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Educator Materials Demonstration HIV Protease Inhibitors

February3,2017

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HIVPROTEASEINHIBITORSOVERVIEW

Thisdemonstrationispartofaseriesofactivitiesanddemonstrationsfocusingonvariousaspectsofthehumanimmunodeficiencyvirus(HIV)lifecycle.

HIVisaretrovirus,atypeofvirusthatintegratesitsgenomeintothehostcell’sgenome.(Twootheractivitiesfocusonthereversetranscriptionandintegrationstepsoftheviralreplicationcycle.)HIVhasgenescommontoallretroviruses(thegenesgag,pol,andenv)thatencodestructuralproteinsandenzymes,aswellasgenesthatareuniquetoitsviralstructureandfunction.Thisactivityfocusesonthegagandpolgenes.ThesetwogenesaretranscribedintoasingleRNAmolecule,whichisthentranslatedtoproduceasinglepolyprotein.TheGag-PolpolyproteiniscleavedbytheHIVproteaseenzymetogeneratesixproteinsessentialforassemblingthevirusparticle.

Duringthisactivity,youwilldemonstratethemechanismbywhichtheGag-PolpolyproteinisproducedandcleavedbyHIVprotease.ThedemonstrationmodelsthisprocessandalsoshowshowinhibitingHIVproteaseactivitypreventsthevirusfromcreatingmatureproteins.ProteaseinhibitorsareaclassofdrugsusedtotreatHIVinfection.

KEYCONCEPTSANDLEARNINGOBJECTIVES

• HIVRNAcanbetranslatedintopolyproteins;post-translationalcleavageofthesepolyproteinsresultsinthegenerationoffunctionalviralproteins.

• OneofthedrugsdevelopedagainstHIVinterfereswiththefunctionofanHIVenzyme,aprotease,responsibleforcleavingHIVpolyproteins.

Studentswillbeableto

• applytheconceptsofproteinsynthesisandenzymefunctiontoviralreplicationandtheHIVlifecycle.

CURRICULUMCONNECTIONS

Curriculum Standards

NGSS(2013) HS-LS1-1

APBiology(2013) 3.B.1;3.C.3;4.A.1;4.B.1

KEYTERMS

virus,genome,DNA,mRNA,gene,transcription,translation,enzyme,ribosome,inhibitor,protease,protein,capsid,reversetranscriptase,integrase,polymerase,5’cap,3’tail

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Educator Materials Demonstration HIV Protease Inhibitors

February3,2017

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TIMEREQUIREMENTS

IfstudentsarefamiliarwiththeHIVlifecycle,thedemonstrationitselftakesapproximately15minutes.Preparationofmaterialsshouldtakeabout30minutesthefirsttimeitisdone.

SUGGESTEDAUDIENCE

ThislessonisappropriateforAPBiology.

PRIORKNOWLEDGE

Studentsshouldbefamiliarwiththeprocessesoftranscriptionandtranslationineukaryoticcells.

MATERIALS

• Woodendowel7/8inchindiametertorepresentHIVRNA.

o Thedowelcanbepurchasedatmosthomeimprovementorcraftstoresoronline.

o Thedowelshouldbepreparedaheadofclassbycoloringdifferentpartstoindicatedifferentgenesorgroupsofgenes.

• Rollofcashregistertape2¼”×130ft(57mm×39m)torepresentthehostcell’sribosome.

o Thecashregistertapeisavailableatmostbusinesssupplystoresoronline.

o Thepaperrolledoutofthecashregistertapewillrepresentthepolyproteinbeingsynthesized.

o Besurethatthecentralcoreoftheregistertapefitsoverthewoodendowel.

• Scissorstorepresenttheviralproteaseenzyme.

• Styrofoamcone2¾”×17/8”(69.85mm×47.62mm)torepresenttheproteaseinhibitor.

o TheStyrofoamconescanbefoundintheflowerarrangementdepartmentofmostcraftstoresoronline.

o Theconeshouldbelargeenoughtofitbetweenthebladesofthescissorsandpreventthescissorsfromcutting.

