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www.BioInteractive.org February3,2017
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Educator MaterialsHands-on Activity Viral DNA Integration
OVERVIEW
Thisactivityispartofaseriesofactivitiesanddemonstrationsfocusingonvariousaspectsofthehumanimmunodeficiencyvirus(HIV)lifecycle.
HIVisaretrovirus.Retrovirusesaredistinguishedfromothertypesofvirusesbytwokeystepsinthereplicationcycle:reversetranscriptionandintegration.AfterHIVinfectsahostcell,reversetranscriptionresultsintheproductionofadouble-strandedDNAcopyofthesingle-strandedHIVRNAgenome.IntegrationresultsintheinsertionoftheHIVDNAintoaregionofthehostgenome.
Inthisactivity,studentsmodeltheintegrationstep.Itmaybehelpfulforstudentstocompletethereversetranscriptionactivitybeforecompletingthisactivity.
KEYCONCEPTSANDLEARNINGOBJECTIVES
• Virusescanonlyreplicatewithinhostcells.
• Thereplicationcycleofretrovirusesisdistinguishedfromthatofothervirusesbytwokeysteps:reversetranscriptionandintegration.
• Followinginfection,thegeneticmaterialofaretrovirusbecomespartofthehostcellgenome.
Studentswillbeableto:
• ModelhowviralRNAisintegratedintohostcellDNAbyusingdifferentcolorsofplasticpopbeads.
• ApplyconceptsrelatedtoDNAandRNAsynthesistoviralreplicationprocesses.
CURRICULUMCONNECTIONS
Curriculum Standards
NGSS(2013) HS-LS-1.1;HS-LS-1.2;HS-LS-3.1
APBiology(2013) 3.A.1;3.C.3
IBBiology 6.3
KEYTERMS
Genome,DNA,RNA,reversetranscriptase,integrase,transcription,translation
TIMEREQUIREMENTS
Thisactivityisdesignedtobecompletedina50-minuteclassperiod.
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Educator MaterialsHands-on Activity Viral DNA Integration
SUGGESTEDAUDIENCE
Thisactivityisappropriateforalllevelsofhighschoolbiology.
PRIORKNOWLEDGE
Studentsshouldknowthatvirusesconsistofanucleicacidsurroundedbyaproteincoatandthattheyinfectdifferenttypesofcells.Itwouldbehelpfulforstudentstounderstandthatsomevirusesusetheirownenzymes,aswellashostcellenzymes,toreproduce.
MATERIALS
Forthisactivity,youwillneedpopbeadsofthreedifferentcolorstorepresent:
• thedouble-strandedDNAgenomeofthehostcell(about40to50beadsperstudentorgroup)
• thesingle-strandedRNAgenomeofthevirus(about5to10beads)
• thedouble-strandedDNAcopyofthevirus’sgenome(doublethenumberofbeadsusedfortheRNA)
TEACHINGTIPS
Introduction
• Inthisactivity,studentswillwatchananimationoftheHIVlifecycle(http://www.hhmi.org/biointeractive/hiv-life-cycle).Thereversetranscriptionandintegrationoftheviralgenomearedemonstratedatabouttime1:32,followedbyviralproteinsynthesis,viralassembly,andbudding.TheHIVlifecycleisalsoshownintheClickandLearn“VirusExplorer”(http://www.hhmi.org/biointeractive/virus-explorer)byclickingontheHIV3-Dmodelandthenselectingthereplicationcycleillustration.SpendsometimeinclassreviewingtheHIVlifecycletoensurethatstudentsunderstandthedifferentsteps.[Foradditionalbackgroundonretroviruses,studentscanalsocompletetheHHMI“Click&Learn”resource“RetrovirusesandViralDiversity”(http://www.hhmi.org/biointeractive/retroviruses-and-viral-diversity).]
• HIVinfectsimmunecells.Ifstudentsarenotfamiliarwithdifferenttypesofimmunecells,youcanshowthemashort42-secondclipfromLecture1oftheHHMIHolidayLecture“AIDS:EvolutionofanEpidemic”(http://www.hhmi.org/biointeractive/aids-evolution-epidemic).ClickonChapter14,“Cellsoftheimmunesystem,”andwatchfromtime19:13to19:55.Thischapterprovidesanoverviewoftheimmunesystemanditsassociatedcells.HIVinfectshelperTcellsbybindingtotheCD4receptoronthesecells.
