Characterizing Envelope Region Enhancer Activity in HIV-1
Arden Nasveschuk
Senior Thesis | 2026
CharacterizingEnvelopeRegionEnhancerActivityinHIV-1
ArdenNasveschuk
BostonUniversityAcademy
2March2026
Introduction
Humanimmunodeficiencyvirus(HIV),thecausative pathogenbehindacquiredimmunodeficiencysyndrome(AIDS), haspresentednumerousmedicalandpublichealthchallengessince itsdiscoveryinthe1980s.HIVtargetsimmunecells,weakening theimmunesystemandmakingindividualsmoresusceptibletoa varietyofinfections.Ifleftuntreated,HIVeventuallyprogressesto AIDS,aseriousconditiondefinedascoinfectionofHIVand anotherpathogen.AlthoughthereareeffectivetreatmentsforHIV, itistypicallyincurable.Furthermore,despiterecentadvancements inpreventingandtreatingHIV,theHIVepidemicisstillongoing; asof2024,thereare40.8millionpeoplelivingwithHIVglobally (JointUnitedNationsProgrammeonHIV/AIDS,n.d.).
Additionally,therewereapproximately1.3millionnewHIV infectionsin2024.HIV-relatedinfectionsanddeathsareexpected toincreaseinthecomingyearsduetotheshutdownoftheUnited States’foreignaidprogramsaddressingtheHIVepidemic, includingtheU.S.President’sEmergencyPlanforAIDSRelief (PEPFAR);theUnitedNationsestimatestherewillbe6.6million additionalHIVinfectionsand4.2millionadditionalAIDS-related deathsbetween2025and2029duetothelossofPEPFARand otheraidprograms(JointUnitedNationsProgrammeon HIV/AIDS,2025).Thisunderscorestheongoingseverityofthe HIVepidemicandtheneedforcontinuedresearchintoHIV pathologyandtreatment.
HIVbelongstothefamily Retroviridae,agroupofRNA virusescharacterizedbytheiruseofreversetranscriptasetomake complementaryDNA(cDNA)copiesoftheirgenomethatarethen integratedintothehostcellgenome(Krebsetal.,2002).HIV typicallyrefersspecificallytoHIV-1–thevirusthatmakesupthe vastmajorityofglobalHIVinfectionsandover99.9%ofHIV infectionsintheUnitedStates–asopposedtoHIV-2,amuchrarer
virus(Peruskietal.,2020).MatureHIV-1viralparticleshavean exteriorlipidbilayerderivedfromthehostcellmembranethat containsviralglycoproteins(Figure1).Interiortothemembraneis thematrix,aproteinaceousshellthatenclosestheviralcontents.At thecenterofthevirusistheviralcoreenclosedbyaconical capsid,whichcontainstheviralRNAalongwithnucleocapsid proteins,integrase,andreversetranscriptase.TheHIV-1genomeis approximately10kilobaseslong(Figure2).Atthestartandendof thegenomearethe5’and3’longterminalrepeats(LTRs),which eachcontaina5’anda3’untranslatedregion(UTR)aswellasa seriesofrepeatedbasepairs(Krebsetal.,2002).The5’LTRacts asthecanonicalpromoterfortheHIVgenome,bindingbothhost celltranscriptionalproteinsaswellasviralregulatoryproteinsto initiateandmodulatetranscription;the3’LTRprimarilyregulates polyadenylationoftranscripts.Inadditiontotheseregulatory sequences,theHIVgenomecontainstheGroup-specificantigen (Gag)region,whichencodescapsidproteinssuchastheprecursor p55myristoylatedproteinanditsderivatives;thepolymerase(pol) region,whichencodestheviralenzymesreversetranscriptase, integrase,andprotease;theenvelope(env)region,whichencodes theviralglycoproteinsgp120andgp41;aswellasvariousother regionsencodingforregulatoryproteins(LosAlamosNational Laboratories,n.d.).
Figure1:DiagramofmatureHIV-1viralparticles(Rossietal., 2021).
