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RESEARCHOpenAccessTheHR2polymorphismN140IintheHIV-1gp41combinedwiththeHR1V38AmutationisassociatedwithalesscytopathicphenotypeFrancescCunyat1,SilviaMarfil1,ElisabetGarcía1,ValentinaSvicher2,NuriaPérez-Alvárez3,4,MartaCurriu1,CarloFedericoPerno2,BonaventuraClotet1,3,JuliàBlanco1andCeciliaCabrera1*AbstractBackground:Resistancetothefusioninhibitorenfuvirtide(ENF)isachievedbychangesinthegp41subunitoftheHIVenvelopeglycoprotein(Env).
SpecificENF-associatedmutationalpathwayscorrelatewithimmunologicalrecovery,evenaftervirologicalfailure,suggestingthattheacquisitionofENFresistancealtersgp41pathogenicity.
Totestthishypothesis,wehavecharacterizedtheexpression,fusioncapability,inductionofCD4+TcelllossandsingleCD4+Tcelldeathof48gp41proteinsderivedfromthreepatientsdisplayingdifferentaminoacids(N,TorI)atposition140thatdevelopedaV38AmutationafterENF-basedtreatment.
Results:Inallcases,intra-patientcomparisonofEnvisolatedpre-orpost-treatmentshowedcomparablevaluesofexpressionandfusogeniccapacity.
Furthermore,EnvwitheitherNorTatposition140inducedcomparablelossesofCD4+T-cells,irrespectiveoftheresiduepresentatposition38.
Conversely,EnvacquiringtheV38Amutationina140IbackgroundinducedasignificantlyreducedlossofCD4+Tcellsandlowersingle-celldeaththandidtheirbaselinecontrols.
Noalteredabilitytoinducesingle-celldeathwasobservedintheotherclones.
Conclusions:Overall,primarygp41proteinswithbothV38AandN140IchangesshowedareducedabilitytoinducesinglecelldeathanddepleteCD4+Tcells,despitemaintainingfusionactivity.
ThespecificityofthisphenotypehighlightstherelevanceofthegeneticcontexttothecytopathiccapacityofEnvandtheroleofENF-resistancemutationsinmodulatingviralpathogenicityinvivo,furthersupportingthehypothesisthatgp41isacriticalmediatorofHIVpathogenesis.
Keywords:HIV,gp41,enfuvirtide,singlecelldeath,fusogenicityBackgroundHIVinfectioncausesaprogressivedepletionofCD4+Tcells,whichleadstothedevelopmentofAIDS[1,2].
AlthoughCD4+TcelllossinHIVinfectionisamultifa-cetedprocess[3-5],thedeathofbystanderCD4+TcellsseemstobeoneofthemaincontributorstoHIV-inducedpathogenesis[6-8].
VariousmechanismshavebeenproposedtoexplainthedestructionofbystanderCD4+Tcells,includingapoptosis,autophagyorabortiveinfection[6,8-11].
TheHIVenvelope(Env)glycoprotein,whichmediatesviralentryintothehostcellbyfusionoftheviralandhostcellmembranes(reviewedin[12-14]),isoneoftheviralfactorsinvolvedinthedeathofbothinfected[15]andbystandercells[7,8,16].
TheEnvcom-plexiscomposedoftwonon-covalentlylinkedsubunits,namely,thesurfaceglycoprotein(gp120)andthetrans-membraneglycoprotein(gp41),andisdisplayedashet-erotrimersonthesurfaceofvirionsandinfectedcells[14,17-20].
Viralentryisamultistepphenomenon:theinteractionofgp120withthehostcellsurfaceCD4-receptor,andeitherCCR5orCXCR4coreceptorenablesgp41subunitstotriggerhemifusionevents,therebyleadingtofusion.
TheHIVgp41isaclassictype1fusionproteinthatcontainsthreedomains:anectodo-main,amembrane-spanningdomain,andalongintra-cytoplasmicsegment.
Theectodomainofgp41consists*Correspondence:ccabrera@irsicaixa.
es1IrsiCaixa-HIVACAT,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol(IGTP),HospitalGermansTrias,UniversitatAutònomadeBarcelona,Badalona08916Barcelona,Catalonia,SpainFulllistofauthorinformationisavailableattheendofthearticleCunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/152012Cunyatetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
ofanN-terminalfusionpeptidefollowedbytwocon-servedcoiled-coildomainsthatarereferredtoasC-andN-terminalheptadrepeats(HR1andHR2),whichareconnectedbyanon-helicalloopregion.
TheseHRinter-actwitheachotherinaleucinezipper-likefashiontomediatemembranefusion[21].
SyntheticpeptidesthatbindtooneoftheHRmotifsinterferewiththeirinter-actionandthusinhibitviralentry[22,23].
Enfuvirtide(ENF,T-20)isthefirstpeptideapprovedforclinicaluseinHIVsalvagetherapy.
Thisdrugisa36-aminoacidpeptidethatwasdesignedbasedontheamino-acidsequenceoftheHR2domainofthegp41subunit.
ThispeptidepreventstheHR1-HR2interactionbybindingtotheHR1domain[22,24,25].
Thetherapeu-ticbenefitsofENFtherapyhavebeendemonstratedbyincreasesinCD4+Tcellcountsandasignificantreduc-tioninHIVRNAlevels[26-28].
Nevertheless,ENF-resistantHIV-1variantsrapidlyemergeunderdrugpressurewhenvirusreplicationisnotcompletelysup-pressed[29-31].
SequenceanalysisofENF-resistantviralpopulationsrevealedtheacquisitionofmutationswithintheHR1domainatpositions36-38(GIV)[29,30],whichwereassociatedwithareductioninviralinfectivity,probablyasaconsequenceofimpairedinteractionbetweenHR1andHR2[32,33].
However,certaincom-pensatorymutationswithinHR2mayariseandrestoreviralinfectivity[29,32,34-37].
Despitevirologicalfailure,specificmutations(theclusterV38A+N140I)havebeenassociatedwithanincreaseinCD4+Tcellcounts[38-40].
TheEnvglycoproteinplaysacrucialroleinthedeple-tionofCD4+Tcellsbyinducingthedeathofsinglebystandercells,whichismediatedbygp41[41,42].
Therefore,changesingp41thatemergeunderENFpressurecouldinduceachangeintheviralpathogeni-city.
Althoughsite-directedpointmutationsatposition38ingp41havebeenshowntoexhibitdeficiencyincell-to-cellfusionactivityandapoptosisinductioninvitroandinahumanizedmousemodel[43,44],itisimportanttonotethatthegeneticbackgroundhasbeenprovenrelevantforfunctionalevaluationoftheENF-resistantEnvsbecausetheremaybecompensatorychangesthatrestoretheinfectivityofthevirus[32,34,36,37,45,46].
Theobjectiveofthecurrentstudywastoevaluatethepathogenicityofseveralpatient-derivedgp41proteinsisolatedfromhighlyexperiencedpatientsreceivinganENF-containingsalvagetherapyandwhetherchangesatposition38and140ingp41haveanimpactinthebio-logicalpropertiesofpatient-isolatedEnvs.
Ourresultsindicatethattheprimarygp41Envproteins,withbothV38AandN140Ichanges,inducedlowerlevelsofsin-gle-celldeathanddepletionofCD4+Tcells,althoughtheyretainedcell-to-cellfusionactivity.
