mlwww.cfjingling.com

Salehetal.
Retrovirology(2016)13:49DOI10.
1186/s12977-016-0284-7RESEARCHHIVintegrationandtheestablishmentoflatencyinCCL19treatedrestingCD4+TcellsrequireactivationofNFκBSuhaSaleh1,2,HaoK.
Lu1,2,VanessaEvans1,2,DavidHarisson3,JinglingZhou3,AnthonyJaworowski2,3,GeorginaSallmann1,2,KareyY.
Cheong1,2,TaliaM.
Mota1,SurekhaTennakoon1,2,ThomasA.
Angelovich3,JennyAnderson1,2,AndrewHarman4,AnthonyCunningham4,LachlanGray2,3,MelissaChurchill2,3,5,JohnsonMak6,HeidiDrummer2,3,5,DimitriosN.
Vatakis7,8,SharonR.
Lewin1,2,3andPaulU.
Cameron1,2,3*AbstractBackground:EradicationofHIVcannotbeachievedwithcombinationantiretroviraltherapy(cART)becauseofthepersistenceoflong-livedlatentlyinfectedrestingmemoryCD4+Tcells.
WepreviouslyreportedthatHIVlatencycouldbeestablishedinrestingCD4+TcellsinthepresenceofthechemokineCCL19.
TodefinehowCCL19facilitatedtheestablishmentoflatentHIVinfection,theroleofchemokinereceptorsignallingwasexplored.
Results:InrestingCD4+Tcells,CCL19inducedphosphorylationofRAC-alphaserine/threonine-proteinkinase(Akt),nuclearfactorkappaB(NF-κB),extracellular-signal-regulatedkinase(ERK)andp38.
Inhibitionofthephosphoinositol-3-kinase(PI3K)andRas/Raf/Mitogen-activatedproteinkinase/ERKkinase(MEK)/ERKsignallingpathwaysinhibitedHIVintegration,withoutsignificantreductioninHIVnuclearentry(measuredbyAlu-LTRand2-LTRcircleqPCRrespec-tively).
InhibitingactivationofMEK1/ERK1/2,c-JunN-terminalkinase(JNK),activatingprotein-1(AP-1)andNF-κB,butnotp38,alsoinhibitedHIVintegration.
WealsoshowthatHIVintegrasesinteractwithPin1inCCL19-treatedCD4+TcellsandinhibitionofJNKmarkedlyreducedthisinteraction,suggestingthatCCL19treatmentprovidedsufficientsig-nalstoprotectHIVintegrasefromdegradationviatheproteasomepathway.
InfectionofCCL19-treatedrestingCD4+TcellswithmutantstrainsofHIV,lackingNF-κBbindingsitesintheHIVlongterminalrepeat(LTR)comparedtoinfec-tionwithwildtypevirus,ledtoasignificantreductioninintegrationbyupto40-fold(range1–115.
4,p=0.
03).
Thiswasincontrasttoonlyamodestreductionof5-fold(range1.
7–11,p>0.
05)infullyactivatedCD4+Tcellsinfectedwiththesamemutants.
Finally,wedemonstratedsignificantdifferencesinintegrationsitesfollowingHIVinfectionofunactivated,CCL19-treated,andfullyactivatedCD4+Tcells.
Conclusions:HIVintegrationinCCL19-treatedrestingCD4+TcellsdependsonNF-κBsignallingandincreasesthestabilityofHIVintegrase,whichallowsubsequentintegrationandestablishmentoflatency.
Thesefindingshaveimpli-cationsforstrategiesneededtopreventtheestablishment,andpotentiallyreverse,latentinfection.
Keywords:HIVlatency,CD4+Tcells,Integration,NF-κB,Chemokinesignalling2016TheAuthor(s).
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0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
OpenAccessRetrovirology*Correspondence:paul.
cameron@unimelb.
edu.
auSuhaSaleh,HaoK.
Lu,SharonR.
LewinandPaulU.