TEACHINGTIPS

Beforeconductingthedemonstration

• IfstudentsarenotfamiliarwiththestructureofHIV,itwillbehelpfulforthemtobecomefamiliarwiththebasics.HIVisanenvelopedretrovirusthatconsistsofanRNAgenome,aviralcapsid,andanoutermembrane,orenvelope,withembeddedproteins.ToseethestructureofHIV,visittheinteractive“VirusExplorer”athttps://www.hhmi.org/biointeractive/virus-explorer.ShowstudentstheHIVcrosssectionandpointouttothemtheHIVcapsidprotein(encodedbythegaggene)andtheintegrase,reversetranscriptase,andproteaseenzymes(encodedbythepolgene).Alsopoint

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Educator Materials Demonstration HIV Protease Inhibitors

February3,2017

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outtheHIVRNAgenome.NotethatthefullHIVgenomeisencodedonasinglestrandofRNA.However,eachvirusparticlecontainstwoseparate,identicalRNAstrands.

• StudentsshouldalsobefamiliarwiththekeyeventsintheHIVlifecycle.Mainly,whenHIVinfectsacell,theHIVRNAgenomeisreverse-transcribedintoDNA,whichisthenintegratedintothehumangenome.ThisDNAcopyoftheHIVgenomeisthentranscribedintoRNAbythehostcellmachinery.TheHIVRNAcanbeincorporatedintonewvirusparticlesortranslatedintoproteins.AnanimationoftheHIVlifecycleisavailableathttps://www.hhmi.org/biointeractive/hiv-life-cycle.

• Studentsmaywonderwhetherhumancellsalsoproducepolyproteins.Humancellsdonotproducepolyproteins.Inhumancells,mRNAsaretypicallytranslatedintosingleproteins.However,severalRNAvirusesproducepolyproteins.Becausethismechanismisnotusedbyhostcellsandutilizesviralenzymes,theseenzymesmakegoodtargetsforantiviraldrugs.

Aftercompletingthedemonstration

• Aftercompletingtheactivity,youmaywanttoshowstudentsananimationdemonstratingproteaseinhibitionavailableathttp://www.hhmi.org/biointeractive/protease-inhibitors.

• StudentsmaywonderhowproteaseinhibitorsrelatetootherdrugsusedtotreatHIV.OtherdrugsareavailablethattargetdifferentstepsintheHIVlifecycle:viralentry,reversetranscription,integration,andproteincleavage.Thefirstsuchdrug,azidothymidine,whichinhibitstheviralreversetranscriptaseenzyme,wasapprovedbytheFoodandDrugAdministrationin1987.Thefirstproteaseinhibitor,saquinavir,wasapprovedin1995.Asof2015,eightproteaseinhibitorswerecommerciallyavailabletotreatHIV.BecauseHIVmutatesrapidlyduetothelackofproofreadingcapacityofreversetranscriptase,resistancecaneasilydevelopagainstanydrug.Tocombatresistance,doctorstypicallygivepatientsseveraldifferentantiretroviraldrugs.Thiscombinationtreatmentlowersthechancesofthevirusbecomingresistantbecausethevirusmustbecomeresistanttomultipledrugsthattargetdifferentaspectsofitslifecycle.

• Althoughhumancellsdonotproducepolyproteins,theydoproduceproteases.Proteasesmodifyproteinsoncetheyaremade.Manyproteinsthatareexportedfromthecytoplasmaresynthesizedbyribosomesasproproteins(orpreproteins)locatedontheroughendoplasmicreticulumandsubsequentlycleavedbyproteasestobecomefunctionalproteins.Forexample,proteasesactivateproproteinssuchasproinsulinandbloodclottingfactors.Proteasesarealsoresponsiblefordestroyingproteinsoncetheyarenolongerneeded.

