanceofthisalteredintegrationdistributionisunclear.
TogaingreaterinsightintothesestepsoftheHIV-1lifecycle,wehavetakenadvantageof
thedifferencesbetweenwildtype(WT)andN74DmutantCAtodesignanovelhighthrough
putscreeningassaywherebyCD4+T-cellsareco-infectedwithWTandN74Dsinglecycle
HIV-1vectors,whichareidenticalexceptfortheCApointmutation.TheWTvirusexpresses
GFPwhereasN74DexpressesmCherry.Compoundlibrariesarescreenedtofindselectivehits
thatinhibitWTmorethanN74D.Thesehitsarethenusedtoidentifytargetmoleculesthat
affect—directlyorindirectly—theinteractionbetweenhostfactorsandHIV-1CAandimpair
earlyeventsofinfection.
Here,usingthisnovelscreeningapproach,wefoundthatthecardiacglycosidedigoxin
inhibitsWTmorepotentlythanN74Dvirus.Weconfirmedthatdigoxininhibitedviralgene
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006460 July20,2017 2/28
AfunctionallinkbetweenHIV-1integrationandT-cellactivation
expressionbytargetingtheNa+/K+ATPase[26]butthisdidnotexplainitsselectivity.To
determinethebasisfortheselectivityofdigoxinweperformed,inparallel,RNAseqandhigh-
throughputintegrationsiteanalysisincellstreatedwiththedrug.Theresultsshowedthat
digoxindown-regulatesgenesinvolvedinT-cellactivationandcellmetabolism.Wefound
thatWTvirus,butnotN74Dvirus,preferentiallyintegratesintosuchgenesandisaffectedby
theirrepression.Therefore,usingdigoxintoacutelyperturbspecificgeneexpressionpath-
ways,weuncoveredafunctionallinkbetweenHIV-1integrationpreference,viralgeneexpres-
sionandT-cellactivationandmetabolism.
Results
DigoxininhibitsinfectionbyWTvirusmorepotentlythanN74Dvirus
Wedesignedanovelhighthroughputscreeningworkflowtoinvestigatetheearlyeventsof
HIV-1replicationthatareinfluencedbyCA(Fig1A).JurkatCD4+T-cellswereco-infected
withtwoVSV-GpseudotypedsinglecycleHIV-1vectors,onecontainingWTCAandexpress-
ingGFP(WT-GFP),andanothercontainingtheCAN74DmutationandexpressingmCherry
(N74D-CHE).WeaimedtoidentifysmallcompoundsthatinhibitedWTvirusinfectionat
least50%ofcontrol(DMSO)withlittleinhibitionofN74Dvirusinfection("selectivehits").
TherationaleforthisassaywasbasedonthedifferentusageofhostcellfactorsbyWTand
N74Dviruses[4,7,27]:selectivehitswerelikelytotargeteithertheWTCAproteinitselfor
hostcellfactorsinteractingpreferentiallywithWToverN74Dvirus,directlyorindirectly.
Afteroptimization,theassayhadaZ’scoreofffi0.7,andthenumberofcellsinfectedwitheach
typeofvirus,andcellsurvival,werebothdetermined24hourspost-infectionbydualcolour
flowcytometry.TheNationalInstituteofNeurologicalDiseasesandStrokesmall-compound
librarywasusedtoperformthescreeningatafinalconcentrationof1μM(S1File).This
librarycontains1041compoundsandincludesmanyFDA-approveddrugs.Wedetectedeigh-
teenselectivehitsbelongingtodifferentchemicalandpharmacologicalclasses(S1FileandS1
Table):fourwereanti-inflammatory,hintingthatWTandN74Dvirusmaybedifferentially
susceptibletoinnateimmuneresponses[28]andtwowerecardiacglycosidessharingasimilar
chemicalstructure:digoxinanddigitoxin.
Wefocusedonthecardiacglycosidesbecauseofthelowprobabilityofidentifyingby
chancetwohitswiththesamephenotypethatshareasimilarchemicalstructure.Furthermore,
digoxinwasreportedpreviouslytoexertpotentantiretroviralactivitybyreducingHIV-1gene
expression[29,30],validatingourscreening.However,becauseofthedifferentdesign,previ-
ousscreeningsdidnotdetecttheCA-dependentselectivityofdigoxin,whichisthefocusof
thisstudy.
