Download Educator Materials Demonstration HIV Protease Inhibitors HIV

Demonstration
HIV Protease Inhibitors
Educator Materials
HIVPROTEASEINHIBITORS
OVERVIEW
Thisdemonstrationispartofaseriesofactivitiesanddemonstrationsfocusingonvariousaspectsofthe
humanimmunodeficiencyvirus(HIV)lifecycle.
HIVisaretrovirus,atypeofvirusthatintegratesitsgenomeintothehostcell’sgenome.(Twoother
activitiesfocusonthereversetranscriptionandintegrationstepsoftheviralreplicationcycle.)HIVhas
genescommontoallretroviruses(thegenesgag,pol,andenv)thatencodestructuralproteinsand
enzymes,aswellasgenesthatareuniquetoitsviralstructureandfunction.Thisactivityfocusesonthegag
andpolgenes.ThesetwogenesaretranscribedintoasingleRNAmolecule,whichisthentranslatedto
produceasinglepolyprotein.TheGag-PolpolyproteiniscleavedbytheHIVproteaseenzymetogenerate
sixproteinsessentialforassemblingthevirusparticle.
Duringthisactivity,youwilldemonstratethemechanismbywhichtheGag-Polpolyproteinisproducedand
cleavedbyHIVprotease.ThedemonstrationmodelsthisprocessandalsoshowshowinhibitingHIV
proteaseactivitypreventsthevirusfromcreatingmatureproteins.Proteaseinhibitorsareaclassofdrugs
usedtotreatHIVinfection.
KEYCONCEPTSANDLEARNINGOBJECTIVES
•
HIVRNAcanbetranslatedintopolyproteins;post-translationalcleavageofthesepolyproteins
resultsinthegenerationoffunctionalviralproteins.
•
OneofthedrugsdevelopedagainstHIVinterfereswiththefunctionofanHIVenzyme,aprotease,
responsibleforcleavingHIVpolyproteins.
Studentswillbeableto
•
applytheconceptsofproteinsynthesisandenzymefunctiontoviralreplicationandtheHIVlife
cycle.
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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Demonstration
HIV Protease Inhibitors
Educator Materials
TIMEREQUIREMENTS
IfstudentsarefamiliarwiththeHIVlifecycle,thedemonstrationitselftakesapproximately15minutes.
Preparationofmaterialsshouldtakeabout30minutesthefirsttimeitisdone.
SUGGESTEDAUDIENCE
ThislessonisappropriateforAPBiology.
PRIORKNOWLEDGE
Studentsshouldbefamiliarwiththeprocessesoftranscriptionandtranslationineukaryoticcells.
MATERIALS
•
•
Woodendowel7/8inchindiametertorepresentHIVRNA.
o
Thedowelcanbepurchasedatmosthomeimprovementorcraftstoresoronline.
o
Thedowelshouldbepreparedaheadofclassbycoloringdifferentpartstoindicate
differentgenesorgroupsofgenes.
Rollofcashregistertape2¼”×130ft(57mm×39m)torepresentthehostcell’sribosome.
o
Thecashregistertapeisavailableatmostbusinesssupplystoresoronline.
o
Thepaperrolledoutofthecashregistertapewillrepresentthepolyproteinbeing
synthesized.
o
Besurethatthecentralcoreoftheregistertapefitsoverthewoodendowel.
•
Scissorstorepresenttheviralproteaseenzyme.
•
Styrofoamcone2¾”×17/8”(69.85mm×47.62mm)torepresenttheproteaseinhibitor.
o
TheStyrofoamconescanbefoundintheflowerarrangementdepartmentofmostcraft
storesoronline.
o
Theconeshouldbelargeenoughtofitbetweenthebladesofthescissorsandpreventthe
scissorsfromcutting.