Cameroncontributedequallytothiswork1DohertyInstituteforInfectionandImmunity,TheUniversityofMelbourne,Melbourne,AustraliaFulllistofauthorinformationisavailableattheendofthearticlePage2of12Salehetal.
Retrovirology(2016)13:49BackgroundThemajorbarriertoHIVcureinpatientsreceivingcom-binationantiretroviraltherapy(cART)isthepersistenceoflong-lived,latentlyinfected,restingmemoryCD4+Tcells[1].
UnderstandinghowHIVlatencyisestablishedandmaintainediscriticaltothedevelopmentofnovelapproachestoeradicateHIV.
Directinfectionofrest-ingCD4+Tcellsinvitroisinefficient[2,3].
Incontrast,HIVintegrationandlatencyisestablishedinrestingCD4+Tcellsinvivo[4],intonsilexplants[5],orfol-lowingco-culturewithendothelialcells[6]ordendriticcells[7],orfollowingculturewithchemokinesthatbindtothechemokinereceptorsCCR7,CXCR3andCCR6expressedonrestingCD4+Tcells[8,9].
ThechemokineCCR7isexpressedonnaveandcen-tralmemoryCD4+TcellsandculturingwiththeCCR7-ligandsCCL19orCCL21facilitatesentryandintegrationofHIVintotheserestingCD4+Tcellswithminimalvirusproduction.
Inthismodel,restingCD4+Tcells5daypostinfectionrepresentastablelatentlyinfectedpopulationthatrespondtolatency-reversingagents(LRA)inasimilarpatterntoCD4+TcellsfromHIV-infectedindividualsoncART[10].
Wethereforebelievethisisanidealmodeltostudytheearlyeventsrequiredforestablishinglatency.
BindingofchemokinestotheirspecificGi-coupledreceptorsleadstoactivationoftheRhoA/GTPase,phos-phatidylinositol3-kinase(PI3K)andphospholipaseC(PLC)pathways[11].
BindingofHIVglycoprotein(gp)120totheHIVco-receptorCXCR4canalsoactivatetheRhoApathwayinrestingCD4+Tcells,activatingcofilinleadingtodepolymerizationofthecorticalactincytoskeleton,thusfacilitatingnuclearentry[3,12].
WedemonstratedsimilarcellularchangeswiththeexogenouschemokineCCL19[9].
However,incontrasttoHIVinfectionofrest-ingCD4+Tcells,wherethereisminimalintegrationofHIV[2,3,9],inchemokinetreatedcellstherewaseffi-cientnuclearlocalizationandHIVintegration[8,9].
ActivationofPI3KleadstochangesinnuclearfactorkappaB(NF-κB),theMitogen-ActivatedProteinkinases(MAPK),extra-cellularsignal-regulatedkinases1and2(ERK1/2)[13],p38,c-JunN-terminalkinase(JNK)[14]andserine/threonineproteinkinase(Akt)phosphoryla-tion(Additionalfile1:FigureS1).
AlthoughNF-κBisapotentactivatorofHIVtranscription,previousstudieshavedemonstratedthatlatentorproductiveinfectionmaydependontherelativeamountofactiveNF-κB.
InTcelllines,highlevelsofNF-κBenhanceHIVtranscrip-tion,butlowlevelsofNF-κBareimportantforHIVinte-gration[15].
Furthermore,generatinglatentinfectionbydirectinfectionofJurkatTcellsrequireslowlevelsofNF-κB[16].
However,theroleofNF-κBinlatencyinpri-maryrestingCD4+Tcellsremainsunknown.
OtherdownstreamsignallingproductsofchemokinereceptorsignallingmayalsoimpactHIVintegrationandlatentinfectionofrestingCD4+Tcells.
Activatingpro-tein-1transcriptionfactors(AP-1)[17]mayplayaroleinestablishingandmaintainingHIVlatency[18].
TheRas/Raf/MEKpathwayhaspreviouslybeenassociatedwiththenuclearimportoftheHIVreversetranscriptasecom-plex(RTC)[19]andthusmayenhanceHIVnuclearentryrequiredforinfectionofrestingCD4+Tcells.