However,themutationV38Ainthecontextofa140Nor140TchangedidnotalterEnvfunctions,underscoringtheimportanceoftheEnvgeneticbackgroundinthemodu-lationofthecytopathiceffectsoftheHIV-1Envglycoproteins.
ResultsanddiscussionPatientsandenvelopeconstructionsInapreviousreport,wecharacterizedgp41proteinsderivedfrom13heavilypre-treatedHIV-1-infectedpatientsreceivinganENF-containingsalvagetherapy[29].
Severaldrugresistance-associatedmutationsweredetectedalongtheentiregp41ectodomain,mainlymap-pingintheHR1domainatpositions36,38and43.
ClinicalfindingshavesuggestedthatcertainENF-resis-tantmutantsarisingduringsalvagetherapy,specifically,theclusterV38A+N140I,areassociatedwithanincreaseinCD4+cellcounts,evenaftervirologicalfailure[38-40,43,44].
Wethereforereasonedthatgp41proteinsderivedfrompatientswithdifferentcombinationsofaminoacidsatpositions38and140couldhavedifferentpathogeniceffects.
Wechosetostudythegp41proteinsderivedfromthreepatients:patients1,9and10,whohadmutationsassociatedwithENFresistanceatposi-tion38inthegp41viralproteinbutdifferedintheaminoacidfoundatposition140[29].
Twoplasmasamplesfromeachpatient,whichwerecollectedatbaselineandduringtreatment,wereusedtoconstructgp160hybridproteins(allbearingthegp120fromanNL4-3virusandthegp41derivedfromthepatients).
Table1summarizesthecharacteristicsofthepatientsatthetimepointsofviralRNAisolation.
Atleast15recombinantexpressionplasmidswereconstructedfromeachsample,andallrecombinantplasmidswerefullysequencedtoverifythatthegp120sequencepresentwasconservedamongtheclonesandthattheNL4-3wtsequenceremainedunchanged(datanotshown).
Theaminoacidsatpositions38and140ofgp41weresubse-quentlydetermined,and48recombinantplasmidswerefinallyselected(Figure1).
Amongtheseplasmids,13cloneswerederivedfrompatient9,containinganaspar-agineatposition140(140N)andthewild-type(wt)aminoacidatposition38(38V)oranalanineatposi-tion38(38A)(n=5andn=8,respectively);19cloneswerederivedfrompatient10,whocontainedthesubsti-tutionN140Tandthewt38V(n=10)ortheV38Amutation(n=9);andfinally,16cloneswerederivedfrompatient1,whohadthepolymorphismN140Iandthewtaminoacidatposition38(n=10)ortheV38Amutation(n=6,Figure1).
Sinceweclonedthefullgp41proteinpresentinvivo,wewereabletoidentifyotherchangesthroughoutthegp41proteininadditiontothechangesatpositions38and140.
ChangeswerefoundprimarilyintheHR2domain,butalsoupstreamCunyatetal.
Retrovirology2012,9:15http://www.
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com/content/9/1/15Page2of11oftheHR1domain,intheHR1domainandintheloopsection.
MostoftherecombinantplasmidsconstructedfromsequencesobtainedduringENFtreatmentcarriedtheV38AmutationastheonlychangeassociatedwithENFresistance,althoughfourclonesderivedfromthe140NpatientcarriedtheN42Tmutation,andthreeothersshowedtheN126Kmutation.
Theanalysisofacaveolin-1bindingmotifinthegp41protein,whichhasbeenrecentlyreportedtoaffectHIV-1pathogenesis[47],showedthatonlyoneplasmidfromthepatientharboringthe140NbackgroundcarriedanMtoVsub-stitutionatposition115.
Theremainingplasmidscon-structedwithsequencesfromthispatientandalloftherecombinantconstructsfromtheothertwopatientsshowednochangesinthisregionbeforeoraftertreat-ment(Figure1).
Ourcloningapproach,usingonlythegp41ofthepatientinsteadoftheentireEnv(gp41+gp120),allowsustospecificallyassesstheeffectofTable1Characteristicsofthethreepatientsreceivinganenfuvirtide-containingsalvagetherapywhensampleswerecollectedPatientaSampleWeeksonENFtreatmentPlasmaviralload(copies/mL)CD4+cellcount(cells/μl)No.
ofexpressionplasmidsconstructed140N(9)140N03663574915V38A140N2485367008N140T(10)N140T01414971310V38AN140T4332794139N140I(1)N140I03347014510V38AN140I12108061506aThepatientIDsfromapreviouswork[29]areindicatedinparentheses.
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HR1HR2N140N140TN140ICav-1BindingDomainPosition38Position140Figure1Thesequencealignmentofthegp41genefromtherecombinantenvelope-expressingplasmidsconstructedfrompatientsamples.
Thealignmentofthegp41ectodomainsequence(HR1andHR2domains)fromtheselectedrecombinantplasmidsderivedfromthreepatientswhofailedanENF-basedtreatmentandcarriedaV38Amutation(HR1box)withtheaminoacidI,NorTatposition140(HR2box).
TheCav-1bindingdomainintheHR2regionishighlighted.
Cunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page3of11certainENFresistancemutationsongp41pathogenesis,avoidingadditionaleffectsofchangesingp120proteinitselfandimportantlyavoidinganyinterferenceoftheCXCR4ortheCCR5coreceptoruse,whichisamajordeterminantofEnvpathogenicitybothinvivoandinvitro[48,49].
Ifviruseswithdifferentcoreceptorusagehadbeencompared,wewouldnothavebeenabletodiscerntheEnvsubunitresponsiblefortheobserveddifferences.
CellsurfaceexpressionofHIV-1recombinantenvelopeglycoproteinsAftertheoptimizationofthetransfectionandthecellsurfacestainingprocedures(CunyatF,CurriuMetal.
,JBiomolecularScreening,inpress),HeLacellsweretran-sientlytransfectedwiththe48recombinantEnv-expres-singplasmids,and24hourspost-transfection,thecellsurfaceexpressionofEnvwasanalyzedusingaprimaryantibodyagainstthegp120subunit(2G12).
Ananti-gp120antibodywasused,insteadofananti-gp41anti-body,toavoidartifactsduetochangesinthegp41pro-teinthatcouldaffectthebindingoftheantibody.
AlltestedEnvswereexpressedonthecellsurfacewithexpressionlevelsrangingfrom2.
7%to29.
9%ofpositivecells(Figure2A).
Whenaninter-patientcomparisonwasperformedbygroupingalloftheEnvsobtainedfromeachpatientirrespectiveofthetimepoint,asignif-icantlydifferentpercentageofEnv-expressingcellswasobservedbetweenplasmidsconstructedfromthe140N-andN140T-carryingpatients.
ThelowestpercentageofEnv-positivecellswasobservedforthe140Nconstructs(mean=11.
40+/-3.
9),whereasthehighestpercentageofEnv-expressingcellswasobservedfortheN140Tclones(mean=15.
91+/-4.
4)(Figure2A).
However,whenweanalyzedthepercentageofcellsexpressingtheEnvconstructsobtainedfromthesamepatientbycom-paringclonesdisplayingornottheV38Amutation(anintra-patientcomparisonafterandbeforetreatment,respectively),theEnvexpressionwassimilarinallcases(Figure2A).