Toconfirmourscreeningresults,were-examinedtheselectivityofdigoxininsinglecycle
infectiousassaysusingWT-GFPorN74D-CHEHIV-1vectors.DigoxininhibitedWTmore
thanN74Dviruswhereastheintegraseinhibitorraltegravirinhibitedeachvirusequally(Fig
1B).Tocontrolthattheobservedphenotypedidnotdependonthespecificfluorescentmarker
expressedbyeachvector(GFPorCHE),wereversedthemarkersandinfectedJurkatcells
withWT-CHEandN74D-GFPvectors.First,wetitratedbothvectorsintheabsenceofdigoxin
todeterminethelinearrangeofinfectionandchoseanMOIof0.1–0.2(S1AFig).Thenwe
infectedJurkatcellsinthepresenceofdigoxin(S1BFig).Inagreementwithourprevious
results,theN74DvectorwaslesssensitivetodigoxinrelativetoWT(S1BFig).Hencetheselec-
tiveresponsetodigoxinwasindependentofthefluorescentmarker.Wesoughttoconfirm
digoxinselectivityusingareplicationcompetentvirus.Tothisend,atitrationwasperformed
ontheluciferaseJurkatreportercellline(1G5cells)[31]usingHIV-1NL4.3WTorN74D,
whichshowedthatWTNL4.3replicatedmarginallybetterthanNL4.3N74D(S1CFig).Next,
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AfunctionallinkbetweenHIV-1integrationandT-cellactivation
Fig1.DigoxininhibitsHIV-1infectionmorepotentlythanN74Dvirus.(A)Schematicdepictionofthehigh
throughputscreen;WTHIV-1vectorexpressesGFPandN74DmutantHIV-1vectorexpressesmCherry.Jurkatcells
areco-infectedwithbothvirusesandalibraryofsmallcompoundsisscreenedin384wellplates.Cellsareanalysed
bydual-colourflowcytometry.Leftplotdepictsanon-hitcompound,middleplotdepictsaselectivehitandtheright
plotdepictsanon-selectivehit.(B)Jurkatcellswereco-infectedwithWTandN74DvectorsatanMOIof0.1inthe
presenceoftheindicatedcompoundsandanalysed24hlater.RepresentativedotplotsfromtheFACSanalysisfor
control(DMSOonly),digoxin(300nM)orraltegravir(100nM)treatedcells.(C)1G5Jurkatluciferaseindicatorcells
wereinfectedatthesameMOIwithNL4.3WTorwithNL4.3N74Dinthepresenceoftheindicatedconcentrationsof
digoxin.Luminescencewasmeasured48hoursafterinfection;dataarerepresentativeoftwoexperimentsperformed
intriplicate.ErrorbarsrepresentSEM.(D,E)MemoryCD4+T-cellswerestimulatedviaCD3/CD28Absfor3days
andinfectedwithVSV-GpseudotypedHIV-1LAIΔenvWT(D)orN74Dmutant(E),cellswereculturedinmedia
containingIL-2inthepresenceorabsenceoftheindicateddosesofdigoxinforadditional40hours.Thepercentageof
viable,infected(GFP+)cellswasdeterminedbyflowcytometry.Bargraphsrepresentmean±SDof9donors.
Friedmantestp-valuesareindicatedonthegraphs:*,p<0.05.