PROCEDURE

BackgroundInformation

TheHIVgenomecontainsninegenes.Threeofthem(gag,pol,andenv)arecommontoallretrovirusesandtheothersareuniquetoHIV(seeFigure1forasimplifiedschematic).Inthisactivity,wewillfocusonthegagandpolgenes.Thegaggeneencodesthreeproteinsthatcomprisetheviralcapsid.Thepolgeneencodesthreeenzymesthatareessentialforviralgenomereplicationandintegrationintothehostgenome(reversetranscriptase,protease,andintegrase).WhenHIVDNAisintegratedintothehumangenome,itis

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transcribedtoproduceaprimaryRNAthatcaneitherbecomepartofnewvirusesorbetranslatedintovariousdifferentproteins.OneoftheproteinsthataregeneratedisaGag-Polpolyprotein.

Figure1.SimplifiedillustrationoftheHIVgenomeandtheproductionoftheGag-Polpolyprotein.TheHIVDNAintegratedintothehostgenomeisrepresentedbythecoloredrectanglesandthehostgenomeisthethinnerline.TheHIVDNAcontainsseveralgenesthatencodestructuralandregulatoryproteinsandenzymes.Thisillustrationshowsthethreemaingenes—gag,pol,andenv—thatareconservedamongallretroviruses.Thecodingregionofthevirusisflankedbynoncodingregionscalledlongterminalregions(orLTRs)thatcontainregulatoryelementsfortranscription.TheHIVDNAistranscribedintoasingleRNAmoleculethatisabout9,700nucleotideslongandthatcanbetranslatedtoproduceapeptideGag-Polpolyprotein.HIVproteasethencleavestheGag-PolpolyproteintoproducetheproteinsthatmakeuptheHIVcapsidandtheenzymesprotease,reversetranscriptase,andintegrase.(TheHIVRNAcanalsobesplicedtoproduceseveraldifferentRNAsthatcanthenbetranslatedintovariousotherproteins,includingregulatoryproteinsandenvelopeproteins.However,thisactivityonlyfocusesonproductionoftheGag-Polpolyprotein.)The5'capand3'tailarecomponentscommontoalleukaryoticmRNAs.The5'capprotectsthemRNAfromdegradationandallowsthecell’sribosometobindtothemRNAtostarttranslation.The3'tailalsoprotectsthemRNAfromdegradation.

Preparingthedowel

ThedowelrepresentsHIVRNA.Usingdifferentcolormarkersorpaint,colorinanareaonthedowelforeachofthekeyproteinregions(Figure2).

Figure2.Exampleofwhatthedowelshouldlooklike.

gag pol envhostgenome

Double-strandedHIVDNAintegratedintohostgenome:

Transcription(bythehostcell’sRNApolymerase)

gag pol env5'cap+UTR UTR+3'tail

Single-strandedHIVRNA:

TranslationofGag/Polpolyprotein(bythehostcell’sribosomes)

Proteincleavage(byHIVprotease)

HIVcapsidproteins

protease

reversetranscriptase

integrase

HIVGag-Polpolyprotein:

HIVproteins:

severalgenes severalgenes

untranslatedregion(UTR)

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Educator Materials Demonstration HIV Protease Inhibitors

February3,2017

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Table1.SuggestedsectionsformarkingtheRNAdowel.

Region Genes Color Approximatelengthondowel

(mm)

5’capandUTR

nocolor 60

gag blue 220

pol black 310

vf/vpr/tat/rev/vpu yellow 100

env green 200

tat/rev/nef red 30

UTRand3’tail

nocolor 60

Note:Youcanpickanycolors;thesearejustsuggestions.Thegenesvf/vpr/tat/rev/vpureceiveasinglecolorforsimplicity.Thesameistruefortat/rev/nef.UTRstandsforuntranslatedregion;thisisaportionoftheRNAthatisnottranslatedintoprotein.The5'capand3'tailarecomponentscommontoalleukaryoticmRNAs.Thelengthofdifferentcoloredsegmentsonthedowelroughlycorrespondtothelengthsofgenes.

Conductingthedemonstration

Part1.ModelingGag-PolPolyproteinSynthesis

1.ShowstudentsthedowelandexplainthatitrepresentsHIVRNAthatwastranscribedbytheviralDNAintegratedintothehostgenome.Explainthateachcolorrepresentsadifferentgeneorclusterofgenes.Pointoutthegagandpolgenes(blueandblackcolorinFigure2).Thesetwogenescanbetranslatedasasinglepolyprotein.ThepolyproteinisthencleavedbytheHIVproteaseintoseveralproteins.Youwillmodelthisprocess.