Figure2:ConfigurationoftheHIV-1genomebasedonthe subtypeBreferencesequenceHXB2.TatandRevexonsare shownshadedingray,whileotherregionsandgeneproductsare showninwhite(LosAlamosNationalLaboratories,n.d.).
AIDSwasfirstidentifiedin1981throughtheriseof Kaposi’ssarcomaandpneumocystiscariniipneumonia—bothrare conditionsassociatedwithimmunosuppression—inyoung,
otherwisehealthymenlivinginUSmetropolitanareas(DavidJ. SencerCDCMuseum,2024).In1983,HIVwasidentifiedasthe causativepathogenbehindAIDS(Schmid,2018).Initially, HIV/AIDSwasanuntreatableterminalcondition;ofthe593cases ofHIVreportedtotheCentersforDiseaseControlandPrevention (CDC)betweenJune1981andSeptember1982,41%ofthose casesresultedindeathbylateSeptember1982(Centersfor DiseaseControlandPrevention,1982).However,in1987,the reversetranscriptaseinhibitorazidothymidine(AZT) was approvedbytheFoodandDrugAdministration(FDA)asthefirst antiretroviraldrugtotreatHIV(NationalInstituteofAllergyand InfectiousDiseases,2025).In1996,proteaseinhibitorswere approvedbytheFDAasanewclassofantiretroviraldrugswhich, incombinationwithreversetranscriptaseinhibitors,candurably suppressHIVindefinitelyprovidedtreatmentisnotinterrupted (NationalInstituteofAllergyandInfectiousDiseases,n.d.).New classesofantiretroviraldrugsdevelopedsince1996,including non-nucleosidereversetranscriptaseinhibitors,entryandfusion inhibitors,andintegraseinhibitorshaveimprovedtheeffectiveness ofantiretroviraltherapy(ART).TheseHIVtreatmentshave dramaticallyimprovedthelifeexpectancyofpeoplelivingwith HIV;whereasinthe1980s,mostpeoplewithHIVdiedofAIDS withinafewyears,ARThasextendedthelifeexpectancyof peoplewithHIVtonear-normalprovidedthattheybeginART withsufficientlyhighwhitebloodcellcounts(Trickeyetal., 2023).ThisshiftfromHIVasanacutediseasetoHIVasachronic conditionhascreatednovelchallengesintreatingHIV.
OnesuchchallengeinmanagingchronicHIVissystemic inflammation;HIVinfectionisassociatedwithavarietyofchronic non-communicableinflammatorydiseases,includingheartdisease andneurocognitivedisorders.HIVinfectioniscorrelatedwitha 1.5totwofoldincreaseinacutemyocardialinfarctionrateswhen
adjustedforcommonriskfactors(Triantetal.,2007).Furthermore, peoplewithHIVhaveanelevatedriskofdevelopingacute coronarysyndrome(ACS)—whichincludesunstableanginaand myocardialinfarctions—againwithinayearafteraninitialACS episode(Boccaraetal.,2010).Increasedriskofmyocardial infarctioniscorrelatedwithlowerCD4+Tcellcountsandhigher viralRNAlevels,suggestingalteredimmuneresponsesmay contributetotheriskofadversecardiovasculareventsinpeople livingwithHIV(Drozdetal.,2018).HIVisalsoassociatedwith neurocognitivedysfunction(Rosenthal&Tyor,2019).AsHIV becomesamorechronicconditionandasthepopulationofpeople withHIVages,therehasbeenanincreaseinHIV-associated neurocognitivedeficits(HAND).Estimatesforthepercentageof peoplelivingwithHIVwithHANDrangefrom33%to52% dependingonthestudy(Rosenthal&Tyor,2019).HIVisbelieved topenetratethecentralnervoussystemearlyininfection,andsince ARThaspoorpenetrationoftheCNSduetotheblood-brain barrier,neuroinflammationcontinuesevenwithundetectableviral RNAlevels.HANDisassociatedwithelevatedmarkersof monocyteactivation,whichsuggestsalinkbetweeninflammatory responsesandneurocognitivedisordersinHIV(Abutaleb& Feinstein,n.d.;Burdoetal.,2011).Ultimately,theconfluenceof bothcardiovascularandneurologicalinflammatoryconditionsin peoplelivingwithHIVsuggeststhepresenceofunderlying inflammatoryresponsesinchronicHIVinfection.