Inadditiontodeterminingthepercentageofpositivecells,theEnvexpressionlevels,whichcouldplayanimportantroleinEnvpathogenesis,wereevalu-ated.
TherewerenodifferencesintheEnvexpressionlevelsbetweenconstructscontainingwtgp41andthosecontainingthe38Amutationfromthesamepatient,asdeterminedbythegeometricmeanfluorescenceinten-sity(datanotshown),ortherelativefluorescenceinten-sity[50],whichisameasureofthetotalEnvexpression(Figure2B).
Thus,theintra-patientcomparisonssuggestthattheexpressionofEnvdoesnotchangeuponacqui-sitionofthe38Amutation,andthelevelofEnvexpres-sionisanintrinsiccharacteristicoftheparticularEnvcarriedbyeachinfectedpatient.
TheseresultsallowedustoanalyzethecytopathiceffectsofEnvwithoutcor-rectingforcellsurfaceexpressionofEnv.
AnalysisoftheEnvproteinfusogenicityThefunctionoftheHIVEnvglycoproteinistofacilitatetheentryoftheviralnucleocapsidintothetargetcell.
ThisprocesshasanimportantroleinHIVpathogenesis,andthefusogenicactivityofHIVEnvhaslongbeenassociatedwithcytopathiceffects[50,51]bothinvitroandinvivo[52-54].
Inagreementwiththisproperty,ithasbeendescribedthatsingle-amino-acidmutationsintheectodomain(V38A/E)ortransmembraneregionsofgp41reducecell-to-cellfusionactivityofthevirus[43,55].
AnumberofdifferentassaysystemshavebeenreportedtomeasuretheHIVenvelopeactivity.
How-ever,wehavedescribedtheimportanceofselectinganappropriatecelllinetoexpressEnvwhenthecytopathicpropertiesofclinicallyderivedgp41glycoproteinsinvitroareevaluated(CunyatF,CurriuMetal.
,JBiomole-cularScreening,inpress).
Inthisstudy,twoenvelope-expressingeffectorcelllines(293TandHeLacells)werecomparedtoevaluatetheactivityofpatient-derivedEnvs:fusion,absolutecelllossandsinglecelldeath.
Theresultsshowedadifferentialbehaviourbetweenbothcelllines.
293Teffectorcellsseemtohavearapidfor-mationofthefusionpore,generatinghighlevelsoffusionandlowerlevelsofsinglecelldeath.
Incontrast,HeLacellswouldfuseslowly,inducinggreaterextentsofsinglecelldeath.
Thus,HeLacellsshouldbeprefer-entiallyusedfortheevaluationofcelldeathparameters,andthe293Tcelllineshouldbeusedwhenenvelopeswithlowfusogeniccapacityareevaluated.
Basedonthisrecommendation,allofourrecombinantEnvswerefirstexpressedin293Tcells,andassayedforfusionactivity.
DetectablefusionwasobservedforallEnv,showingfusionlevelsoverthe50%whencomparedwithanNL4-3wtEnv(datanotshown).
SinceallourEnvwerefusogenic,weusedHeLacellastheeffectorcelllineinourassays.
Thesecellsweretransientlyco-transfectedwiththeEnv-andpcTat-expressingplasmidsandcocul-turedwiththereporterTZM-blcells.
Aftersixhoursofcoculture,theluminescenceinthesamplewasmea-sured,andtherelativefusioncapacityofeachrecombi-nantEnvwascalculatedincomparisontothefusionvaluesobtainedusingtheNL4-3wtEnv,whichwasusedasacontrol(100%).
TheleveloffusionoftheEnvsobtainedfromdifferentpatients(inter-patientanalysis)showedsignificantdifferences,underscoringthattheviruseachpatientcarriesmayhaveanEnvwithadis-tinctfusogeniccapacitywhich,inthiscase,isdeter-minedbygp41.
DifferencesinfusionwerenotcorrelatedwiththeexpressionlevelofEnvonthecellsurfacebecauseahigherexpressionleveldidnotresultCunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page4of11ABFigure2TheexpressionoffunctionalrecombinantenvelopesontransfectedHeLacells.
Env-expressingplasmidsconstructedfromthreepatientscarryinganaminoacidN,TorIatposition140with(V38A)withoutsubstitutionsatposition38ofgp41weretransfectedintoHeLacells.
ThesurfaceexpressionofEnvwasanalyzed24hourspost-transfectionbystainingthecellswiththe2G12antibody.
EnvexpressionwasdeterminedbythepercentageofEnv-positivecellsinaninter-patientanalysisusingtheclonesconstructedfromeachpatientorinanintra-patientanalysisusingclonesconstructedfrombaselineandaftertreatment(V38A)samples(A).
ThetotallevelofEnvexpressionwasdeterminedbycalculatingtherelativefluorescenceintensity(RFI=%ofEnv-positivecells*geometricmeanfluorescenceofEnv-positivecells)inanintra-patientanalysis(B).
Baselinesamples(whiteboxes)andV38Asamples(grayboxes).
Theboxesrepresentthemedianandinterquartilerangeofthevalues.
ThemedianvalueswerecomparedusinganonparametricMannWhitneytest.
*p95%CD4+Tcellsasdeterminedbyflowcytometry.
TheisolatedCD4+Tcellswereincubatedovernightat37°CinRPMImediasupplementedwith10%ofFCSpriortouse.
AllofthemediawerepurchasedfromInvitrogen(Madrid,Spain).
TheCXCR4antagonistJM-2987(hydrobromidesaltofAMD-3100)[59]andtheCCR5antagonistTAK-779[60,61]wereobtainedthroughtheNIHAIDSResearchandReferenceProgram.
Thebroadlygp120neutralizingantibody2G12andthesecondaryantibodygoatanti-HumanIgGwereobtainedfromPolymun(Vienna,Aus-tria)andJacksonImmunoResearchLaboratories(Penn-sylvania,USA),respectively.
TheTatexpressionplasmidpcTatwasobtainedthroughtheNIHAIDSResearchandReferenceReagentProgram[62].
Cunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page8of11ThecelltrackerDichloro-DimethylAcridin-One(DDAO)waspurchasedfromMolecularProbes(Invitro-gen,Madrid,Spain).
ThecationicfluorescentdyePropi-diumIodide(PI)andthepotentiometricmitochondrialprobeDIOC6(3)werepurchasedfromSigma(Madrid,Spain)andInvitrogen,respectively.
PlasmidconstructionTheRNAfromtheplasmasampleswasisolatedbeforeandaftertheinitiationofENFtreatmentusingtheQIAmpViralRNAkit(Qiagen).
Full-lengthenv/revgeneswereamplifiedthroughRT-PCRusingspecificprimersaspreviouslydescribed[63].
AsubsequentnestedPCRwascarriedoutusingPlatinumTaqDNAPolymeraseHighFidelity(Invitrogen)toobtainafragmentcorrespondingtothegp41protein(primersMluF2andRNANestedRcorre-spondingtonucleotides7726-7747and8882-8904oftheHIVHXB2numberingsystem,respectively).
Afragmentcorrespondingtothegp120proteinwasamplifiedfromaNL4-3plasmid(primersRNANestedFandMluR2corre-spondingtonucleotides5954-5983and7727-7747oftheHIVHXB2numberingsystem,respectively).