https://doi.org/10.1371/journal.ppat.1006460.g001
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AfunctionallinkbetweenHIV-1integrationandT-cellactivation
wetestedtheeffectofdigoxinbytitratingthedrugon1G5cellsinfectedwitheitherNL4.3WT
orNL4.3N74Dandreadingluciferase48hourspost-infection,whencelltoxicityanddiffer-
encesinreplicationbetweenthetwoviruseswerebothminimal.Intheseconditions,WT
NL4.3waslesssensitivetodigoxinthanNL4.3N74D(Fig1C).Wealsotestedtheselectivityof
digoxininprimarymemoryCD4+T-cells.PBMCswereobtainedfrom9healthydonors,
memoryT-cells(CD3+CD4+CD45RA-)weresortedbynegativeselection,stimulatedusing
CD3/CD28AbsandinfectedatanMOIof0.1withaVSV-GpseudotypedsinglecycleHIV-1
LAIwithadeletioninenvandexpressingGFPinplaceofnef(HIV-1LAIΔenv)[1].Thirty-six
hourspost-infection,thepercentageofGFP+cellswasmeasuredbyflowcytometryaftergat-
ingforthelivecellpopulation.Intheabsenceofdigoxin,WTandN74DHIV-1LAIΔenv
virusesshowedsimilarinfectionlevels(S1DFig).Inthepresenceofdigoxin,WTHIV-1was
inhibitedmorestronglythanN74Datconcentrationsgreaterthan200nM(Fig1Dand1E),in
agreementwiththeresultsinJurkatcells.Therefore,weconcludedthatthehigh-throughput
screenidentifieddigoxinasagenuinelyselectiveantiretroviral.
Identificationofthedigoxintargetresponsibleforantiretroviralactivityin
CD4+T-cells
ApreviousreportshowedthattheNa+/K+ATPaseisthemaintargetofdigoxinmediatingthe
antiretroviraleffectin293Tcells[30].However,digoxinhastwoknowntargets,thenuclear
hormonereceptorRORγ/γt,(alsoknownasRORC),whichisexpressedinasubsetofCD4
+T-cellsandinnatelymphoidcells[32,33]andtheα1-subunitoftheNa+/K+ATPase,whichis
ubiquitouslyexpressed[34,35,36].Therefore,inCD4+T-cells,digoxinmighthavemorethan
onetargetresponsibleforitsantiretroviralactivity.Theco-crystalstructureofRORγ/γtbound
todigoxinshowedthatthesugarmoietyofdigoxiniscriticalforstabilizingthedrugintothe
ligand-bindingpocketofRORγ/γt[37].Thusdigoxinderivativeslackingtwoormoresugar
moieties,suchasdigoxigeninorouabain,willonlybindtotheNa+/K+ATPasebutnotto
RORγ/γt[36,38];conversely,thelactoneringofdigoxinisimportantforhigh-affinitybinding
totheNa+/K+ATPase[34]hencemodificationofthelactoneringin20,22-dihydrodigoxin-
21-23-diol(Dig(dhd))substantiallyreducesitsaffinityfortheNa+/K+ATPasebutnotfor
RORγ/γt[35,36].WesynthesizedDig(dhd)andtesteditininfectionassaysinJurkatcells
usingHIV-1LAIΔenv.At36hpost-infection,cellswereanalysedbyflowcytometry;digoxin
showedthetypicalbiphasicinhibitorycurve,andataconcentrationof100nMsuppressed
WTHIV-1infectionwithoutaffectingcellviability(Fig2A).Dig(dhd)phenocopieddigoxin,
showingthesametypicalbiphasiccurve,howeveritshowedadrasticdropinantiretroviral
potency(Fig2B).
Thisindicatedthat,inCD4+T-cells,themaintargetofdigoxinmediatingthepotentanti-
retroviralactivitywastheNa+/K+ATPase.Tofurthertestthispoint,weexpressedinJurkat
cellsthemurineNa+/K+ATPase(mATPase),whichisnotsusceptibletodigoxininhibition
[39].CellsexpressingthemATPase,orcontrolJurkatcells,wereinfectedwithHIV-1LAIΔenv
inthepresenceofdigoxinorouabain.Theantiretroviralactivityofdigoxinwasdrastically
lowerinJurkatcellsexpressingthemATPase(Fig2C).Incontrast,digoxinandouabain
potentlyinhibitedHIV-1LAIΔenvinfectionincontrolcells(Fig2C).Theseresultsestablished
that,similarto293T-cells[30],theNa+/K+ATPasewasthemaindigoxintargetinCD4+T-
cells.