Onepossiblemechanismdrivingthischronicinflammation istheaccumulationofdefectiveproviraltranscripts;multiple factorscontributetothegenerationofalargepoolofdefective provirusesinpeoplelivingwithHIV,thetranscriptionofwhichis suspectedtocontributetochronicinflammationthroughmultiple distinctmechanisms(Kilroyetal.,2024).Adefectiveprovirusisa pieceofviralDNAintegratedintothehostcellgenomethatis
unabletoproduceafunctionalvirus.Defectiveprovirusesare generatedthroughmultiplemechanisms,includingerroneous reversetranscription,cellularrestrictionfactors,andDNAdamage responses.Reversetranscriptaselacksproofreadingcapabilities andispronetotemplateswitching,whichcanleadto hypermutationandinternaldeletionsintheHIVgenomethat renderitdefective.Furthermore,restingCD4+Tcells, monocyte-derivedmacrophages(MDMs),andmacrophages derivedfrominducedpluripotentstemcellsallcontainrestriction factorsthatmaylimitreversetranscription.Forexample,the restrictionfactorSAMHD1reducesthenucleotideconcentrationin cells,possiblycontributingtodefectiveproviruses.Additionally, DNAdamagerepairinvolvedinDNAintegrationisanother error-proneprocessthatcouldcontributetothedefectivereservoir. Cellswithdefectiveprovirusesmayescapeselectiveimmune pressures,allowingfortheexpansionofthisreservoirovertime. Thus,thereareseveralmechanismsthatcontributetothe generationofdefectiveproviraltranscripts,includingbutnot limitedtoerroneousreversetranscription,thepresenceofcellular restrictionfactors,andDNAdamageresponsepathways.
ThemRNAsgeneratedfromdefectiveprovirusesare hypothesizedtocontributetochronicinflammationinpeople livingwithHIV.PeoplelivingwithHIVundergoingARTcontinue tohaveelevatedlevelsofproinflammatorycytokinesthatsuggest persistentinflammationonthecellularlevel,whichisconsistent withtheacceleratedaging,increasedriskofadversecardiac events,andneurologicaldisordersassociatedwithlong-termHIV infection(Kilroyetal.,2024).Therearemultiplepotential explanationsforthisphenomenon.First,defectiveviralRNAsmay serveasintracellularpathogenassociatedmolecularpatterns (PAMPs)thatactivatepathogenrecognitionreceptors(PRRs), whichdriveinflammation.Second,defectiveprovirusesmaylack
mechanismstoevadetheinnateimmunesysteminmyeloidcells duetoalteredsplicingpatterns.Forexample,unsplicedRNAsthat containintronscantriggerinnateimmuneresponsesviaactivation ofmitochondrialantiviralsensingprotein-mediatedinterferon responsesinmacrophagesandthroughcytosolicsensing mechanismsinmicroglia(Akiyamaetal.,2021;Ramaswamyet al.,2025).Third,humanendogenousretroviruses(HERVs)are knowntocompriseroughly8%ofthehumangenome,butare typicallysilencedthroughepigeneticfactorsorothermechanisms. However,HIVmayupregulatetranscriptionofHERVs,providing anothersetofPAMPsthatcouldcontributetoinflammation. Regardlessoftheexactmechanism,defectiveproviraltranscripts likelycontributetocellular-levelchronicinflammatoryresponses inpeoplelivingwithHIV.