Thepurifiedgp41andgp120products,whichoverlappedeachotherin22bases,werecombinedbyPCRandpurifiedtoobtaintherecombinantEnvs(gp120fromNL4-3andgp41frompatients).
AdirectionalcloningreactionwasperformedtoinsertthefragmentintotheplasmidexpressionvectorpcDNA.
3.
1D/V5/His-TOPO(Invitrogen),andseveraltransformedbacterialcolonieswereselectedforeachsam-ple.
Allrecombinantplasmidsweresequencedusingspeci-ficprimers,theBigDyeTerminatorv3.
1cyclesequencingkit(AppliedBiosystems)andanautomaticDNASequen-cer(3100GeneticAnalyzer).
Thesequenceswereedited(usingSequencher,v4.
7,fromtheGeneCodesCorpora-tion,AnnArbor,MIandGeneDoc,v2.
6,software),andtherecombinantplasmidswiththerequiredmutationswereselected.
TransfectionsHeLacellswereplatedatadensityof8*105cells/wellinsix-wellplatesandallowedtogrowovernight.
Thecellsweretransientlytransfected(usingLipofectamine2000Reagent,Invitrogen,Spain)with1.
3μgoftheEnv-expressingplasmidsforthecocultureswithprimarycellsorwerecotransfectedwiththeEnv-expressingplas-midsand2.
7μgofpcTatforthefusionassays.
Twenty-fourhourspost-transfection,thecellswerecollectedforfurtheranalyses.
Asnegativecontrols,cellsweremock-transfected(withthepcDNA3.
1vector)ortransfectedwithpcTatalone.
EnvelopeexpressionTwenty-fourhourspost-transfection,cellmembraneexpressionoftheEnvglycoproteinwasassessedbyflowcytometryafterindirectstainingwiththeanti-gp120monoclonalantibody2G12(4μg/ml)for20minat37°C,followedbystainingwithphycoerythrin-labeledgoatanti-humanIgG(RTfor15min).
Thecellswerewashed,fixedin1%formaldehydeandanalyzedbyaFACSLSRIIflowcytometer.
ThedatawereanalyzedusingFACSDivasoftware(BDBiosciences).
Mock-trans-fectedcellswereusedasanegativestainingcontrol.
ThepercentageofEnv-positivecellsandthegeometricmeanfluorescenceintensity(geoMFI)ofthesecellswereconsideredasindividualparametersorusedtocalculatetherelativefluorescenceintensity(RFI=%ofEnv-posi-tivecells*geoMFIofEnv-positivecells),asdescribedpreviously[50].
Cell-to-CellFusionassaysTwenty-fourhourspost-transfection,Env/pcTat-andpcTat-transfectedHeLacellswerecoculturedwiththereportercelllineCD4+/CCR5+/CXCR4+TZM-blfor6hoursin96-wellplatesinthepresenceorabsenceoftheCXCR4andCCR5co-receptorinhibitorsJM-2987andTAK-779(1μg/ml),respectively.
Thefusioneffi-ciencyofeachclonewasquantifiedbyassessingtheluminescenceofthecells(Britelitekit,PerkinElmer)withaLuminoskanAscentluminometer(Labsystems,Spain).
Envelope-induceddeathinprimaryCD4+Tcells:absolutecelllossandbystanderapoptosisEnv-inducedcytopathiceffectswereevaluatedusingacoculturesystemofEnv-expressingHeLacellsaseffec-torcellsandlabeledprimaryCD4+Tcellsastargetcells.
TheprimaryCD4+Tcellswerestainedwiththefarredcelltracker,DDAO(10μg/mL),for1hourat37°C.
Env+HeLacellsandCD4+/DDAO+Tcellswerecoculturedfor24hoursintheabsenceandpresenceoftheinhibitor,JM-2987(1μg/mL),andwerestainedwithDiOC6(3)(40nM)andPI(5μg/mL)for1hourat37°C.
Labeledmicrobeads(BeadsPerfectCount,Invitrogen)wereaddedtothestainedcoculturetoquantifytheabsolutecellloss,andflowcytometrywasperformedbyaFACSLSRIIflowcytometer.
ThedatawereanalyzedbytheFACSDivasoftware(BDBiosciences).
StatisticalanalysesThedatawerecomparedusingnon-parametricMann-Whitneytests.
AllstatisticalanalyseswereperformedusingGraphPadPrism,version5.
01,forWindows(Graph-PadSoftware,SanDiego,California,USA).
AP-valueof0.
05wasconsideredtobesignificantforthesestudies.
AcknowledgementsThisworkwassupportedbytheFISproject07/0418(toCC),theSpanishAIDSnetwork,"RIS,RedTemáticaCooperativadeInvestigaciónenSIDACunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page9of11(RD06/0006)"andtheCHAINEuropeanConsortium.
C.
CabreraandJ.
BlancoareresearchersfromFundacióInstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujolsupportedbytheHealthDepartmentoftheCatalanGovernment(GeneralitatdeCatalunya).
F.
CunyatissupportedbytheFISproject07/0418andVSissupportedbygrantsfromCHAIN,CollaborativeHIVandAnti-HIVDrug-ResistanceNetwork,IntegratedProjectno.
223131,fundedbytheEuropean-CommissionFramework-7Program.
MCissupportedbyaRIScontract.
ThisworkispartofthePhDthesisofF.
CunyatatUniversitatAutònomadeBarcelona,Barcelona,Spain.
Authordetails1IrsiCaixa-HIVACAT,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol(IGTP),HospitalGermansTrias,UniversitatAutònomadeBarcelona,Badalona08916Barcelona,Catalonia,Spain.
2DepartmentofExperimentalMedicine.
Universityof"TorVergata,"Rome,Italy.
3LluitacontralaSIDAFoundation,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol,HospitalUniversitariGermansTriasiPujol,UniversitatAutònomadeBarcelona,08916Badalona,Barcelona,Spain.
4StatisticsandOperationResearchDepartment,UniversitatPolitècnicadeCatalunya,Barcelona,Spain.
Authors'contributionsFC,JBandCCtogetherdesignedthisstudy.
FC,EGandMCperformedtheplasmidconstructions,thefusogenicityandthecelldepletionassays.
FCandNP-Aperformedthestatisticalanalysis.
FC,VS,CP,JBandCCdraftedandeditedthismanuscript.
SMwasresponsibleforsequencingtheEnvs.
Allauthorshavereadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
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doi:10.
1186/1742-4690-9-15Citethisarticleas:Cunyatetal.
:TheHR2polymorphismN140IintheHIV-1gp41combinedwiththeHR1V38Amutationisassociatedwithalesscytopathicphenotype.
Retrovirology20129:15.
Cunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page11of11
SpecificENF-associatedmutationalpathwayscorrelatewithimmunologicalrecovery,evenaftervirologicalfailure,suggestingthattheacquisitionofENFresistancealtersgp41pathogenicity.
Totestthishypothesis,wehavecharacterizedtheexpression,fusioncapability,inductionofCD4+TcelllossandsingleCD4+Tcelldeathof48gp41proteinsderivedfromthreepatientsdisplayingdifferentaminoacids(N,TorI)atposition140thatdevelopedaV38AmutationafterENF-basedtreatment.
Results:Inallcases,intra-patientcomparisonofEnvisolatedpre-orpost-treatmentshowedcomparablevaluesofexpressionandfusogeniccapacity.