Inthepre-sentstudy,weaskedwhetherthePI3Ksignallingpathway,inducedbyligationofCCL19toCCR7,playedakeyroleintheestablishmentoflatentinfectioninCCL19-treatedrestingCD4+Tcells.
WedemonstratedthatactivationoftheNF-κBpathwayiscriticalforefficientintegrationofHIVinCCL19-treatedrestingCD4+TcellsandthatthesitesofHIVintegrationdependedontheactivationstateofthecellatthetimeofinfection.
ResultsDoseresponseofCCL19onrestingCD4+TcellsignallingWefirstdeterminedthedoseresponseofCCL19onactivationofdownstreamsignallinginrestingCD4+Tcells.
Afteronly5minoftreatmentwithCCL19,wesawastrongincreaseinthelevelofphosphorylatedAktandNF-κBwithalldoses(range30–300nM)ofCCL19(Additionalfile2:FigureS2A).
However,weobservedadoseresponseofCCL19onthelevelofphosphorylatedERK.
Webelievethatthismaybeduetodifferentkineticofactivation.
Indeed,treatmentofrestingCD4+TcellswithvariousdosesofCCL19for15minledtophospho-rylationofAkt,NF-κB,ERKandlowlevelsofJNK(Addi-tionalfile2:FigureS2B).
SinceverylowlevelsofCCL19(10–100nM)arerequiredforprimingofrestingCD4+TcellsforlatentinfectionwithHIV[8],weused30and100nMofCCL19forsubsequentinfectionandimmuno-blottingexperiments,respectively.
InhibitionofCCL19inducedsignallingbypharmacologicalinhibitorsActivationofCCR7hasbeenshowntoinducemultiplemodulesofsignallingleadingtocellsurvival,chemot-axis,endocytosisandothers(seereviewin[11,20]).
TodeterminewhichpathwayofCCL19-mediatedsignallingwasimportantforestablishmentoflatency,pharmaco-logicalagentsthatinhibitvarioussignallingmoleculeswereused(Additionalfile1:FigureS1).
InhibitionofCCL19-inducedphosphorylationofAkt,NF-κB,ERKandp38wereobservedusingspecificinhibitorstoPI3K(LY294002andWortmannin),NF-κB(SC-514)(Fig.
1a,c-I,c-II);MEK1/ERK1/2(PD980509),p38(SB203580)(Fig.
1b,c-III,c-IV);andabroad-spectruminhibitorofAktandNF-κB(Bay11-7082)(Fig.
1a,c-II).
Phosphoryla-tionofJNKwasnotconsistentlyobservedinresponsetoPage3of12Salehetal.
Retrovirology(2016)13:49Fig.
1InhibitionoftheCCL19-mediatedsignallingbypharmacologicalinhibitors.
RestingCD4+Tcellsweretreatedfor1hwithapredetermined(see"Methods")concentrationofinhibitortoPI3K(LY294002andWortmannin),NF-κB(Bay11-7082andSC-514),JNK(SP600125),ERK(PD980509),AP-1(SR11302)andp38(SB203580)priortotheadditionofCCL19(100nM)for15min.
Cellswerelysedandthelevelofspecificphosphorylatedproteinswasmeasuredusingimmunoblotting.
a,bRepresentativeimmunoblotsfromtwodifferentdonorstreatedwithvariousinhibitors.
GAPDHimmunoblotwasusedascontrolforequalproteinloading.
cDensitometryofvariousphosphorylatedproteinsinthepresenceorabsenceofinhibi-torsinCCL19-treatedrestingCD4+Tcells.
ValueswerenormalisedtobothGAPDHandunactivatedcontrol.
Datarepresentmean±SDfrom2to3experimentsPage4of12Salehetal.
Retrovirology(2016)13:49CCL19.
SincephosphorylationofJNKisrequiredforcellmigration[21],thelackofpJNKinCCL19-treatedcellsislikelyduetotimingofdetection,asCD4+Tcellmigra-tionisnormallymeasuredatleast3haftertheadditionofCCL19[22].