TargetingtheNa+/K+ATPasewithdigoxindidnotimpairHIV-1reversetranscription
(S2BFig),nuclearentry(S2CFig)orintegration(S2DFig)butreducedviralmRNAlevels
(S2E–S2GFig),inagreementwithpreviousreports[29,30].Lairdetal.showedthatdigoxin
inhibitsexpressionfromaHIV-1plasmidDNAtransfectedinto293Tcells[30],which
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AfunctionallinkbetweenHIV-1integrationandT-cellactivation
Fig2.Na+/K+ATPaseisthedigoxintargetmediatingantiretroviralactivityinCD4+T-cells.(A)Jurkatcellswere
infectedwithVSV-GpseudotypedHIV-1LAIΔenv atanMOIof0.1inthepresenceoftheindicateddosesofdigoxin
GFP
andanalysedbyflowcytometry36hourspost-infection.Thechemicalstructureofdigoxinisshownatthetop.(B)Same
asforpanel(A)butcellswereinfectedinthepresenceofDig(dhd).Dig(dhd)chemicalstructureisshownatthetop.In
panelsAandBerrorbarsindicateSD,N=3independentexperiments.(C)Jurkatcellsstablyexpressingthemouse
Na+/K+ATPase(mATPase),oranemptyvector(vector),wereinfectedwithHIV-1LAIΔenv atanMOIof0.1inthe
GFP
presenceoftheindicatedconcentrationsofcardiacglycosidesdigoxinorouabain,bothNa+/K+ATPaseantagonists.
Thepercentageofinfected,GFP+cellswasdeterminedbyFACS24hpost-infection.Dataarerepresentativeof3
independentexperiments.
https://doi.org/10.1371/journal.ppat.1006460.g002
suggeststhatHIV-1integrationmaynotbenecessary.TransfectionofJurkatcellsisextremely
inefficienthencetotestiftheselectivephenotypeofdigoxinwasmaintainedintheabsenceof
integration,wetitrateddigoxininJurkatcellspre-exposedtoahighdose(20μM)ofraltegravir
(apotentintegrationinhibitor[40])andinfectedthemwithWTorN74DHIV-1LAIΔenv
(S2HFig).Intheseconditions,residualviralgeneexpressionmostlycomesfromnon-inte-
gratedviralgenomes,whicharelostduringprolongedpassageinculture[41,42].Asexpected,
inthepresenceofraltegravir,infectionbybothWTandN74Dviruseswasreducedat48h
post-infection(S2HFig)andwasreducedmuchfurther10dayspost-infection(S2IFig).This
suggestedthatGFPexpressionat48hmostlycamefromnon-integratedviralgenomes.Nota-
bly,inthepresenceofraltegravir,digoxinappearedtobelesspotent(IC >300nM)andwas
50
nolongerselective(S2HFig).Thissuggestedthattheselectivephenotypeofdigoxindepends
onintegration.
DigoxinrepressesgenepathwaysinvolvedincellmetabolismandT-cell
activation
TheidentificationofNa+/K+ATPaseasthedigoxintargetdidnotreadilyexplainpreferential
inhibitionofWToverN74Dvirus.Digoxininhibitsviralgeneexpression(S2E–S2GFig)and
[29,30]),yetthepromoter/enhancerregions(LTR)ofWTandN74Dviruseswereidentical,
excludingthattheselectivitycouldbemediatedbyadirecteffectofthedrugontheLTR.
HIV-1WThasagreaterpreferencetointegratewithinornearactivegenesthanN74D
virus[7,23]anddigoxincantriggerchangesincellulargeneexpression[43,44].Wetherefore
hypothesizedthatifWTviruswasmorepronetointegrateintogeneswhoseexpressionwas
perturbedbydigoxinthenthevirusmightbecomemoresusceptibletothedrug.Conversely,if
theN74Dviruswerelesspronetointegrateintogenesperturbedbydigoxinthenthisvirus
wouldbelesssusceptibletothedrug.Totestthishypothesiswecarriedout,inparallel,global
geneexpressionandintegrationsiteanalysisoncellsthatwereinfectedwithWTorN74D
HIV-1inthepresenceofdigoxinorDMSO.