Furthermore,EnvwitheitherNorTatposition140inducedcomparablelossesofCD4+T-cells,irrespectiveoftheresiduepresentatposition38.
Conversely,EnvacquiringtheV38Amutationina140IbackgroundinducedasignificantlyreducedlossofCD4+Tcellsandlowersingle-celldeaththandidtheirbaselinecontrols.
Noalteredabilitytoinducesingle-celldeathwasobservedintheotherclones.
Conclusions:Overall,primarygp41proteinswithbothV38AandN140IchangesshowedareducedabilitytoinducesinglecelldeathanddepleteCD4+Tcells,despitemaintainingfusionactivity.
ThespecificityofthisphenotypehighlightstherelevanceofthegeneticcontexttothecytopathiccapacityofEnvandtheroleofENF-resistancemutationsinmodulatingviralpathogenicityinvivo,furthersupportingthehypothesisthatgp41isacriticalmediatorofHIVpathogenesis.
Keywords:HIV,gp41,enfuvirtide,singlecelldeath,fusogenicityBackgroundHIVinfectioncausesaprogressivedepletionofCD4+Tcells,whichleadstothedevelopmentofAIDS[1,2].
AlthoughCD4+TcelllossinHIVinfectionisamultifa-cetedprocess[3-5],thedeathofbystanderCD4+TcellsseemstobeoneofthemaincontributorstoHIV-inducedpathogenesis[6-8].
VariousmechanismshavebeenproposedtoexplainthedestructionofbystanderCD4+Tcells,includingapoptosis,autophagyorabortiveinfection[6,8-11].
TheHIVenvelope(Env)glycoprotein,whichmediatesviralentryintothehostcellbyfusionoftheviralandhostcellmembranes(reviewedin[12-14]),isoneoftheviralfactorsinvolvedinthedeathofbothinfected[15]andbystandercells[7,8,16].
TheEnvcom-plexiscomposedoftwonon-covalentlylinkedsubunits,namely,thesurfaceglycoprotein(gp120)andthetrans-membraneglycoprotein(gp41),andisdisplayedashet-erotrimersonthesurfaceofvirionsandinfectedcells[14,17-20].
Viralentryisamultistepphenomenon:theinteractionofgp120withthehostcellsurfaceCD4-receptor,andeitherCCR5orCXCR4coreceptorenablesgp41subunitstotriggerhemifusionevents,therebyleadingtofusion.
TheHIVgp41isaclassictype1fusionproteinthatcontainsthreedomains:anectodo-main,amembrane-spanningdomain,andalongintra-cytoplasmicsegment.
Theectodomainofgp41consists*Correspondence:ccabrera@irsicaixa.
es1IrsiCaixa-HIVACAT,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol(IGTP),HospitalGermansTrias,UniversitatAutònomadeBarcelona,Badalona08916Barcelona,Catalonia,SpainFulllistofauthorinformationisavailableattheendofthearticleCunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/152012Cunyatetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
ofanN-terminalfusionpeptidefollowedbytwocon-servedcoiled-coildomainsthatarereferredtoasC-andN-terminalheptadrepeats(HR1andHR2),whichareconnectedbyanon-helicalloopregion.
TheseHRinter-actwitheachotherinaleucinezipper-likefashiontomediatemembranefusion[21].
SyntheticpeptidesthatbindtooneoftheHRmotifsinterferewiththeirinter-actionandthusinhibitviralentry[22,23].
Enfuvirtide(ENF,T-20)isthefirstpeptideapprovedforclinicaluseinHIVsalvagetherapy.
Thisdrugisa36-aminoacidpeptidethatwasdesignedbasedontheamino-acidsequenceoftheHR2domainofthegp41subunit.
ThispeptidepreventstheHR1-HR2interactionbybindingtotheHR1domain[22,24,25].
Thetherapeu-ticbenefitsofENFtherapyhavebeendemonstratedbyincreasesinCD4+Tcellcountsandasignificantreduc-tioninHIVRNAlevels[26-28].
Nevertheless,ENF-resistantHIV-1variantsrapidlyemergeunderdrugpressurewhenvirusreplicationisnotcompletelysup-pressed[29-31].
SequenceanalysisofENF-resistantviralpopulationsrevealedtheacquisitionofmutationswithintheHR1domainatpositions36-38(GIV)[29,30],whichwereassociatedwithareductioninviralinfectivity,probablyasaconsequenceofimpairedinteractionbetweenHR1andHR2[32,33].
However,certaincom-pensatorymutationswithinHR2mayariseandrestoreviralinfectivity[29,32,34-37].
Despitevirologicalfailure,specificmutations(theclusterV38A+N140I)havebeenassociatedwithanincreaseinCD4+Tcellcounts[38-40].
TheEnvglycoproteinplaysacrucialroleinthedeple-tionofCD4+Tcellsbyinducingthedeathofsinglebystandercells,whichismediatedbygp41[41,42].
Therefore,changesingp41thatemergeunderENFpressurecouldinduceachangeintheviralpathogeni-city.
Althoughsite-directedpointmutationsatposition38ingp41havebeenshowntoexhibitdeficiencyincell-to-cellfusionactivityandapoptosisinductioninvitroandinahumanizedmousemodel[43,44],itisimportanttonotethatthegeneticbackgroundhasbeenprovenrelevantforfunctionalevaluationoftheENF-resistantEnvsbecausetheremaybecompensatorychangesthatrestoretheinfectivityofthevirus[32,34,36,37,45,46].
Theobjectiveofthecurrentstudywastoevaluatethepathogenicityofseveralpatient-derivedgp41proteinsisolatedfromhighlyexperiencedpatientsreceivinganENF-containingsalvagetherapyandwhetherchangesatposition38and140ingp41haveanimpactinthebio-logicalpropertiesofpatient-isolatedEnvs.
Ourresultsindicatethattheprimarygp41Envproteins,withbothV38AandN140Ichanges,inducedlowerlevelsofsin-gle-celldeathanddepletionofCD4+Tcells,althoughtheyretainedcell-to-cellfusionactivity.
However,themutationV38Ainthecontextofa140Nor140TchangedidnotalterEnvfunctions,underscoringtheimportanceoftheEnvgeneticbackgroundinthemodu-lationofthecytopathiceffectsoftheHIV-1Envglycoproteins.
ResultsanddiscussionPatientsandenvelopeconstructionsInapreviousreport,wecharacterizedgp41proteinsderivedfrom13heavilypre-treatedHIV-1-infectedpatientsreceivinganENF-containingsalvagetherapy[29].
Severaldrugresistance-associatedmutationsweredetectedalongtheentiregp41ectodomain,mainlymap-pingintheHR1domainatpositions36,38and43.
ClinicalfindingshavesuggestedthatcertainENF-resis-tantmutantsarisingduringsalvagetherapy,specifically,theclusterV38A+N140I,areassociatedwithanincreaseinCD4+cellcounts,evenaftervirologicalfailure[38-40,43,44].
Wethereforereasonedthatgp41proteinsderivedfrompatientswithdifferentcombinationsofaminoacidsatpositions38and140couldhavedifferentpathogeniceffects.
Wechosetostudythegp41proteinsderivedfromthreepatients:patients1,9and10,whohadmutationsassociatedwithENFresistanceatposi-tion38inthegp41viralproteinbutdifferedintheaminoacidfoundatposition140[29].