However,theeffectofJNK(SP600125)andAP-1(SR11302)inhibitorsonCCL19-treatedcellswereindirectlyobserved(Fig.
1c).
SP600125markedlyinhibitedphosphorylationofAkt,butincreasedp38phosphorylation,whileSR11302inhibitedthephospho-rylationofbothERKandp38.
Asexpected,specificinhibitionofPI3K(LY294002andWortmannin)andNF-κBactivation(SC-514)hadmini-maleffectonCCL19-mediatedphosphorylationofERKandp38(Fig.
1a).
Similarly,inhibitionoftheRas/Raf/MEKsignallingpathwayhadminimaleffectsonphos-phorylationofAktandNF-κB(Fig.
1b),demonstratingthedichotomyofthesetwopathwaysinCCR7-signalling.
CCL19mediatedHIVintegrationisrestrictedbyinhibitorsofbothPI3KandRas/Raf/MEKsignallingpathwaysWeusedinhibitorsofthePI3Kpathway,todetermineifactivationofthispathwaywascriticalforHIVintegrationinCCL19-treatedrestingCD4+Tcells(Fig.
2).
InHIV-infectedCCL19-treatedrestingCD4+Tcells,treatmentofcellspriortoinfectionwiththeinhibitorsofPI3K,LY294002andWortmannin,significantlyreducedHIVintegrationasmeasuredbyAlu-LTRPCR,andreducedthelevelsof2-LTRcirclesbacktothelevelofunactivatedcells(Fig.
2b,c).
TodefinethespecificpathwaysdownstreamofthechemokinereceptorimportantforHIVintegration,wenexttestedinhibitorsofp38,MEK1/ERK1/2,JNK,AP-1andNF-κB,priortoinfectionofCCL19-treatedrestingCD4+Tcells.
HIVintegrationwasreducedtoundetect-ablelevelsinthepresenceofinhibitorstoMEK1/ERK1/2,JNK,AP-1andNF-κB(Fig.
2d).
Incomparison,thep38inhibitorSB203580hadamildinhibitory(butnotsignifi-cant)effectonHIVintegration(Fig.
2d),despitebeingabletofullyinhibitp38phosphorylation(Fig.
1).
Theseinhibi-torsalsoreducedthelevelof2-LTRcirclesbacktounac-tivatedcellslevel,althoughthereductiondidnotreachstatisticalsignificance(Fig.
2e).
Theeffectoftheseinhibi-torsonHIVintegrationand2-LTRcircleswerenotlikelyduetothecytotoxicityofthesedrugs(Additionalfile3:FigureS3).
Wefoundthatthebroad-spectruminhibitorofAktandNF-κB,Bay11-7082,washighlytoxictorestingCD4+Tcellsandwasremovedfromfurtheranalysis.
Pin1isacellularfactorrequiredforHIVintegrationinCCL19treatedcellsSinceJNKactivationphosphorylatesandstabi-lizesHIVintegraseviaaninteractionwithpeptidylFig.
2TreatmentwithPI3KandRas/Raf/MEKinhibitors,eliminatesHIVintegrationinCCL19-treatedrestingCD4+Tcells.
aRestingCD4+T-cellswerepre-incubatedwithinhibitorsofspecificsignallingpath-waysfor1hbeforeadditionofCCL19,PHA-IL2orDMSOandtheninfectedwithHIVNL4-3for2handculturedwithmediacontainingIL2(1U/mL)forupto4daysfollowinginfection.
HIVintegrationwasmeasuredbyqPCRforAlu-LTR(b,d)andnuclearentrywasmeasuredbyqPCRfor2-LTRcircles(c,e).
ExperimentswerealsoperformedinthepresenceofinhibitorstoPI3K(LY294002andWortmannin;b,c).
d,eFurtherexperimentswereconductedinthepresenceofinhibi-torsofp38(SB203580),ERK1/2(PD980509),JNK(SP600125),AP-1(SR11302),orNF-κB(SC-514)activation.
Eachcolumnrepresentsthemeancopynumberandthesymbolsrepresentindividualdonors.
