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www.BioInteractive.org Educator Materials Demonstration HIV Protease Inhibitors February 3, 2017 Page 1 of 7 HIV PROTEASE INHIBITORS OVERVIEW This demonstration is part of a series of activities and demonstrations focusing on various aspects of the human immunodeficiency virus (HIV) life cycle. HIV is a retrovirus, a type of virus that integrates its genome into the host cell’s genome. (Two other activities focus on the reverse transcription and integration steps of the viral replication cycle.) HIV has genes common to all retroviruses (the genes gag, pol, and env) that encode structural proteins and enzymes, as well as genes that are unique to its viral structure and function. This activity focuses on the gag and pol genes. These two genes are transcribed into a single RNA molecule, which is then translated to produce a single polyprotein. The Gag-Pol polyprotein is cleaved by the HIV protease enzyme to generate six proteins essential for assembling the virus particle. During this activity, you will demonstrate the mechanism by which the Gag-Pol polyprotein is produced and cleaved by HIV protease. The demonstration models this process and also shows how inhibiting HIV protease activity prevents the virus from creating mature proteins. Protease inhibitors are a class of drugs used to treat HIV infection. KEY CONCEPTS AND LEARNING OBJECTIVES • HIV RNA can be translated into polyproteins; post-translational cleavage of these polyproteins results in the generation of functional viral proteins. • One of the drugs developed against HIV interferes with the function of an HIV enzyme, a protease, responsible for cleaving HIV polyproteins. Students will be able to • apply the concepts of protein synthesis and enzyme function to viral replication and the HIV life cycle. CURRICULUM CONNECTIONS Curriculum Standards NGSS (2013) HS-LS1-1 AP Biology (2013) 3.B.1; 3.C.3; 4.A.1; 4.B.1 KEY TERMS virus, genome, DNA, mRNA, gene, transcription, translation, enzyme, ribosome, inhibitor, protease, protein, capsid, reverse transcriptase, integrase, polymerase, 5’ cap, 3’ tail

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Page 1: Demonstration HIV Protease Inhibitors Educator Materials · HIV Protease Inhibitors February 3, 2017 Page 1 of 7 HIV PROTEASE INHIBITORS OVERVIEW This demonstration is part of a series

www.BioInteractive.org

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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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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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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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.


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