hands-on activity viral dna integration educator materials · viral dna integration overview this...

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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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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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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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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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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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.

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