ThedetectionlimitfortheAlu-LTRwas300copies/106cellsandisshownasadashedline.
*p95%,asassessedbyflowcytometry.
Celllysis,immunoblotting(IB)andimmunoprecipitation(IP)TomeasuretheeffectofCCL19onactivationofthePI3Ksignallingpathway,wefirstdeterminedthedoseandkineticsofCCL19onkeysignallingproteinsphos-phorylationinrestingCD4+Tcells.
Inbrief,freshlyiso-latedrestingCD4+TcellswereculturedovernightinRF1[RPMI1640mediasupplementedwith1*penicil-lin–streptomycin–glutamine(LifeTechnologies,Carls-bad,CA)and1%heat-inactivatedfetalbovineserum(BovogenBiologicals,Melbourne,Australia)].
Onthefollowingday,fivemillionviablecellsperconditionwasstimulatedwithvariousdosesofCCL19(30,100,200and300nM)forexactly5or15minandimmediatelylysedwithRIPAbuffer(1%NP40,0.
5%SDS,1mMDTT,10mMTris–HClpH7.
6,150mMNaCl,2mMEDTA)containingHaltproteaseandphosphataseinhib-itorcocktail(ThermoFisherScientific,Waltham,MA).
Unstimulatedorphorbol12-myristate13-acetate(PMA,10g/mlSigma-Aldrich,St.
LouisMO)andionomycin(2M,Sigma-Aldrich)stimulatedcellswereusedascontrols.
ProteinconcentrationsofcelllysatesweremeasuredbytheBradfordproteinassay(Bio-Rad,Hercules,CA).
Totalcelllysates(50μg)wereresolvedona10%SDS-polyacrylamidegel,transferredontonitrocellulosemembrane,andprocessedfurtherforimmunoblotingaspreviouslydescribed[9].
Themembranewassequen-tiallyprobedwith1:1000ofanti-p-NF-κB(ser529,BDBiosciences,SanJose,CA),anti-p-Akt,anti-p-JNK,anti-p-ERK1/2,anti-p-p38,andanti-glyceraldehyde3-phosphatedehydrogenase(GAPDH;allfromCellsig-nallingTechnologies,Danvers,MA),andre-probedwitheitherhorseradishperoxidase-conjugatedanti-mouseoranti-rabbitsecondaryantibodies(CellSignallingTech-nologies).
Thesignaldensitywasanalysedusingelec-trochemiluminescence(ECL)plusWesternblottingdetectionreagents(GEHealthcare,UK)andMF-Chemi-BISchemiluminescenceimaging(DNRBio-Imagingsys-tems,JerusalemIsrael).
Immunoprecipitationexperimentswereperformedusing200goftotalcelllysatefromCCL19orPHA-IL2_treatedCD4+TcellsinthepresenceorabsenceofJNKinhibitor,SP600125(10M,Sigma-Aldrich).
PrimaryantibodiestoPin1(SantaCruzBiotechnologyInc,Dallas,TX)wasaddedandincubatedovernightat4°Cfollowedbytheadditionofprotein-G-sephrosebeads(Genscript,Piscataway,NJ)foranother3h.
Afterwashingthebeads,thesamplesweresubjectedtoIBanalysisusinganti-HIVintegraseantibody(Abcam,Cambridge,UK).
InhibitionofPI3KsignallingpathwayusingpharmacologicalinhibitorsFreshlyisolatedpurifiedCD4+Tcellswererestedover-nightinRF1andincubatedinthepresenceorabsenceofeitherPI3KinhibitorsLY294002(50M)andWortman-nin(100nM);p38inhibitorSB203580(5M);MEK1/ERK1/2inhibitorPD980509(50M);JNKinhibitorSP600125(10M,AllfromSigma-Aldrich);NF-κBinhibitorsSC-514(100M)andBay11-7082(10M,Calbiochem,SanDiego,CA);orAP-1inhibitorSR11032(1M,TocrisBiosciences,Bristol,UK)for1h.
Cells(5million)weretreatedwith100nMofCCL19for15min,lysedandimmunoblottedasdescribedabove.