Kuniholmetal.identifieddefectiveHIV-1transcripts originatingfromanintragenictranscriptionalelement(2021). CanonicalHIV-1transcripts—ortranscriptsoriginatingfromthe5’ LTR—alwayscontaintheuniversalsplicedonor(U5)region. Kuniholmetal.identifiedtranscriptsinCD4+TcellsandMDMs thatcontaintheenveloperegionoftheHIV-1genomebutlackthe U5region.Thesetranscriptscontainonlyroughlythelatterhalfof theHIVgenome.Thisfindingimpliestheexistenceofa non-canonicalintragenictranscriptionalelementthatservesasan alternativepromoterforHIV-1.Notably,theseU5deficient transcriptswerealsoidentifiedinasubsetofpeoplewithHIVon ART,suggestingthattheymaybeclinicallyrelevant.Kuniholmet al.thenidentifiedaspecificsubsectionoftheenveloperegionof theHIVgenomethatcouldexplainthesedefectivetranscripts.This envelope-regionpromoterispostulatedtocontributetothe accumulationofdefectiveproviraltranscripts,whichinturnmay contributetochronicinflammatoryresponsesinpeoplelivingwith HIV.
Whetherthisenvelope-regionintragenictranscriptional element(ITE)alsobehavesasanenhancerintheHIVgenomeis anongoingquestionthathasimplicationsforunderstandingthe mechanismsdrivingHIVviralreplicationandchronic inflammation.LuciferasereporterassaysinvolvingtheITEwere usedtoevaluatethepotentialenhanceractivityofthisenvelope regionsequenceandtomorebroadlycharacterizeits transcriptionalactivity.Theresultsshowthat,comparedtoother regionsoftheHIVgenome,theenvelope-regionITEupregulates transcriptionofluciferasewhenclonedinfrontofaminimal promoterandluciferasereportergene.IftheITEregionbehaved strictlyasapromoter,onewouldexpectluciferaseexpressionto decreasecomparedtocontrolsequencesduetointerference betweenpromoters.Thus,theupregulationofluciferaseexpression whentheITEisclonedinfrontofaminimalpromotersuggests thatthisregionbehavesasanenhancer.Thisfindingfurther illuminatesthemechanismsdrivingtheaccumulationofdefective proviraltranscriptsinpeoplelivingwithHIV,withthegoalof ultimatelyaddressingHIV-associatedchronicinflammation.
Methods
Sequences
Theenvelope-regionITEwasdefinedasTile14in Kuniholmetal.’sidentificationoftranscriptionallyactiveHIV-1 sequencesinNL43(2021).Asequencefromthepolyprotein(pol) regioncorrespondingtoTile5inthisdatasetwaschosenasa controlsequencetotheITEbecauseofitslackoftranscriptional activity.
PlasmidGeneration
ModifiedplasmidsweregeneratedusingNEBuilderQ5 High-FidelityDNAPolymerasePCRamplificationofbothNL43 backbonesequencesandaminimalpromoterdrivenfirefly luciferasevector.Theluciferasevectorcontainedaminimal promoterinfrontofaluc2luciferasereporter,aswellasan ampicillinresistancegeneandarestrictionsitebeforetheminimal promoter(Figure3).PCRamplificationoftheITE,polsequence, andtheminimalpromotervectorwasconductedaccordingtoQ5 High-FidelityDNAPolymerasePCRProtocol(NewEngland Biolabs,n.d.).Forpolsequenceamplification,Region5Forward withSacIRestrictionEnzymeandRegion5ReversewithXhoI RestrictionEnzymeprimersasdefinedbyKuniholmetal.were utilized(2021).Likewise,forITEamplification,Region14 ForwardwithSacIRestrictionEnzymeandRegion14Reverse withXhoIrestrictionenzymewasused.
Figure3:Theuneditedluc2minimalpromoterdrivenfirefly luciferasevector.NotetheSacIandXhoIrestrictionsitesadjacent totheminimalpromoter.
Amplificationwasconfirmedusinggelelectrophoresis. Sampleswererunonagelcontaining0.8%agarand1μLofgel redper10mlofagarsolutionat85Vfor40minutes.QuickLoad Purple1kbLadderwasusedtoconfirmthatthelengthofthe amplifiedregionswasconsistentwithexpectedlengths.Plasmids werethenassembledusingarestrictionenzymedoubledigestand T4ligation.TherestrictionenzymesSacI-HSandXhoIwereused accordingtotheNEBclonerRestrictionEnzymeDoubleDigestion protocol(n.d.).Therestrictionsiteslocatednexttotheminimal promoterontheminimalpromotervector,andappropriate restrictionsitesoneithersideofthepolregionandITEinserts, allowedforeffectiveconstructionofthisplasmid(Figure4). LigationwasperformedusingNewEnglandBiolabs’Ligation ProtocolwithT4DNALigase(n.d.).