TEACHINGTIPS
Beforeconductingthedemonstration
•
IfstudentsarenotfamiliarwiththestructureofHIV,itwillbehelpfulforthemtobecomefamiliar
withthebasics.HIVisanenvelopedretrovirusthatconsistsofanRNAgenome,aviralcapsid,and
anoutermembrane,orenvelope,withembeddedproteins.ToseethestructureofHIV,visitthe
interactive“VirusExplorer”athttps://www.hhmi.org/biointeractive/virus-explorer.Showstudents
theHIVcrosssectionandpointouttothemtheHIVcapsidprotein(encodedbythegaggene)and
theintegrase,reversetranscriptase,andproteaseenzymes(encodedbythepolgene).Alsopoint
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Demonstration
HIV Protease Inhibitors
•
•
Educator Materials
outtheHIVRNAgenome.NotethatthefullHIVgenomeisencodedonasinglestrandofRNA.
However,eachvirusparticlecontainstwoseparate,identicalRNAstrands.
StudentsshouldalsobefamiliarwiththekeyeventsintheHIVlifecycle.Mainly,whenHIVinfectsa
cell,theHIVRNAgenomeisreverse-transcribedintoDNA,whichisthenintegratedintothehuman
genome.ThisDNAcopyoftheHIVgenomeisthentranscribedintoRNAbythehostcellmachinery.
TheHIVRNAcanbeincorporatedintonewvirusparticlesortranslatedintoproteins.Ananimation
oftheHIVlifecycleisavailableathttps://www.hhmi.org/biointeractive/hiv-life-cycle.
Studentsmaywonderwhetherhumancellsalsoproducepolyproteins.Humancellsdonotproduce
polyproteins.Inhumancells,mRNAsaretypicallytranslatedintosingleproteins.However,several
RNAvirusesproducepolyproteins.Becausethismechanismisnotusedbyhostcellsandutilizes
viralenzymes,theseenzymesmakegoodtargetsforantiviraldrugs.
Aftercompletingthedemonstration
•
•
•
Aftercompletingtheactivity,youmaywanttoshowstudentsananimationdemonstrating
proteaseinhibitionavailableathttp://www.hhmi.org/biointeractive/protease-inhibitors.
StudentsmaywonderhowproteaseinhibitorsrelatetootherdrugsusedtotreatHIV.Otherdrugs
areavailablethattargetdifferentstepsintheHIVlifecycle:viralentry,reversetranscription,
integration,andproteincleavage.Thefirstsuchdrug,azidothymidine,whichinhibitstheviral
reversetranscriptaseenzyme,wasapprovedbytheFoodandDrugAdministrationin1987.Thefirst
proteaseinhibitor,saquinavir,wasapprovedin1995.Asof2015,eightproteaseinhibitorswere
commerciallyavailabletotreatHIV.BecauseHIVmutatesrapidlyduetothelackofproofreading
capacityofreversetranscriptase,resistancecaneasilydevelopagainstanydrug.Tocombat
resistance,doctorstypicallygivepatientsseveraldifferentantiretroviraldrugs.Thiscombination
treatmentlowersthechancesofthevirusbecomingresistantbecausethevirusmustbecome
resistanttomultipledrugsthattargetdifferentaspectsofitslifecycle.
Althoughhumancellsdonotproducepolyproteins,theydoproduceproteases.Proteasesmodify
proteinsoncetheyaremade.Manyproteinsthatareexportedfromthecytoplasmaresynthesized
byribosomesasproproteins(orpreproteins)locatedontheroughendoplasmicreticulumand
subsequentlycleavedbyproteasestobecomefunctionalproteins.Forexample,proteasesactivate
proproteinssuchasproinsulinandbloodclottingfactors.Proteasesarealsoresponsiblefor
destroyingproteinsoncetheyarenolongerneeded.