Unstimu-latedorphorbol12-myristate13-acetate(PMA,10g/mlSigma-Aldrich)andionomycin(2M,Sigma-Aldrich)stimulatedcellswereusedascontrols.
ProteinintensitywasanalysedusingdensitometryofgelimagesusingFIJIsoftware[47].
Plasmidsandvirusproduction293Tcells(ATCC,Manassas,VA)weretransfectedaccordingtothemanufacturer'sinstructions(FuGene;RocheDiagnostics,Indianapolis,IN)withtheplasmidcodingforCXCR4(X4)-usingvirusNL4-3(kindlysup-pliedbyProf.
DamianPurcell,UniversityofMelbourne,Australia).
ViralplasmidswithmutationsintheNF-κBbindingsitesintheHIVlongterminalrepeat(LTR)werekindlyprovidedbyDr.
MonsefBenkirane(InstituteofHumanGenetics,Montpellier,France[48])andincludedthosewitheitherasinglemutation(GGG→CTC)atposition105(site1)orposition92(site2),oratbothsites(doublemutant)[49].
Followingtransfection,cul-turesupernatantswereconcentratedoversucrosegradi-entsandassessedforreversetranscriptase(RT)activity[8].
InfectionofrestingCD4+TcellsRestingCD4+Tcellswereisolatedbymagneticbeaddepletionandcellsorting,aspreviouslydescribed[8].
PurifiedcellswereincubatedwithCCL19(30nM)for24hpriortoinfection.
Whenpharmacologicalinhibi-torsofPI3Ksignallingwereused,inhibitorswereadded1hpriortotheadditionofCCL19.
Cellsactivatedfor2dayswithphytohemagglutinin(PHA,10μg/ml;Sigma)andIL2(10IU/ml,RocheDiagnostics)orleftunstimu-latedwereusedascontrols.
CellsweretheninfectedwithNL4-3virus,orwithNL4-3thathadamutationinoneorbothNF-κBbindingsitesat1reversetranscriptase(RT)countperminute(CPM)/cellfor2hat37°CandwashedbeforefurtherculturewithIL2(1IU/ml).
Page10of12Salehetal.
Retrovirology(2016)13:49CytotoxicityassayToaddressthetoxicityofthepharmacologicalagentsused,freshlyisolatedrestingCD4+TcellsweretreatedwiththesamedoseofpharmacologicalinhibitorsinthepresenceofCCL19(30nM)for48h.
Cellswerethenwashedandculturedforafurther72h.
Thepercentageofviablecellsonday2andday5wasdeterminedbyflowcytometryusinglivefixableviabilitydyeeFluor780(eBioscience,SanDiego,CA),accordingtomanufactur-er'sinstruction.
QuantificationofHIVinfectionFollowinginfection,RTwasquantifiedincellculturesupernatantaspreviouslydescribed[8].
IntegratedHIVDNAand2-LTRcircleswerequantifiedusingrealtimePCR(iCycler,Bio-Rad,Hercules,CA)aspreviouslydescribed[9,50].
Smallinterfering(si)RNAnucleofectionRestingCD4+Tcellsweretransfectedwithpeptidylpro-lyl-isomerase(Pin1)specificsiRNA(sc-36230-SH;SantaCruz,BiotechnologyInc,Dallas,TX)orcontrol(scram-bled)siRNA(sc-37007,SantaCruzBiotechnologyInc),at100pmolperreactionusingnucleofection(AmaxaHumanTcellNucleofectorkit,Lonza,Cologne,Ger-many)accordingtothemanufacturer'sinstruction.
Pin1siRNAsequences:5′-GUCAGAUGCAGAAGCCAUU-3′,5′-CCGAAUUGUUUCUAGUUAG-3′,and5′-UCCU-CUGUUCAGUCGCAAA-3′.
KnockdownofPin1proteinwasconfirmedbyImmunoblotusinganti-Pin1antibody(SantaCruzBiotechnologyInc).