Figure4:Configurationofthepromoterdrivenfireflyluciferase vectorswithHIVenvelope-regionITE(left)andpolregion(right) inserts.minPreferstotheminimalpromoter.
PlasmidSelection
Modifiedplasmidswereselectedthroughtransformation into E. coli TOP10s.Bacterialcellswerethawedonicebefore transformation.Asmallamount(1uL)ofDNAwasthenaddedto bacterialcells.Thecellswereelectroporatedandresuspendedin SuperOptimalbrothwithCataboliteexpression(SOC).The outgrowthwasincubatedinashakerat~225rpmand37°Cfor1 hourandthenspreadontoLBagarplatescontainingcarbenicillin andincubatedat37°Cfor24hours.Colonieswerethenselected andincubatedinLBbrothcontainingcarbenicillininashakerat 37°Cfor24hours.Sincetheplasmidscontainedampicillin resistancegenes,anycoloniesthatgrewincarbenicillin-containing mediahadbeeneffectivelytransformed.Plasmidswerethen isolatedfromcellsusinganinvitrogenPureLinkQuickPlasmid Miniprepkit.Thepropersequencingandorientationofthe plasmidswasconfirmedusingPlasmidsource.
TissueCulture
ThisexperimentutilizedHEK293Ts.Stockcellswere thawedandwashedtwicewithphosphatebufferedsaline(PBS). CellswerethenresuspendedinDMEM(containing10%fetal bovineserum,1%penicillin-streptomycin,and1%L-glutamine)at aconcentrationof1x10^5cellspermilliliterandtransferredtoa T75flask.Cellswereincubatedat37°Cand5%carbondioxide. Cellsweresplit1:10onalternatingdaysbyremovingoldmedia, washingcellswithPBS,detachingcellsfromtheflaskwith 0.025%trypsinandethylenediaminetetraacetic acid,andseeding cellsinanewT75.
Transfection
HEK293Tsonapproximatelypassage4wereseededina white-walled96-wellplate.Cellswereseededin100μLof DMEMmediaperwellataconcentrationof2x10^5cellsper milliliter.Cellswerethenincubatedat37°Cand5%carbon dioxidefor20hoursuntiltheyreachedapproximately70% confluency.HEK293TsweretransfectedusingLipofectamine3000. ReagentsweredilutedwithOptiMEMandaddedtosamples accordingtoanadaptedversionofLipofectamine3000Reagent Protocol(invitrogen,2016).Conditionsforthistransfection includedpolregion-luciferaseandrenilla,ITE-luciferaseand renilla,polregion-luciferaseonly,ITE-luciferaseonly,minimal promotor-luciferaseandrenilla,minimalpromotor-luciferaseonly, renillaonly,andcellsonly.VaryingamountsofplasmidDNAwas addedtoaccountforthecopynumberofeachplasmid.90ngof DNAwasaddedtoeachITEconditionsincetheITE-luciferase plasmidwasthelargest.79ngofDNAwasaddedtoeachpol region-luciferasecondition,and72.6ngofluciferasevectorDNA withnoinsertwasaddedtoeachminimalpromotor-luciferase condition.Thesequantitieswerecalculatedtonormalizetothesize ofeachplasmidandthecorrespondingrateoftranscription.10ng ofrenillaluciferasewasaddedtoeachoftherenillapositive conditions.TheamountsofOptiMEM,Lipofectamine3000,and P3000werethenscaledtoaccountforthesevaryingamountsof DNA.(Forthecellsonlyandrenillaonlycontrolconditions,these quantitieswerecalculatedasifthewellscontained100ngof DNA.)AftertheadditionofLipofectamine3000,thesolutions wereincubatedfor15minutesandthenaddedtoplated HEK293Ts,withthreewellspercondition.