• Amoreextensivereviewoftheimmunesystemisavailableasa“ClickandLearn”resourceentitled“CellsoftheImmuneSystem”(http://www.hhmi.org/biointeractive/cells-immune-system).ThisClickandLearnoffersastudentworksheetthatcanbecompletedashomework.
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Educator MaterialsHands-on Activity Viral DNA Integration
Procedure
• StudentswillneedtoworkthroughtheClickandLearn“VirusExplorer”(http://www.hhmi.org/biointeractive/virus-explorer)tocompletequestions1–5inthestudentworksheetbeforedoingtheviralDNAintegrationactivity.ThisClickandLearnandthethreequestionscanbecompletedashomeworkbeforeclass.
• Providestudentswiththreedifferentcolorsofpopbeadsandaskthemtomakemodelsthatrepresentsingle-strandedviralRNA,double-strandedviralDNA,anddouble-strandedhostcellDNAgenomes.Studentswillneedatleast40to50popbeadsofonecolor(forthehostDNA),5to10fortheviralRNAgenome,and10to20fortheDNAcopyoftheviralgenome.Studentsmayworkindividually,inpairs,orinsmallgroups.
• Asanalternativetousingpopbeads,it’salsopossibletousepipecleanersandregularponybeads.
• Whenreviewingstudentwork,besurethatthesingle-strandedviralRNAmodelisthesamelengthasthedouble-strandedviralDNAcopyandthatbothviralgenomemodelsareshorterthanthehostcellDNApop-beadstrands.Askstudentstotryagainiftheirmodelsdonotshowthisdistinction.Pointouttostudentsthatthesearerelativesizes.Theactualhumangenomehasover3millionnucleotides,whereastheHIVRNAgenomeislessthan10,000nucleotideslong.
• Onepointthatyoumaywanttomentiontostudents,dependingontheirlevel,isthatwhenthereversetranscriptasesynthesizesthedouble-strandedDNAmolecule,theresultingmoleculeactuallyisn’tidenticaltotheRNAgenome;theprocessgeneratesaslightlylongermoleculewithlongterminalrepeats(LTR)ateachend.TheLTRsequenceisnotfoundintheRNAgenomebutisgeneratedduringtheprocessofreversetranscription.(Thisstepisnotincludedintheanimations.)TheLTRisimportantforexpressionoftheviralgenes.
• StudentsmayhavenoticedintheanimationthateachHIVparticlecontainstwocopiesoftheHIVgenome.Eachcopyisasingle-strandedRNAmolecule.Humancellsalsohavetwocopiesofthegenome;eachcopyisadouble-strandedDNAmolecule.
Extensionactivities
• Asanextensionactivity,showashort58-secondclipfromLecture2oftheHHMIHolidayLecture“AIDS:EvolutionofanEpidemic”(http://www.hhmi.org/biointeractive/aids-and-hiv-life-cycle).ClickonChapter5,“Howvirusescausedisease,”andwatchfromtime5:57to6:55.Dr.BruceWalkerexplainshowavirus“actsasacommandothattakesoveramachinetoolfactoryandturnsitintoabombfactory.”AskstudentshowHIVdoesthatbasedonwhattheylearnedthroughthisactivity,usingtheintegrationoftheviralgenomeasanexample.
• Aftercompletingthisactivity,studentsmayfinditinterestingtoresearchantiretroviraldrugs.Discusswithstudentshowunderstandingthemechanismbywhichavirusreplicatescanhelpidentifydrugtargets.Explaintothemthatavarietyofantiretroviraldrugsareavailableandthe
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Educator MaterialsHands-on Activity Viral DNA Integration
drugscanbeeffectivebytargetingdifferentpartsofthereplicationprocess.Studentsmaybeparticularlyinterestedinthedrugsthattargetintegrasesincetheynowhaveabetterunderstandingoftheprocessofintegration.ThreesuchdrugsthatpreventthevirusfromintegratingintothehostDNAareraltegravir,elvitegravir,anddolutegravir.
ANSWERKEY
1.DrawadiagramandexplainthebasiccharacteristicsofHIV.