PROCEDURE
BackgroundInformation
TheHIVgenomecontainsninegenes.Threeofthem(gag,pol,andenv)arecommontoallretrovirusesand
theothersareuniquetoHIV(seeFigure1forasimplifiedschematic).Inthisactivity,wewillfocusonthe
gagandpolgenes.Thegaggeneencodesthreeproteinsthatcomprisetheviralcapsid.Thepolgene
encodesthreeenzymesthatareessentialforviralgenomereplicationandintegrationintothehostgenome
(reversetranscriptase,protease,andintegrase).WhenHIVDNAisintegratedintothehumangenome,itis
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Demonstration
HIV Protease Inhibitors
Educator Materials
transcribedtoproduceaprimaryRNAthatcaneitherbecomepartofnewvirusesorbetranslatedinto
variousdifferentproteins.OneoftheproteinsthataregeneratedisaGag-Polpolyprotein.
hostgenome
gag
severalgenes
severalgenes
Double-strandedHIVDNAintegratedintohostgenome:
pol
env
untranslatedregion(UTR)
Transcription(bythehostcell’sRNApolymerase)
Single-strandedHIVRNA:
5'cap+UTR
gag
HIVGag-Polpolyprotein:
pol
env
UTR+3'tail
TranslationofGag/Polpolyprotein(bythehostcell’sribosomes)
Proteincleavage(byHIVprotease)
HIVproteins:
protease
HIVcapsidproteins
integrase
reversetranscriptase
Figure1.SimplifiedillustrationoftheHIVgenomeandtheproductionoftheGag-Polpolyprotein.TheHIVDNA
integratedintothehostgenomeisrepresentedbythecoloredrectanglesandthehostgenomeisthethinnerline.The
HIVDNAcontainsseveralgenesthatencodestructuralandregulatoryproteinsandenzymes.Thisillustrationshows
thethreemaingenes—gag,pol,andenv—thatareconservedamongallretroviruses.Thecodingregionofthevirusis
flankedbynoncodingregionscalledlongterminalregions(orLTRs)thatcontainregulatoryelementsfortranscription.
TheHIVDNAistranscribedintoasingleRNAmoleculethatisabout9,700nucleotideslongandthatcanbetranslated
toproduceapeptideGag-Polpolyprotein.HIVproteasethencleavestheGag-Polpolyproteintoproducetheproteins
thatmakeuptheHIVcapsidandtheenzymesprotease,reversetranscriptase,andintegrase.(TheHIVRNAcanalsobe
splicedtoproduceseveraldifferentRNAsthatcanthenbetranslatedintovariousotherproteins,includingregulatory
proteinsandenvelopeproteins.However,thisactivityonlyfocusesonproductionoftheGag-Polpolyprotein.)The5'
capand3'tailarecomponentscommontoalleukaryoticmRNAs.The5'capprotectsthemRNAfromdegradationand
allowsthecell’sribosometobindtothemRNAtostarttranslation.The3'tailalsoprotectsthemRNAfrom
degradation.
Preparingthedowel
ThedowelrepresentsHIVRNA.Usingdifferentcolormarkersorpaint,colorinanareaonthedowelfor
eachofthekeyproteinregions(Figure2).
Figure2.Exampleofwhatthedowelshouldlooklike.
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Demonstration
HIV Protease Inhibitors
Educator Materials
Table1.SuggestedsectionsformarkingtheRNAdowel.
Region
Genes
Color
Approximate
lengthon
dowel
(mm)
5’cap
and
UTR
nocolor
60
gag
blue
220
pol
black
310
vf/vpr/tat/rev/vpu
yellow
100
env
green
200
tat/rev/nef
red
30
UTR
and3’
tail
nocolor
60
Note:Youcanpickanycolors;thesearejustsuggestions.Thegenesvf/vpr/tat/rev/vpureceiveasinglecolorfor
simplicity.Thesameistruefortat/rev/nef.UTRstandsforuntranslatedregion;thisisaportionoftheRNAthatisnot
translatedintoprotein.The5'capand3'tailarecomponentscommontoalleukaryoticmRNAs.Thelengthof
differentcoloredsegmentsonthedowelroughlycorrespondtothelengthsofgenes.