TheDual-GloLuciferaseAssaySystemwasutilizedtoread outluciferasereporters.Theprotocolfromthemanufacturerfor
thisexperimentwasmodifiedslightly;50μLofmediawas removedfromthewellsandthen50μLofDual-GloReagentwas addedtothewells.Likewise,50μLofDual-GloStop&Glo reagentwasaddedafterreadingfireflyluciferase(Promega,2025). Thismodificationwasmadetoconservereagentsandlikelyhadno effectontheefficacyoftheexperiment.
Results
Inordertodeterminethecis-regulatoryactivityofthe enveloperegion,wegeneratedplasmidswiththeITEclonedin frontofaminimalpromoterandluciferasereportergene.Higher luciferaseluminescencecorrespondstohigherlevelsofluciferase geneexpression,whichindicatesincreasedtranscriptionalactivity. Thisexperimentusedplasmidconstructsthatcontainedeitherthe polregioncontrolsequenceortheITEsequenceclonedinfrontof aminimalpromoterandfireflyluciferasereporter.Because adjacentpromoterstypicallyinterferewitheachother,increased luciferasereporterreadoutintheITE-luciferaseplasmidsample comparedtothepolregion-luciferasecontrolsamplewould suggestthattheITEsequencebehavesasanenhancerratherthan solelyasapromoter.
Sincerelativeluminescencemustbestandardizedto backgroundlevelsofexpression,Renillaluciferaseconstructswere usedinsomeexperimentalconditions;thisenabledthe luminescenceoffireflyluciferasetobestandardizedtoRenilla luciferasebackground.
Therelativefireflyluciferaseluminescenceofthe ITE-luciferase+renillaconditionwasgreaterthanthe luminescenceofthepolregion-luciferase+renillaconditionforall samples(Table1).Thepolregion-luciferaseonlyand ITE-luciferaseonlyconditionswereusedtoconfirmthatthe additionofRenillaluciferasedidnotinhibittheexpressionof
fireflyluciferase;thehigherluminescenceintheRenillaconditions suggeststheRenillaluciferaseconstructdidnotinterferewiththe fireflyluciferaseconstruct(Table1).Additionally,firefly luciferasereadoutforboththepolregion-luciferase+renillaand ITE-luciferase+renillaconditionswassignificantlylowerthanthe minimalpromoter-luciferase+Renillaconditionreadout(Table1). Thissuggeststhatcloninganysequenceinfrontoftheminimal promoter-luciferaseconstructdecreasesluciferaseexpression, regardlessofthetranscriptionalactivityofthatsequence. However,thiseffectcanbedisregardedbecauseitislikelytobe theidenticalforboththeITE-luciferaseandpolregion-luciferase conditions.
Table1:Fireflyluciferasereporterreadout.Relativeluminescence units(RLU)forfireflyluciferaseineachexperimentalcondition.
Condition
Renillaluciferasereadoutprovidesamechanismwith whichtostandardizefireflyluciferasereadoutforconditions
containingbothfireflyluciferaseandRenillaluciferase.Whilethe Renillaluciferasereadoutfortheminimalpromoter-luciferase+ Renillaconditionwasabnormallyhigh,theRenillaluciferase readoutwascomparabletothefireflyluciferasereadoutforthis condition(Table2).Furthermore,asexpected,theRenilla luciferasereadoutwassimilaracrossthepolregion-luciferase+ renillaandITE-luciferase+renillaconditions.Thissuggeststhat theRenillaluciferasereadoutisreliableandcanbeusedto standardizefireluciferaseexpression.
Table2:Renillaluciferasereporterreadout.RLUforRenilla luciferaseineachexperimentalcondition.