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AfunctionallinkbetweenHIV-1integrationandT-cellactivation
WeconductedthreeindependentexperimentsinJurkatcellsinfectedwithWTorN74D
LAIΔenv,ensuringthatinfectionlevelswerewithinthelinearrange(S1EFig),andextracted
totalRNAandDNAfromeachsample36hpost-infection(S3Fig).TheRNAwasusedfor
RNAseqandtheDNAwasusedtoperformhigh-throughputintegrationsiteanalysis.Total
RNAwaspreparedforsequencingfollowingtheIlluminaTruSeqmRNAprotocoland
sequencedonanIlluminaNextseqtoyieldanaverageof15millionreadspersample.One
sample(DMSOWTexperiment1)didnotpasstheRNAqualitycontrolandcouldnotbe
sequenced.FollowingalignmentandremovalofPCRduplicates,normalizedreadcountswere
analysedusingGeneSpring.Atotalof2,826geneswerefounddifferentiallyregulated(2-fold
ormore,p<0.05)bydigoxin(S2File).Clusteranalysisdemonstratedaclearandsignificant
distinctionbetweentreatedanduntreatedsamplesbutnosignificantdistinctionbetweencells
infectedwitheitherWTorN74Dviruses(S4Fig).
NormalisedRNAreadcountswerestudiedforgenepathwayenrichmentusingIngenuity
PathwayAnalysis(IPA)(www.qiagen.com/ingenuity).Toidentifychangesingeneexpression
likelytobeofbiologicalrelevance,weappliedacut-offfoldchange(cid:21)4withap-valueof<0.05
(MannWhitneytestafterBenjamini-Hochbergfalsediscoveryrate).Usingthisfilter,we
founddigoxinup-regulated221anddown-regulated336genesthatcouldbeunequivocally
mapped(S2File).Withintheup-regulatedgenegroup,IPAshowedthatthemainbiological
functionsaffectedbydigoxin,intermsofbothnumberofparticipatinggenesandsignificance,
weretranscriptionofRNAandDNA,chromatinremodelling,cellcycleprogressionandcell
deathandsurvival(Fig3A,3BandS3File).Aprominentup-regulatednetworkwasAP-1,
includingJun,FosandATF3;Junwasoneofthemostup-regulatedgenes,increasing60fold
indigoxin-treatedcells(S2File).Suchstrongup-regulationofJunmayberelatedtoasurvival
responsetostressinducedbydigoxin[45].
Withinthedown-regulatedgenegroup,themainbiologicalfunctionsaffectedbydigoxin
weremetabolism,inparticularofcholesterol,lipidsandcarbohydrates,antigenpresentation,
T-cellsignallingandactivationofleukocytes(Fig3Cand3DS4File).
WTHIV-1preferentiallyintegrateswithinorneargenessusceptibleto
silencingbydigoxin
Tovalidatethehypothesisthattheselectivityofdigoxinoccursattheintegrationstage,we
examinedtheintegrationprofileofsinglecycleWTandN74DHIV-1LAIΔenvindigoxin-
treatedanduntreatedJurkatcells.Weanalysedintegrationsiteselectionusingahigh-through-
putmethodwerecentlydeveloped[46,47].Weobtainedbetween15,000and56,000unique
proviralintegrationsite(UIS),dependingonthesample(S5AFigandDasaset5).Nosignifi-
cantdifferencesinthenumberofUISwereobservedbetweencontrolanddigoxin-treated
samples(S5AFig).ThiswasinagreementwiththeAlu-LTRqPCRresults,whichdemon-
stratedthatdigoxindidnotinhibitHIV-1integration(S2CFig).Furthermore,noclonal
expansionwasdetected(comparetotalclonestoshearsitesinS5AFig)becauseinfectedcells
wereexamined36hourspost-infection.