Twoplasmasamplesfromeachpatient,whichwerecollectedatbaselineandduringtreatment,wereusedtoconstructgp160hybridproteins(allbearingthegp120fromanNL4-3virusandthegp41derivedfromthepatients).
Table1summarizesthecharacteristicsofthepatientsatthetimepointsofviralRNAisolation.
Atleast15recombinantexpressionplasmidswereconstructedfromeachsample,andallrecombinantplasmidswerefullysequencedtoverifythatthegp120sequencepresentwasconservedamongtheclonesandthattheNL4-3wtsequenceremainedunchanged(datanotshown).
Theaminoacidsatpositions38and140ofgp41weresubse-quentlydetermined,and48recombinantplasmidswerefinallyselected(Figure1).
Amongtheseplasmids,13cloneswerederivedfrompatient9,containinganaspar-agineatposition140(140N)andthewild-type(wt)aminoacidatposition38(38V)oranalanineatposi-tion38(38A)(n=5andn=8,respectively);19cloneswerederivedfrompatient10,whocontainedthesubsti-tutionN140Tandthewt38V(n=10)ortheV38Amutation(n=9);andfinally,16cloneswerederivedfrompatient1,whohadthepolymorphismN140Iandthewtaminoacidatposition38(n=10)ortheV38Amutation(n=6,Figure1).
Sinceweclonedthefullgp41proteinpresentinvivo,wewereabletoidentifyotherchangesthroughoutthegp41proteininadditiontothechangesatpositions38and140.
ChangeswerefoundprimarilyintheHR2domain,butalsoupstreamCunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page2of11oftheHR1domain,intheHR1domainandintheloopsection.
MostoftherecombinantplasmidsconstructedfromsequencesobtainedduringENFtreatmentcarriedtheV38AmutationastheonlychangeassociatedwithENFresistance,althoughfourclonesderivedfromthe140NpatientcarriedtheN42Tmutation,andthreeothersshowedtheN126Kmutation.
Theanalysisofacaveolin-1bindingmotifinthegp41protein,whichhasbeenrecentlyreportedtoaffectHIV-1pathogenesis[47],showedthatonlyoneplasmidfromthepatientharboringthe140NbackgroundcarriedanMtoVsub-stitutionatposition115.
Theremainingplasmidscon-structedwithsequencesfromthispatientandalloftherecombinantconstructsfromtheothertwopatientsshowednochangesinthisregionbeforeoraftertreat-ment(Figure1).
Ourcloningapproach,usingonlythegp41ofthepatientinsteadoftheentireEnv(gp41+gp120),allowsustospecificallyassesstheeffectofTable1Characteristicsofthethreepatientsreceivinganenfuvirtide-containingsalvagetherapywhensampleswerecollectedPatientaSampleWeeksonENFtreatmentPlasmaviralload(copies/mL)CD4+cellcount(cells/μl)No.
ofexpressionplasmidsconstructed140N(9)140N03663574915V38A140N2485367008N140T(10)N140T01414971310V38AN140T4332794139N140I(1)N140I03347014510V38AN140I12108061506aThepatientIDsfromapreviouswork[29]areindicatedinparentheses.
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.
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K.
.
N.
.
.
.
.
N.
TN.
140T-38A.
30A.
Q.
F.
A.
.
.
.
RS.
.
D.
I.
.
.
.
LT.
.
.
T.
.
.
.
.
.
K.
.
.
.
W.
K.
.
N.
.
.
.
.
N.
TN.
140T-38A.
32A.
Q.
F.
A.
.
.
.
RS.
.
D.
I.
.
.
.
LT.
.
.
T.
.
.
.
.
.
K.
.
.
.
W.
K.
.
N.
.
.
.
.
N.
TN.
140I-WT.
1T.
.
.
.
A.
.
G.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
2T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
6T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
7F.
T.
.
.
.
A.
.
.
.
.
TYDH.
.
N.
.
.
.
.
D.
.
K.
.
.
.
.
.
SA.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
H.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
8T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
17T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
NV.
.
.
.
.
T.
.
.
I.
.
.
.
.
AH.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
18.
.
A.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
NV.
.
.
.
.
T.
.
.
I.
.
.
.
.
AH.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
20T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
21T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-WT.
22D.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
ST.
.
.
.
.
T.
.
.
I.
.
.
.
.
.
K.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-38A.
4A.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
NV.
.
.
.
.
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.
.
I.
.
.
.
.
AH.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-38A.
8A.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
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.
.
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.
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.
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.
.
.
.
K.
.
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.
.
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.
.
.
.
.
.
.
D.
.
N.
140I-38A.
16A.
I.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
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.
.
.
.
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.
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.
.
.
.
AH.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
140I-38A.
19A.
T.
.
.
.
A.
.
.
.
.
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.
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.
.
.
.
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.
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.
.
.
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.
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.
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.
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.
.
.
.
.
K.
.
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.
.
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.
.
.
.
.
.
.
D.
.
N.
140I-38A.
34A.
T.
.
.
.
A.
.
.
.
.
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.
N.
.
.
.
.
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.
.
.
.
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.
.
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.
.
.
.
AH.
.
M.
.
.
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.
.
.
.
.
.
.
D.
.
N.
140I-38A.
35A.
T.
.
.
.
A.
.
.
.
.
SYDH.
.
N.
.
.
.
.
D.
NV.
.
.
.
.
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.
.
I.
.
.
.
.
AH.
.
M.
.
.
K.
.
.
.
.
.
.
.
D.
.
N.
HR1HR2N140N140TN140ICav-1BindingDomainPosition38Position140Figure1Thesequencealignmentofthegp41genefromtherecombinantenvelope-expressingplasmidsconstructedfrompatientsamples.
Thealignmentofthegp41ectodomainsequence(HR1andHR2domains)fromtheselectedrecombinantplasmidsderivedfromthreepatientswhofailedanENF-basedtreatmentandcarriedaV38Amutation(HR1box)withtheaminoacidI,NorTatposition140(HR2box).
TheCav-1bindingdomainintheHR2regionishighlighted.
Cunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page3of11certainENFresistancemutationsongp41pathogenesis,avoidingadditionaleffectsofchangesingp120proteinitselfandimportantlyavoidinganyinterferenceoftheCXCR4ortheCCR5coreceptoruse,whichisamajordeterminantofEnvpathogenicitybothinvivoandinvitro[48,49].
Ifviruseswithdifferentcoreceptorusagehadbeencompared,wewouldnothavebeenabletodiscerntheEnvsubunitresponsiblefortheobserveddifferences.
CellsurfaceexpressionofHIV-1recombinantenvelopeglycoproteinsAftertheoptimizationofthetransfectionandthecellsurfacestainingprocedures(CunyatF,CurriuMetal.
,JBiomolecularScreening,inpress),HeLacellsweretran-sientlytransfectedwiththe48recombinantEnv-expres-singplasmids,and24hourspost-transfection,thecellsurfaceexpressionofEnvwasanalyzedusingaprimaryantibodyagainstthegp120subunit(2G12).
Ananti-gp120antibodywasused,insteadofananti-gp41anti-body,toavoidartifactsduetochangesinthegp41pro-teinthatcouldaffectthebindingoftheantibody.
AlltestedEnvswereexpressedonthecellsurfacewithexpressionlevelsrangingfrom2.
7%to29.
9%ofpositivecells(Figure2A).