Condition
Thefoldchangeinluminescencebetweenthefirefly luciferasereadoutandtheRenillaluciferasereadoutwascalculated forthepolregion-luciferase+renilla,ITE-luciferase+renilla,and minimalpromoter-luciferase+renillaconditions(Table3).Firefly luciferasereadoutwasdividedbyRenillaluciferasereadoutfor
eachsample,andthesequantitieswereaveragedacrosstechnical replicatesforeachexperimentalcondition.Foldchange calculationsstandardizetheluminescenceofeachwellandenable furtherdataanalysis.Theaveragefoldchangeinluminescencewas greaterfortheITE-luciferase+renillaconditioncomparedtothe polregion-luciferase+renillaandminimalpromoter+renilla conditions(Table3).ThissuggeststhattheITEincreased luciferaseexpressioncomparedtoboththepolregion-luciferase controlconditionandtheminimalpromoter-luciferasecontrol condition.Furthermore,asexpected,theaveragefoldchangein luminescencebetweenfireflyluciferaseandRenillaluciferasewas similarforthepolregion-luciferase+renillaandminimal promoter-luciferase+renillaconditions(M=1.17andM=1.22, respectively).Thissuggeststhatthepolregionhadnoadditional transcriptionalactivitycomparedtothepromoterandfurther confirmsthereliabilityoftheassay.
Table3:Standardizedrelativeluminescenceunitsoffirefly luciferaserelativetoRenillaluciferase.
Minimalpromoter-luciferase+
Thefoldchangeinluminescencebetweenfireflyand Renillaluciferasewasplottedforboththepolregion-luciferase+ renillaconditionandtheITE-luciferase+renillacondition(Figure 5).Analysisshowedthattherewasasignificantdifferencein
relativeluminescencebetweenthepolregionandtheITE(paired t-test,p<0.001).
Figure5:Foldchangeinpolregionluminescence(blue)andITE luminescence(purple)relativetoRenillaluciferase.
Thesignificantincreaseinstandardizedfireflyluciferase expressionintheITE-luciferase+renillaconditioncomparedto thepolregion-luciferase+renillaconditionindicatesthattheITE upregulatestranscriptionwhenclonedinfrontofaminimal
promoter.Sinceadjacentpromoterstypicallyinterferewitheach other,causingeitheradownregulationofexpressionornochange inexpression,thisfindingsuggeststhattheITEbehavesasan enhancer.
Discussion
TheseresultsillustratethatcloningtheITEinfrontofa minimalpromoterandluciferasereportergeneincreasedluciferase geneexpressionrelativetopol-regioncontrolsequences.The averagefoldchangeinfireflyluciferaseluminescencerelativeto RenillaluciferaseoftheITEsampleswassignificantlygreaterthan theaveragefoldchangeofthecontrolsamples(Table3andFigure 5,p<0.001).ThesefindingsillustratethattheITEhas transcriptionalactivitywhenclonedinfrontofaminimal promoter,whichfurthercorroboratespreviousfindingsthatthis regionistranscriptionallyactive(Kuniholmetal.2021).More specifically,theseresultssuggestthattheITEactsasa cis-regulatoryelementintheHIVgenome.IftheITEwassolelya promoter,theresultswouldlikelyhaveshowneithernochangeor adecreaseinluciferaseexpressioncomparedtocontrolsequences duetointerferencewiththeminimalpromoter.Theincreasein luciferaseexpressionintheITEplasmidscomparedtocontrol plasmidsthereforesuggeststhattheITEbehavesasa cis-regulatoryelement.