2.Slidetherollofcashregistertapeontothedowel.Explainthattherollrepresentstheribosome.Theribosomerecognizesthe5'capsequenceofthemRNAandstartstranslation.

3.Haveoneortwostudentvolunteersholdthedowel,oneateachend.

4.Askanothervolunteertoslowlymovetherollofcashregistertapealongthedowel.Startunrollingthetapeasyoumovetheribosomeoverthestartofthegaggene(thebluesection).

5.Astheregistertape(ribosome)movesalongthedowel(RNA),it“translates”thepolyprotein,whichisrepresentedbyunrollingthepaper(Figure3).

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Figure3.ModelingthetranslationofHIVRNA.ThedowelrepresentsmRNAandthecashregistertaperepresentstheribosome.Starttranslation(whichmeansunrollingthetape)atthebeginningofthegaggene,whichismarkedinbluehere.ThepapercomingofftheregistertaperepresentstheGag-Polpolyprotein.

6.Whentherolloftapereachestheendofthegaggene,stopandmarktheendoftheGagproteinonthepaper(Figure4).

7.Continuetounrollthetapeuntilyoureachtheendofthepolgene(blacksection).Asyoureachtheend,ripthepapertoendtranslation.

Figure4.ContinuingtotranslatetheGag-Polpolyprotein.MarktheendofthegaggenewithalineonthewhiterollofpaperandcontinuetotranslatetheHIVmRNAuntilyoureachtheendofthepolgene(coloredblackhere).

8.ThewhitepaperyouproducedrepresentstheGag-Polpolyprotein.

Part2:ModelingcleavageoftheGag-Polpolyprotein(proteaseactivity)

9.AftertheGag-Polpolyproteinhasbeentranslated,theHIVprotease(scissors)cutsthepolyproteinintoseparateproteinstogeneratematureproteinsthatmakeupnewviralparticles(Figure5).Thegaggeneencodestheproteinsthatmakeupthecapsid,theproteincoatingaroundtheHIVviralgenome.Thepolgeneencodesthreeenzymes:protease,reversetranscriptase,andintegrase.

Pol Gag

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Figure5.ModelingthecleavageoftheGag-Polpolyproteinintosixproteins.Thefirstthreeproteins,encodedbythegaggene,arepartoftheHIVcapsid.Theotherthree,encodedbythepolgene,aretheHIVenzymesprotease,reversetranscriptase,andintegrase.

Part3:Modelingtheproteaseinhibitor

Todemonstratetheactionofaproteaseinhibitor,repeatsteps1through8above.Then,beforestep9,wedgeaStyrofoamconebetweenthebladesofthescissors(Figure6).Thisactionmodelshowaninhibitorymoleculemaybindtheactivesiteofproteaseenzymeandpreventitfrombindingtoitstarget(inthiscase,theregistertaperepresentingtheHIVpolyproteinGag-Pol).Iftheactionoftheproteaseisblocked,thentheGag-Polpolyproteinisnotseparatedintoindividualproteinsandcannotgeneratenewviruses.

Figure6.Modelingtheactionoftheproteaseinhibitor.Thescissorsrepresenttheproteaseenzyme,thebladesofthescissorsrepresenttheactivesite,andtheStyrofoamconerepresentstheproteaseinhibitordrug.

AUTHORS

WrittenbyPatriciaNolanBertino,Scotia-GlenvilleHighSchool,andAnthonyBertino,CanandaiguaAcademy.JamesBlankenship,PhD,CornellUniversity,advisedontheproject.PhotosandadditionalwritingbyMaryColvard.

EditedbyLeahM.Cataldo,PhD,BuckinghamBrowne&NicholsSchool;KarenGulliver,consultant;andLauraBonetta,PhD,HHMI.ReviewedbyMelissaCsikari,HHMI.

ScientificreviewbyAllenBateman,PhD,DebbyWalser-Kuntz,PhD,CarletonCollege,MunirSyed,PhD,HartwickCollege.