Conductingthedemonstration
Part1.ModelingGag-PolPolyproteinSynthesis
1.ShowstudentsthedowelandexplainthatitrepresentsHIVRNAthatwastranscribedbytheviralDNA
integratedintothehostgenome.Explainthateachcolorrepresentsadifferentgeneorclusterofgenes.
Pointoutthegagandpolgenes(blueandblackcolorinFigure2).Thesetwogenescanbetranslatedasa
singlepolyprotein.ThepolyproteinisthencleavedbytheHIVproteaseintoseveralproteins.Youwillmodel
thisprocess.
2.Slidetherollofcashregistertapeontothedowel.Explainthattherollrepresentstheribosome.The
ribosomerecognizesthe5'capsequenceofthemRNAandstartstranslation.
3.Haveoneortwostudentvolunteersholdthedowel,oneateachend.
4.Askanothervolunteertoslowlymovetherollofcashregistertapealongthedowel.Startunrollingthe
tapeasyoumovetheribosomeoverthestartofthegaggene(thebluesection).
5.Astheregistertape(ribosome)movesalongthedowel(RNA),it“translates”thepolyprotein,whichis
representedbyunrollingthepaper(Figure3).
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Demonstration
HIV Protease Inhibitors
Educator Materials
Figure3.ModelingthetranslationofHIVRNA.Thedowel
representsmRNAandthecashregistertaperepresents
theribosome.Starttranslation(whichmeansunrolling
thetape)atthebeginningofthegaggene,whichis
markedinbluehere.Thepapercomingofftheregister
taperepresentstheGag-Polpolyprotein.
6.Whentherolloftapereachestheendofthegaggene,stopandmarktheendoftheGagproteinonthe
paper(Figure4).
7.Continuetounrollthetapeuntilyoureachtheendofthepolgene(blacksection).Asyoureachtheend,
ripthepapertoendtranslation.
Pol
Gag
Figure4.ContinuingtotranslatetheGagPolpolyprotein.Marktheendofthegag
genewithalineonthewhiterollofpaper
andcontinuetotranslatetheHIVmRNA
untilyoureachtheendofthepolgene
(coloredblackhere).
8.ThewhitepaperyouproducedrepresentstheGag-Polpolyprotein.
Part2:ModelingcleavageoftheGag-Polpolyprotein(proteaseactivity)
9.AftertheGag-Polpolyproteinhasbeentranslated,theHIVprotease(scissors)cutsthepolyproteininto
separateproteinstogeneratematureproteinsthatmakeupnewviralparticles(Figure5).Thegaggene
encodestheproteinsthatmakeupthecapsid,theproteincoatingaroundtheHIVviralgenome.Thepol
geneencodesthreeenzymes:protease,reversetranscriptase,andintegrase.
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Demonstration
HIV Protease Inhibitors
Educator Materials
Figure5.ModelingthecleavageoftheGag-Polpolyproteinintosixproteins.Thefirstthreeproteins,encodedbythe
gaggene,arepartoftheHIVcapsid.Theotherthree,encodedbythepolgene,aretheHIVenzymesprotease,reverse
transcriptase,andintegrase.
Part3:Modelingtheproteaseinhibitor
Todemonstratetheactionofaproteaseinhibitor,repeatsteps1through8above.Then,beforestep9,
wedgeaStyrofoamconebetweenthebladesofthescissors(Figure6).Thisactionmodelshowaninhibitory
moleculemaybindtheactivesiteofproteaseenzymeandpreventitfrombindingtoitstarget(inthiscase,
theregistertaperepresentingtheHIVpolyproteinGag-Pol).Iftheactionoftheproteaseisblocked,then
theGag-Polpolyproteinisnotseparatedintoindividualproteinsandcannotgeneratenewviruses.
Figure6.Modelingtheactionoftheproteaseinhibitor.Thescissorsrepresenttheproteaseenzyme,thebladesof
thescissorsrepresenttheactivesite,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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