TransfectedcellsweremaintainedinRPMI1640with10%Fetalcalfserumforupto48handstimulatedwitheitherPHA-IL2orCCL19priortoHIVinfectionoranalysedbyIB.
CloningandsequencingofHIVintegrationsitesRestingCD4+TcellswereactivatedwithPHA-IL2,CCL19,orleftunactivatedbeforeinfectionwithHIV.
Cellswereharvestedatday4postinfectionandthelev-elsofviralintegrationwereassessedbyreal-timePCRassayforAlu-LTR[50].
TheidentificationofHIVinte-grationsiteswasdeterminedbyaninversePCRassayaspreviouslydescribed[24].
TheintegrationsitesequencesweremappedontothehumangenomeusingUniversityofCaliforniaSantaCruz(UCSC)GenomeBioinformaticsdatabase.
Allthegenomicfeaturedatasetsweredown-loadedfromtheUCSCgenomedatabase.
Thepatternofthesefeatureswascomparedbetweentheinvitrocondi-tionsandCD4+TcellsfromHIV-infectedpatientsonantiretroviraltherapyfrompublisheddata[25].
Toassesspatternsinrelationtogenomicfeatures,suchasDNAmethylationand/oracetylationpattern,thedistancefromthesegenomicfeatureswascalculatedusingGenomicHyperbrowser(http://hyperbrowser.
uio.
no/hb/).
Geneexpressionintheinvitroinfectedcellswasdeterminedusinggenearrayanalysis[51].
CellswereactivatedwithCCL19foreither6hor72hbeforeinfection,geneexpressionfromthetwotime-pointswerecomparedtounactivatedorPHA-IL2activatedcells.
StatisticalanalysisGraphpadPrismwasusedforstatisticalanalysesandpreparingplots.
AKruskal–Wallistestwasusedforcom-parisonsbetweengroupsandaMann–Whitneytestforanalysiswithingroups.
ForcomparisonsbetweengroupseitheraStudent'sttestoraMann–WhitneyUtestwasused.
Normalizationwasperformedbylogtransforma-tionbeforeanalysis.
ThestatisticalprogramR[51]wasusedforanalysisofgenearrays,clusteranalysisandheatmapgeneration.
AStudent'sttestorMann–Whitneytestwasusedforcom-parisonsbetweenpopulationsandp<0.
05wasconsid-eredsignificant.
Forthesiteofintegration,aFisher'sexacttestwasusedtodeterminethestatisticalsignificancebetweenthegroupswhenexaminingtheproportionofintegrationsitesthatwerenearorfarfromaspecificgenomicfea-ture.
Inaddition,wetreatedthemediandistanceofinte-grationsitesasameasureofassociationforthatgenomicfeature.
Sincethepopulationsofintegrationsitesfailedthenormalitytests,weusedanon-parametricKruskal–WallisANOVAtodeterminesignificance.
WethenusedaDunn'stestwithBonferronicorrectiontodeterminethedifferencebetweeneachgroup.
AdditionalfilesAdditionalfile1:FigureS1.
SignallingpathwaysdownstreamofCCR7.
SchematicrepresentationofthesignallingpathwaysactivatedbyPI3KandRasfollowingchemokineligation.
Thesiteofactionandnamesofspecificinhibitorsareshownasredlines.
Figureisbasedon[20,52–54];andtheKEGGChemokinesignallingpathway;http://www.
genome.
jp/kegg-bin/show_pathwaymap04062.
Additionalfile2:FigureS2.
DoseresponseofCCL19onrestingCD4+Tcells.
RestingCD4+TcellswereincubatedwithvariousconcentrationsofCCL19for5minutes(A)or15minutes(B)andthelevelofintracellularphosphorylatedproteinsexamined.
Celllysateswereassessedbyimmu-noblotingusingantibodytophosphorylatedAkt(pAkt),pNF-κB,pERK,pJNKandloadingcontrolGAPDH.
CellstreatedwithPMAandIonomycinwasusedasapositivecontrol.
Datarepresentimmunoblotsoftwoinde-pendentexperiments.
Additionalfile3:FigureS3.