UISweremappedtohumangenomereferencehg19(S5File);acontrolwasgeneratedin
silicousing100,000non-gapgenomicpositionschosenatrandom,whichwereprocessedin
thesamewayastheexperimentallyobservedUIS.WTandN74Dvirusesintegratedpreferen-
tiallywithinanygene,howeverWTvirusfavouredintegrationwithinanygenemorestrongly
thantheN74Dvirusrelativetotheexpectedrandomdistribution(baseline)(Fig4Aleftpanel
andS5BFig).WTvirushadaclearpreference((cid:25)2foldabovebaseline)tointegratenearany
gene(10Kbfromthetranscriptionalstartsite)whereasN74Dvirusdidnot(Fig4Aright
panel),inagreementwithpreviousobservations[7,23,25].DigoxinreducedWTvirus
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AfunctionallinkbetweenHIV-1integrationandT-cellactivation
Fig3.Digoxinperturbsexpressionofspecificgenepathways.(A)IPA-generateddiagramshowingthe221genesup-regulatedby
digoxinandtheirnetworks.Continuouslinesindicatedirectandexperimentallyvalidatedinteractionsbetweengenes;dashedlinesindicate
experimentallyvalidated,indirectinteractions.Circulararrowsindicateself-activation.Genesthatcouldnotbeassignedtoanetworkare
shownintherighthandcorner.(B)ListofthemainfunctionallyannotatedpathwaysidentifiedbyIPAonthebasisofthe221genesup-
regulatedbydigoxin.Thep-valueforeachpathwayisshownonthex-axis.ThefulldatasetisavailableinS3File.(C)IPA-generated
diagramshowingthe336genesdown-regulatedbydigoxin,sameasforpanel(A).(D)Listofthemainfunctionallyannotatedpathways
identifiedbyIPAonthebasisofthe336genesdown-regulatedbydigoxin.Thep-valueforeachpathwayisshownonthex-axis.Thefull
datasetisavailableinS4File.
https://doi.org/10.1371/journal.ppat.1006460.g003
integrationnearanygeneandincreasedN74Dvirusintegrationnearanygene(p<0.001,
Fisher’sexacttest)butthiseffectwasquitesmall(Fig4A).Intheseconditions,theorientation
oftheprovirusrelativetothecellulargenetranscriptionalstartsitewasunchanged(S5CFig).
Thusdigoxincausedmodest,albeitselective,changesintheintegrationpreferenceofHIV-1
WTandN74Dvirus.Nonetheless,irrespectiveofdigoxintreatment,asignificantgap
remainedbetweenWTandN74Dvirusintegrationpreference,whichwarrantedfurther
investigation.
Toachievegreaterspecificity,weexaminedintegrationpreferencewithinorneargenes
whoseexpressionwaschanged((cid:21)4foldup-ordown-regulated)bydigoxin,accordingtoour
RNAseqresults(S2File).Forgreaterstringency,becauseoftheobservedbiasofWTvirusto
integratenearandwithingenes,aninsilicocontrolforrandomintegrationsiteswithinor
neargeneswasgenerated,whichwasprocessedinthesamewayastheexperimentally
observeddata.ThentheratiobetweenUISwithinorneargenesdownregulatedbydigoxin
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006460 July20,2017 8/28
AfunctionallinkbetweenHIV-1integrationandT-cellactivation
Fig4.WTHIV-1integratesmorefrequentlywithinorneargenesthataresusceptibletodown-regulation
bydigoxin.ThreeseparatealiquotsofJurkatcellswereindependentlyinfectedwithsinglecycleVSV-G
pseudotypedHIV-1LAIΔenvWTorN74DatanMOIof0.2inthepresenceofDMSOor400nMdigoxin.Thirty-six
hourslater,DNAwasextractedandintegrationsiteswereidentifiedandquantified.Sameanalysiswascarried
outforadatasetofrandomintegrationsitesgeneratedinsilico,sampledfromagap-excludedreferenceofthe
humangenome(hg19).Thismatchedrandomcontrolisshownasabluedashedbaseline,pointedbyanarrow.
(A)PlotsforintegrationofHIV-1WTandN74Dwithin(leftpanel)ornear(within10kbofstartsite)(rightpanel)
knowngenes.(B)PlotsforintegrationofWTandN74Dviruswithin(leftpanel)ornear(rightpanel)genes
susceptibletodown-regulationbydigoxinrelativetotheirbaselinebiastointegratewithinornearanygene.(C)
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006460 July20,2017 9/28
AfunctionallinkbetweenHIV-1integrationandT-cellactivation
Sameas(B)butplotsshowintegrationwithin(leftpanel)ornear(rightpanel)genessusceptibletoup-regulation
bydigoxin.Eachbarshowsanaggregateresultfromallthreereplicates,withtherangeofresultsfortheindividual
replicatesshownbythewhiskers.Blackasterisksabovebargraphsdenotestatisticalsignificancebetween
control(DMSO)anddigoxin-treatedsamples.Blueasterisksjustabovethebargraphsdenotestatistical
significanceinthesamplesrelativetobaseline(sameanalysiscarriedontheinsilicosites)andwasassessed
usingtheFisher’sexacttestwithBonferroni’scorrectionformultiplecomparisons(*<0.05,**<0.01,***<
0.001).