Whenaninter-patientcomparisonwasperformedbygroupingalloftheEnvsobtainedfromeachpatientirrespectiveofthetimepoint,asignif-icantlydifferentpercentageofEnv-expressingcellswasobservedbetweenplasmidsconstructedfromthe140N-andN140T-carryingpatients.
ThelowestpercentageofEnv-positivecellswasobservedforthe140Nconstructs(mean=11.
40+/-3.
9),whereasthehighestpercentageofEnv-expressingcellswasobservedfortheN140Tclones(mean=15.
91+/-4.
4)(Figure2A).
However,whenweanalyzedthepercentageofcellsexpressingtheEnvconstructsobtainedfromthesamepatientbycom-paringclonesdisplayingornottheV38Amutation(anintra-patientcomparisonafterandbeforetreatment,respectively),theEnvexpressionwassimilarinallcases(Figure2A).
Inadditiontodeterminingthepercentageofpositivecells,theEnvexpressionlevels,whichcouldplayanimportantroleinEnvpathogenesis,wereevalu-ated.
TherewerenodifferencesintheEnvexpressionlevelsbetweenconstructscontainingwtgp41andthosecontainingthe38Amutationfromthesamepatient,asdeterminedbythegeometricmeanfluorescenceinten-sity(datanotshown),ortherelativefluorescenceinten-sity[50],whichisameasureofthetotalEnvexpression(Figure2B).
Thus,theintra-patientcomparisonssuggestthattheexpressionofEnvdoesnotchangeuponacqui-sitionofthe38Amutation,andthelevelofEnvexpres-sionisanintrinsiccharacteristicoftheparticularEnvcarriedbyeachinfectedpatient.
TheseresultsallowedustoanalyzethecytopathiceffectsofEnvwithoutcor-rectingforcellsurfaceexpressionofEnv.
AnalysisoftheEnvproteinfusogenicityThefunctionoftheHIVEnvglycoproteinistofacilitatetheentryoftheviralnucleocapsidintothetargetcell.
ThisprocesshasanimportantroleinHIVpathogenesis,andthefusogenicactivityofHIVEnvhaslongbeenassociatedwithcytopathiceffects[50,51]bothinvitroandinvivo[52-54].
Inagreementwiththisproperty,ithasbeendescribedthatsingle-amino-acidmutationsintheectodomain(V38A/E)ortransmembraneregionsofgp41reducecell-to-cellfusionactivityofthevirus[43,55].
AnumberofdifferentassaysystemshavebeenreportedtomeasuretheHIVenvelopeactivity.
How-ever,wehavedescribedtheimportanceofselectinganappropriatecelllinetoexpressEnvwhenthecytopathicpropertiesofclinicallyderivedgp41glycoproteinsinvitroareevaluated(CunyatF,CurriuMetal.
,JBiomole-cularScreening,inpress).
Inthisstudy,twoenvelope-expressingeffectorcelllines(293TandHeLacells)werecomparedtoevaluatetheactivityofpatient-derivedEnvs:fusion,absolutecelllossandsinglecelldeath.
Theresultsshowedadifferentialbehaviourbetweenbothcelllines.
293Teffectorcellsseemtohavearapidfor-mationofthefusionpore,generatinghighlevelsoffusionandlowerlevelsofsinglecelldeath.
Incontrast,HeLacellswouldfuseslowly,inducinggreaterextentsofsinglecelldeath.
Thus,HeLacellsshouldbeprefer-entiallyusedfortheevaluationofcelldeathparameters,andthe293Tcelllineshouldbeusedwhenenvelopeswithlowfusogeniccapacityareevaluated.
Basedonthisrecommendation,allofourrecombinantEnvswerefirstexpressedin293Tcells,andassayedforfusionactivity.
DetectablefusionwasobservedforallEnv,showingfusionlevelsoverthe50%whencomparedwithanNL4-3wtEnv(datanotshown).
SinceallourEnvwerefusogenic,weusedHeLacellastheeffectorcelllineinourassays.
Thesecellsweretransientlyco-transfectedwiththeEnv-andpcTat-expressingplasmidsandcocul-turedwiththereporterTZM-blcells.
Aftersixhoursofcoculture,theluminescenceinthesamplewasmea-sured,andtherelativefusioncapacityofeachrecombi-nantEnvwascalculatedincomparisontothefusionvaluesobtainedusingtheNL4-3wtEnv,whichwasusedasacontrol(100%).
TheleveloffusionoftheEnvsobtainedfromdifferentpatients(inter-patientanalysis)showedsignificantdifferences,underscoringthattheviruseachpatientcarriesmayhaveanEnvwithadis-tinctfusogeniccapacitywhich,inthiscase,isdeter-minedbygp41.
DifferencesinfusionwerenotcorrelatedwiththeexpressionlevelofEnvonthecellsurfacebecauseahigherexpressionleveldidnotresultCunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page4of11ABFigure2TheexpressionoffunctionalrecombinantenvelopesontransfectedHeLacells.
Env-expressingplasmidsconstructedfromthreepatientscarryinganaminoacidN,TorIatposition140with(V38A)withoutsubstitutionsatposition38ofgp41weretransfectedintoHeLacells.
ThesurfaceexpressionofEnvwasanalyzed24hourspost-transfectionbystainingthecellswiththe2G12antibody.
EnvexpressionwasdeterminedbythepercentageofEnv-positivecellsinaninter-patientanalysisusingtheclonesconstructedfromeachpatientorinanintra-patientanalysisusingclonesconstructedfrombaselineandaftertreatment(V38A)samples(A).
ThetotallevelofEnvexpressionwasdeterminedbycalculatingtherelativefluorescenceintensity(RFI=%ofEnv-positivecells*geometricmeanfluorescenceofEnv-positivecells)inanintra-patientanalysis(B).
Baselinesamples(whiteboxes)andV38Asamples(grayboxes).
Theboxesrepresentthemedianandinterquartilerangeofthevalues.
ThemedianvalueswerecomparedusinganonparametricMannWhitneytest.
*p95%CD4+Tcellsasdeterminedbyflowcytometry.
TheisolatedCD4+Tcellswereincubatedovernightat37°CinRPMImediasupplementedwith10%ofFCSpriortouse.
AllofthemediawerepurchasedfromInvitrogen(Madrid,Spain).
TheCXCR4antagonistJM-2987(hydrobromidesaltofAMD-3100)[59]andtheCCR5antagonistTAK-779[60,61]wereobtainedthroughtheNIHAIDSResearchandReferenceProgram.
Thebroadlygp120neutralizingantibody2G12andthesecondaryantibodygoatanti-HumanIgGwereobtainedfromPolymun(Vienna,Aus-tria)andJacksonImmunoResearchLaboratories(Penn-sylvania,USA),respectively.
TheTatexpressionplasmidpcTatwasobtainedthroughtheNIHAIDSResearchandReferenceReagentProgram[62].
Cunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page8of11ThecelltrackerDichloro-DimethylAcridin-One(DDAO)waspurchasedfromMolecularProbes(Invitro-gen,Madrid,Spain).
ThecationicfluorescentdyePropi-diumIodide(PI)andthepotentiometricmitochondrialprobeDIOC6(3)werepurchasedfromSigma(Madrid,Spain)andInvitrogen,respectively.
PlasmidconstructionTheRNAfromtheplasmasampleswasisolatedbeforeandaftertheinitiationofENFtreatmentusingtheQIAmpViralRNAkit(Qiagen).