CytotoxicityofsignallinginhibitorsonCD4+Tcells.
RestingCD4+Tcellsweretreatedwithvariousinhibitors(see"Methods"forconcentrationsused)inthepresenceofCCL19andincu-batedfor48h.
Cellswerethenwashedandculturedforanother72h.
Cellviabilitywasdeterminedusinglive/deadstainingandanalysedbyflowcytometer.
Datarepresentsmean±SDoftwoindependentexperiments.
Additionalfile4:FigureS4.
Integrationsiteselectionandgeneactiva-tioninchemokinetreatedcells.
A,GeneexpressionwasdeterminedbyIlluminabeadarrayinunactivated,CCL19-treatedorPHA-IL2activatedPage11of12Salehetal.
Retrovirology(2016)13:49Authors'contributionsPUC,SRL,AJ,DV,SS,HLandJMconceivedanddesignedtheexperiments;SS,HL,GS,DV,DH,KC,ST.
,TA,JZ,AHperformedexperiments;SS,HL,AJ,DV,DH,KC,ST,TA,JZ,JA,AH,TC,LG,MC,HD,PUC,SRLanalysedthedata;AH,TC,LG,MC,JM,HD,contributedreagentsmaterialsandanalysistools;SS,HL,DV,AJ,VE,JA,PUCandSRLwrotethemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
Authordetails1DohertyInstituteforInfectionandImmunity,TheUniversityofMelbourne,Melbourne,Australia.
2DepartmentofInfectiousDiseases,AlfredHealth,MonashUniversity,Melbourne,Australia.
3CentreforBiomedicalResearch,BurnetInstitute,Melbourne,Australia.
4CentreforVirusResearch,WestmeadMillenniumInstitute,UniversityofSydney,Sydney,Australia.
5DepartmentofMicrobiology,MonashUniversity,Melbourne,Australia.
6DepartmentofInfectiousDiseases,SchoolofMedicine,DeakinUniversity,Melbourne,Australia.
7DivisionofHematology–Oncology,DepartmentofMedicine,DavidGeffenSchoolofMedicineatUCLA,LosAngeles,CA,USA.
8UniversityCaliforniaLosAngeles(UCLA)AIDSInstitute,DavidGeffenSchoolofMedicineatUCLA,LosAngeles,CA,USA.
AcknowledgementsWethankthestaffoftheflowcytometryunitattheAlfredMonashResearchandEducationPrecinctforassistancewithsortingandanalysisbyflowcytom-etry.
WewouldliketothanktheUCLA/CFARVirologyCorelaboratoryforPCRsupportneededforHIVintegrationsiteanalysis.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
EthicsstatementTheuseofbloodsamplesfromnormaldonorsforthisstudywasapprovedbytheAlfredHospital(HREC156/11)andMonashUniversity(CF11/1888)HumanResearchandEthicsCommittees.
DonorswererecruitedbytheRedCrossBloodTransfusionServiceasnormalblooddonorsandallprovidedwritteninformedconsentfortheuseoftheirbloodproductsfortheresearch.
FundingsourcesSRLisanAustralianNationalHealthandMedicalResearchCouncil(NHMRC)PractitionerFellow.
ThisworkwassupportedbygrantsfromtheNationalInstitutesofHealth(NIH)U19-AI096109and1R56AI095073-01A1(SRLandPUC),R21DA031036andR21AI106472(DV),theAmericanFoundationforAIDSResearch(SS,PUC,SRL)andtheNHMRC(491154and1002761).
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B,Expressionofindi-vidualgenesatthesiteofHIVintegrationinCCL19-treatedrestingCD4+Tcells(x-axis)comparedtounactivated(y-axis;upperpanel)orPHA-IL2activatedCD4+Tcells(y-axis;lowerpanel).
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Only2genesNDS1andTBCD(blue)werecom-monwiththelistofgeneswithmultipleinsertionsitesreportedinCD4+TcellsfromHIV-infectedpatientsoncART[25–27,32,33].
Genesymbolsforeachoftheinsertionsitesareshownwithineachgroup.
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