Thediagramshouldincludetheenvelope,envelopeproteins,capsid,andRNAgenome.Theycouldalsoincludethereversetranscriptaseandintegraseenzymes.
2.Definetheterm“genome”anddescribetheHIVgenome.
AnswersmayvarybutshouldincludetheinformationthatgenomesaremadeofnucleicacidsandthatgenesareregionsofDNAthatencodeproteinsessentialforfunction.Agenometypicallycontainsalloftheinformationneededtobuildandmaintaintheorganism.TheHIVgenomeisasingle-strandedRNAmolecule.
3.WhatisthehostcellforHIV?HowarethecompositionandsizeofthehostcellgenomedifferentfromthoseofHIV?
HIVinfectshumanimmunecells,andmorepreciselyhelperTcells.Thehumangenomeconsistsofdouble-strandedDNA.ItismadeofDNAandnotRNA,itisdouble-strandedratherthansingle-stranded,anditismuchbiggerthantheHIVgenome.
4.Whatistheenzymeresponsibleforreversetranscription?Isitaviralorhostenzyme?
Theenzymeresponsibleforreversetranscriptioniscalledreversetranscriptase.Itisaviralenzymethatisreleasedintothehostcellafterinfection.
5.WhatistheenzymeresponsiblefortheinsertionoftheviralDNAintothehostgenome?Isitaviralorhostenzyme?
TheviralenzymeintegraseisinvolvedintheinsertionofacopyoftheviralDNAintothehostcellgenome.
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Educator MaterialsHands-on Activity Viral DNA Integration
Table1.ModelsofHostCellGenome,HIVGenome,andReverse-TranscribedViralDNA.
Model HostCellGenome HIVgenome DNAcopyofHIVgenome
Drawadiagramofthemodelyouconstructed
Characteristics(RNAorDNA;single-ordouble-stranded;relativesize)
Double-strandedDNA
Genomesize=relativelylarge
Single-strandedRNA
Genomesize=relativelysmall
Double-strandedDNA
Genomesize=relativelysmall
8.Comparethethreemoleculesyouconstructed.Whatarethekeysimilaritiesanddifferences?
ThehumangenomeismanytimeslargerthantheHIVgenome.Theviralgenomeconsistsofsingle-strandedRNA,whilethehumanhostcellgenomeiscomposedofdouble-strandedDNA.DNAiscomposedofthenucleotidesadenine,guanine,cytosine,andthymine.TheHIVRNAgenomeiscomposedofthenucleotidesadenine,guanine,cytosine,anduracilinplaceofthymine.Reversetranscriptionresultsinadouble-strandedDNAcopyoftheviralgenome.Thismoleculeislikethehostcellgenomeexceptmuchsmallerinsize.Studentsmayalsopointoutthateachgenomecontainsuniquegenesthatencodeproteinsfortheirviralorcellularfunction.However,theviralgenomedoesnotcodeforalltheproteinsthevirusneedstoreproduce;forexample,thevirusreliesonthehostcellmachinerytoproduceitsproteins.
9.AftertheviralDNAisproduced,thenextstepintheHIVlifecycleistheinsertionofviralDNAintothehostcellgenome.Usethedouble-strandedviralDNAandthehostDNApop-beadmodelstoconstructa
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Educator MaterialsHands-on Activity Viral DNA Integration
modelshowinghowtheviralgenomeisintegratedintothehostgenome.Drawadiagramofyourpop-beadconstructionbelow.(Makesuretolabelyourdiagram.)
10.Describethestepsinvolvedintheintegrationprocess.(Thinkaboutwhatyouhadtodowiththebeads.)
Thestepsarebreaking(orcleaving)thedouble-strandedhostDNAandthensplicingintheviralDNA.Moreadvancedstudentswillknowthatchemicalreactionsareinvolvedinbreakingbondsbetweennucleotidesandthencreatingthosebonds.
11.DescribewhathappenstotheviralDNAnowthatitisintegratedintothehostcellgenome.
TheHIVgenomewillbetranscribedintomRNAusingthecellularenzymeRNApolymerase.Upontranscription,theviralmRNAencodingitsstructuralproteinswillbetranslated,processedinthecytoplasm,andtransportedtothecellmembrane,whichiswherenewviralparticlescontainingsingle-strandedHIVgenomeswillbudfromthehostcell.
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.