Full-lengthenv/revgeneswereamplifiedthroughRT-PCRusingspecificprimersaspreviouslydescribed[63].
AsubsequentnestedPCRwascarriedoutusingPlatinumTaqDNAPolymeraseHighFidelity(Invitrogen)toobtainafragmentcorrespondingtothegp41protein(primersMluF2andRNANestedRcorre-spondingtonucleotides7726-7747and8882-8904oftheHIVHXB2numberingsystem,respectively).
Afragmentcorrespondingtothegp120proteinwasamplifiedfromaNL4-3plasmid(primersRNANestedFandMluR2corre-spondingtonucleotides5954-5983and7727-7747oftheHIVHXB2numberingsystem,respectively).
Thepurifiedgp41andgp120products,whichoverlappedeachotherin22bases,werecombinedbyPCRandpurifiedtoobtaintherecombinantEnvs(gp120fromNL4-3andgp41frompatients).
AdirectionalcloningreactionwasperformedtoinsertthefragmentintotheplasmidexpressionvectorpcDNA.
3.
1D/V5/His-TOPO(Invitrogen),andseveraltransformedbacterialcolonieswereselectedforeachsam-ple.
Allrecombinantplasmidsweresequencedusingspeci-ficprimers,theBigDyeTerminatorv3.
1cyclesequencingkit(AppliedBiosystems)andanautomaticDNASequen-cer(3100GeneticAnalyzer).
Thesequenceswereedited(usingSequencher,v4.
7,fromtheGeneCodesCorpora-tion,AnnArbor,MIandGeneDoc,v2.
6,software),andtherecombinantplasmidswiththerequiredmutationswereselected.
TransfectionsHeLacellswereplatedatadensityof8*105cells/wellinsix-wellplatesandallowedtogrowovernight.
Thecellsweretransientlytransfected(usingLipofectamine2000Reagent,Invitrogen,Spain)with1.
3μgoftheEnv-expressingplasmidsforthecocultureswithprimarycellsorwerecotransfectedwiththeEnv-expressingplas-midsand2.
7μgofpcTatforthefusionassays.
Twenty-fourhourspost-transfection,thecellswerecollectedforfurtheranalyses.
Asnegativecontrols,cellsweremock-transfected(withthepcDNA3.
1vector)ortransfectedwithpcTatalone.
EnvelopeexpressionTwenty-fourhourspost-transfection,cellmembraneexpressionoftheEnvglycoproteinwasassessedbyflowcytometryafterindirectstainingwiththeanti-gp120monoclonalantibody2G12(4μg/ml)for20minat37°C,followedbystainingwithphycoerythrin-labeledgoatanti-humanIgG(RTfor15min).
Thecellswerewashed,fixedin1%formaldehydeandanalyzedbyaFACSLSRIIflowcytometer.
ThedatawereanalyzedusingFACSDivasoftware(BDBiosciences).
Mock-trans-fectedcellswereusedasanegativestainingcontrol.
ThepercentageofEnv-positivecellsandthegeometricmeanfluorescenceintensity(geoMFI)ofthesecellswereconsideredasindividualparametersorusedtocalculatetherelativefluorescenceintensity(RFI=%ofEnv-posi-tivecells*geoMFIofEnv-positivecells),asdescribedpreviously[50].
Cell-to-CellFusionassaysTwenty-fourhourspost-transfection,Env/pcTat-andpcTat-transfectedHeLacellswerecoculturedwiththereportercelllineCD4+/CCR5+/CXCR4+TZM-blfor6hoursin96-wellplatesinthepresenceorabsenceoftheCXCR4andCCR5co-receptorinhibitorsJM-2987andTAK-779(1μg/ml),respectively.
Thefusioneffi-ciencyofeachclonewasquantifiedbyassessingtheluminescenceofthecells(Britelitekit,PerkinElmer)withaLuminoskanAscentluminometer(Labsystems,Spain).
Envelope-induceddeathinprimaryCD4+Tcells:absolutecelllossandbystanderapoptosisEnv-inducedcytopathiceffectswereevaluatedusingacoculturesystemofEnv-expressingHeLacellsaseffec-torcellsandlabeledprimaryCD4+Tcellsastargetcells.
TheprimaryCD4+Tcellswerestainedwiththefarredcelltracker,DDAO(10μg/mL),for1hourat37°C.
Env+HeLacellsandCD4+/DDAO+Tcellswerecoculturedfor24hoursintheabsenceandpresenceoftheinhibitor,JM-2987(1μg/mL),andwerestainedwithDiOC6(3)(40nM)andPI(5μg/mL)for1hourat37°C.
Labeledmicrobeads(BeadsPerfectCount,Invitrogen)wereaddedtothestainedcoculturetoquantifytheabsolutecellloss,andflowcytometrywasperformedbyaFACSLSRIIflowcytometer.
ThedatawereanalyzedbytheFACSDivasoftware(BDBiosciences).
StatisticalanalysesThedatawerecomparedusingnon-parametricMann-Whitneytests.
AllstatisticalanalyseswereperformedusingGraphPadPrism,version5.
01,forWindows(Graph-PadSoftware,SanDiego,California,USA).
AP-valueof0.
05wasconsideredtobesignificantforthesestudies.
AcknowledgementsThisworkwassupportedbytheFISproject07/0418(toCC),theSpanishAIDSnetwork,"RIS,RedTemáticaCooperativadeInvestigaciónenSIDACunyatetal.
Retrovirology2012,9:15http://www.
retrovirology.
com/content/9/1/15Page9of11(RD06/0006)"andtheCHAINEuropeanConsortium.
C.
CabreraandJ.
BlancoareresearchersfromFundacióInstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujolsupportedbytheHealthDepartmentoftheCatalanGovernment(GeneralitatdeCatalunya).
F.
CunyatissupportedbytheFISproject07/0418andVSissupportedbygrantsfromCHAIN,CollaborativeHIVandAnti-HIVDrug-ResistanceNetwork,IntegratedProjectno.
223131,fundedbytheEuropean-CommissionFramework-7Program.
MCissupportedbyaRIScontract.
ThisworkispartofthePhDthesisofF.
CunyatatUniversitatAutònomadeBarcelona,Barcelona,Spain.
Authordetails1IrsiCaixa-HIVACAT,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol(IGTP),HospitalGermansTrias,UniversitatAutònomadeBarcelona,Badalona08916Barcelona,Catalonia,Spain.
2DepartmentofExperimentalMedicine.
Universityof"TorVergata,"Rome,Italy.
3LluitacontralaSIDAFoundation,InstitutdeRecercaenCiènciesdelaSalutGermansTriasiPujol,HospitalUniversitariGermansTriasiPujol,UniversitatAutònomadeBarcelona,08916Badalona,Barcelona,Spain.
4StatisticsandOperationResearchDepartment,UniversitatPolitècnicadeCatalunya,Barcelona,Spain.
Authors'contributionsFC,JBandCCtogetherdesignedthisstudy.
FC,EGandMCperformedtheplasmidconstructions,thefusogenicityandthecelldepletionassays.
FCandNP-Aperformedthestatisticalanalysis.
FC,VS,CP,JBandCCdraftedandeditedthismanuscript.
SMwasresponsibleforsequencingtheEnvs.
Allauthorshavereadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
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