https://doi.org/10.1371/journal.ppat.1006460.g004
andallUISwithinorneargeneswascalculated.Notably,WTvirusintegratedmoreoftenthan
N74Dviruswithingenessusceptibletodown-regulationbydigoxin((cid:25)1.5foldabovebaseline)
(Fig4Bleftpanel),andthiseffectwasstronger((cid:25)2.5foldabovebaseline)nearsuchgenes(Fig
4Brightpanel),demonstratingselectivity.ThisdifferencebetweenWTandN74Dviruswas
largeenoughtosuggestbiologicalrelevance.Digoxintreatmenthadasmallyetselective
impactontheintegrationpreferenceofeachvirus(Fig4B).
WTandN74Dvirusesalsointegratedmoreoftenthanmatchedrandomcontrolwithinor
neargenessusceptibletoup-regulationbydigoxin(Fig4C).Notably,however,incontrastto
thesituationinthedown-regulatedgenes,therewasnoWT/N74Dselectivityforintegration
withinup-regulatedgenes,therewasamodestselectivityforintegrationnearup-regulated
genesanddigoxintreatmentenhancedtheintegrationpreferenceofeachvirus(Fig4C).Thus
integrationwithinorneargenessusceptibletoup-regulationbydigoxinwasunlikelyto
explaintheselectivephenotype.
Inlightoftheseresults,wenextexaminedintegrationwithinorneargenesbelongingtothe
twomaingenepathwaysdown-regulatedbydigoxin:T-cellactivationandcellmetabolism,
whichareintimatelyrelatedbothtoeachotherandtoHIV-1infection[48,49,50,51].Basedon
IPA,weidentified59and73genesinvolvedinT-cellactivationandcellmetabolism,respec-
tively(S4File).Intheabsenceofdigoxin,integrationofWTviruswithinT-cellactivation
geneswas(cid:25)1.8-foldabovebaseline,andapproximately4-foldabovebaselinenearsuchgenes
(Fig5A).TheN74Dvirusintegrationpreferencewithinthisgenegroupwassimilartobaseline
whereasintegrationnearthesegeneswas(cid:25)2-foldabovebaseline(Fig5A).Digoxinincreased
N74DvirusintegrationfrequencywithinorneartheT-cellactivationgenesalthoughthisdid
notreachstatisticalsignificance,presumablyduetothelowernumberofintegrationsitesthat
couldbeanalysed(Fig5A).DigoxinhadlittleornoeffectonWTvirusintegrationfrequency
withinornearT-cellactivationgenes,whichremainedmuchhigherthanrandom,irrespective
ofthedrug(Fig5A).Toexplainthisobservation,wespeculatethatWTvirusintegration
withinornearthisgenegroupisfasterthantheeffectofdigoxinontheirexpressionlevels.A
similartrendinWTandN74Dvirusintegrationpreferencewasfoundforthegroupofgenes
relatedtocellmetabolism(Fig5B).Therefore,althoughatotalofonly132geneswereanalysed
intheT-cellactivationandcellmetabolismpathways,WTvirusintegrationwithinornear
thesegeneswasdisproportionallyfrequentbothinabsolutetermsandrelativetotheN74D
virus.
Asacontrolforthisanalysis,wealsoexaminedintegrationpreferenceinthehereditaryand
developmentaldisordergenenetwork(66genes),alsoidentifiedbyIPAamongthegenes
down-regulatedbydigoxin(S6File).Althoughtherewasintegrationpreferenceabovebaseline
inthisgroupofgenes,itwasnotaspronouncedasforT-cellactivationormetabolismgenes
andtherewasnodifferencebetweenWTandN74Dviruses(Fig5C).ThissuggestedthatWT
virushassomedegreeofspecificintegrationtargetingwithincertaingroupsofgenes.
Totestthisnotionfurther,welookedforintegrationhotspotsofWTorN74Dvirusesin
oursamples.Hotspotsweredefinedasgeneshavingmorethan5UISinatleasttwooutof
threeexperiments.Wedetected24hotspotsintotalwithintegrationfrequenciesrangingfrom
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006460 July20,2017 10/28