Asthiswasafairlypreliminarystudy,moreresearchis neededtocorroboratethesefindingsandfurthercharacterizethe intragenictranscriptionalactivityoftheITE.Alimitationofthis studyisthatitdoesnotexaminethetranscriptionalactivityofthe ITEinHIV-infectedcells.Thus,anotherfutureexperimentisa chromatinimmunoprecipitationassay(ChIP)ofHIV-infected CD4+TcellsinconjunctionwithaddPCRanalysisofboth canonicalandnoncanonicalHIVtranscriptsinthesecells.This
experimentwouldrelatemethylationmarksontheHIVgenome withviraltranscriptsisolatedfrominfectedcells.Correlation betweenadecreasemethylationmarksintheenveloperegionand anincreaseinU5deficientproviraltranscriptswouldfurther suggestthattheITEactsasapromoterregioninlymphoidcells. Furthermore,acorrelationbetweenadecreaseinenvelope-region methylationandanincreaseinnormalproviraltranscriptscould suggestthattheITEupregulatesnormalLTR-driventranscription throughenhanceractivity.Anotherfutureexperimentinvolves creatingplasmidconstructswheretheITEandpolregioncontrol sequenceareclonedoppositetheminimalpromoterandrepeating theluciferasereporterassayconductedinthisstudy(Figure6). Theabilitytoupregulateordownregulategeneexpressionevenfar fromthetargetgeneitselfispartoftheclassicdefinitionofan enhancer.Thus,anincreaseinluciferaseexpressioninITEplasmid samplescomparedtocontrolsampleswhentheseregionsare locatedoppositetheminimalpromoterwouldfurthersupportthe hypothesisthattheITEactsasacis-regulatoryelement. Additionally,iftheITEregulatorysequenceisindeedanenhancer, itshouldalsobeabletoactdownstreamofatargetgeneto upregulatetranscription.Thus,anotherpotentialexperimentto furthercharacterizetheregulatoryactivityoftheITEisarepeatof theluciferasereporterassaybutwiththeITEandpolregioncloned downstreamofaminimalpromoter.
Figure6:PlasmidconstructswiththepolregionandITEopposite aminimalpromoter.
Theseresultshaveimplicationsforunderstandingchronic inflammationinHIV.Defectiveprovirusesarethoughtto contributetochronicinflammationthroughmultiplepathways, includingthroughservingaspathogen-associatedmolecular pathwaysthatactivatepathogenrecognitionreceptors; upregulatingtranscriptionofhumanendogenousretroviruses;and lackingmechanismstoevadeimmuneresponsesinmyeloidcells (Kilroyetal.,2024).Intragenictranscriptionalregulationprovides anewmechanismforunderstandingtheaccumulationofdefective proviraltranscriptsthatcontributetochronicinflammation.For example,perhapsdefectiveprovirusesgeneratedfromintragenic transcriptionalactivityincreaseactivationofpathogenrecognition receptorsinhostcells.Additionally,abnormalsplicingofdefective transcriptscouldtriggerspecificimmuneresponsesevadedby intacttranscripts.Intragenictranscriptionalactivitythus illuminatespossiblenewpathwaysdrivingchronicimmune activationinHIVinfection.SinceU5deficienttranscriptswere
previouslyidentifiedinpeoplelivingwithHIV,thisinvitro understandingofITE-driventranslationcantranslatetoclinical understandingofchronicinflammation(Kuniholmetal.,2021). Furthermore,theITEmayalsoplayaroleinupregulatingnormal HIVtranscriptionthatcontributestopersistentlow-levelimmune activationinpeoplelivingwithHIV.Thus,furthercharacterizing theITEisessentialforunderstandingHIVbiochemistryand pathology.
Conclusion
Theenvelope-regionITEincreasesluciferaseexpression comparedtocontrolsequenceswhenclonedinfrontofaminimal promoterandluciferasereportergene,indicatingthatitbehavesas anintragenicenhancer.Futureexperimentstocharacterizethis activityincludeassayswheretheITEisclonedoppositetoor downstreamofaminimalpromoter.Furthermore,chromatin immunoprecipitationcouldbeusedtocorrelateITEactivitywith levelsofbothintactanddefectiveproviraltranscriptsin HIV-infectedcells.Theseexperimentswouldprovideafoundation forbetterunderstandingITEenhanceractivity,includingitsrolein generatingbothintactanddefectiveviraltranscripts.Theenhancer activitypreliminarilyidentifiedinthispaperprovidesapotential newmechanismforunderstandingchronicinflammationinHIV;it ispossiblethat,throughgenerationofintactanddefectiveproviral transcripts,ITEenhanceractivitytriggersimmuneresponsesthat ultimatelycontributetochronicinflammation.Understanding chronicinflammationmechanismsinHIVisessentialasART continuestoextendthelifeexpectancyofpeoplelivingwithHIV. Characterizingthepathwaysbehindinflammatoryresponsescould eventuallyilluminatewaystopreventortreatHIV-associated chronicinflammatoryconditions.
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