Chapter38 Genomic RNA replication.ppt

Chapter38GenomicRNAreplication,Outline,RNA-dependentRNAReplicationRNAreplicationofdsRNAvirusRNAreplicationofssRNAvirusDNA-mediatedRNAreplicationRNAreplicationofretro-virusesRetro-transposonRetro-transcriptioncatalyzedbytelomeraseRetro-transcriptioninthelifecycleofsomeDNAvirusesRetro-transcriptionoccurringintheprokaryotes,RNA-dependentRNAReplication,RNA-dependentRNAreplicase(RdRP)isencodedbyviralgenomicRNAProceedsonlyin53PrimersareseldomrequiredError-proneandhighlymutagenicInsensitiveactinomycinDandsensitivetoRNaseMainlyoccursinthecytoplasmofhostcells,ReplicationofPlus-strandorMinus-strandRNAViruses,RNAreplicationofretro-viruses,DiscoveryRetro-viruses1)Structure2)Life-cycle(takingHIVasanexample)-3)Retro-transcriptaseanditsinhibitorThreeenzymaticactivitiesInhibitors:AZTandothers4)“Cocktailtherapy”Retro-transcriptaseandItsapplication,DiscoveryofRetro-transcription,1960s:HowardTeminknewthatretrovirusgenomeswerecomposedofRNAandobservedthatreplicationwasinhibitedbyactinomycinDthereforeheproposedtheconceptofreversetranscription(TheNobelprizeawardedtoBaltimoreandTemin,1975).1970:TeminandDavidBaltimore(separately)discoveredtheRNA-directedDNApolymerase-"reversetrascriptase",Retrovirusesandoncogenes,AllRNAtumorvirusesareretroviruses.RNAvirusoncogenesarealteredformsofnormalhostgenesthatoccurinthevirusgenome.Examplesofretrovirusesinclude:Roussarcomavirus(RSV)FelineleukemiavirusMousemammarytumorvirusHumanimmunodeficiencyvirus(HIV),Structureofaretrovirus,StructureofHIV,RetroviralGenomicRNA,Identicaltofull-lengthviralmRNA:5-m7GpppNcapstructure3-polyAsplicingsignalsU:unique,e.g.,U5isuniqueto5-endoftheRNAgenomeCis-actingsignalsforreplication:RisadirectlinearrepeatrequiredforstrandtransferPBS:primerbindingsiteforfirststrandsynthesisPPT:polypurinetractprimessecondstrandsynthesis,TheRetrovirusGenome-plusstrandRNA,HIV-1GENOME,Diploid,1XRNA,Size=9,749nteachmoleculetRNA(LYS)primerattachedtoeachRNA5-capand3-PolyAtail;Encodes9proteinsCap-R-U5-PrimerBind-(TENORFs)-U3-R-PolyALTRofPROVIRUS(2XDNA):U3-R-U5=634bpeachLTRU3=453bpR=98bpU5=83bp,Threegenesoccurinmostretroviruses,gag(groupantigen):codestheproteincorepol(polymerase):codesreversetranscriptaseandanenzymeforproviralintegration.env(envelope):codesenvelopeglycoproteins,Lifecycleofaretrovirus,Ch6anim2.mov,TimediagramofHIVlifecyclebyJoeLertola,LifecycleofHIV,HIV-1MembraneFusionMachine,OverviewofReverseTranscription,GenomicRNA,Clue:Differenceinthetwoforms,primer,ViralgenomicRNA,Reversetranscriptase,dsDNA,Result:NewcopyofviralRNAisshorter-lackscontrolsequences,ProblemofusingRNApolIItocopyagenome,RT,ViralRNA,Reversetranscriptase,RU5,U3R,U3RU5,U3RU5,Longterminalrepeatsareformed,POLII,FormationofLTR,Retrovirusescanhaveonlyonepromoter,LTR,LTR,U5,ThereforeonlyonelongRNAcanbemadeThereforemRNArequiresprocessingExplainswhyRNAhastobepositivesense,POLII,ContainedinU3,Reversetranscriptase,Theenzymecontainstwosubunits-p66andp51.Thesmallersubunitisproducedfromthelargersubunitthroughtheactionofaprotease.Despitetheidentityinsequence,however,thesmallersubunitfoldsdifferentlythanthelargesubunit.Thelargesubunitcontainstheenzymaticactivities.Ithasboththe5->3polymeraseactivityandtheRNaseHactivity.Thepolymeraseactivityislocatedinthreedomainsfoldedintothe"palm-fingers-thumb"motifthatiscommonamongpolymerases.TheRNaseHactivityislocatedinaseparatedomain.Thesmallsubunitassociateswiththelargesubunitandprotectsitfromdegradation.Reversetranscriptaseisveryinacurate-Coupledwiththeabsenceofanyproofreadingactivity,thisisthemajorreasonwhytheHIVretrovirusissoeasilyabletodevelopresistancetoanti-retroviraldrugs.Inthelaboratory,reversetranscriptaseisusedtomakecDNAfromeukaryoticmRNAandalsointheRT-PCRprocedure.,Retro-transcriptionoftheHIVRNAgenome,Retro-transcriptionoftheHIVRNAgenome(continued),IntegrationofRetroviralcDNAintohostChromosomalDNAEstablishestheProvirus,Integration:covalentlinkageofdsviralcDNAtohostchromosomalDNA.SiteofintegrationisrandomwithrespecttohostchromosomalDNA.Provirus:permanentgeneticelementintheinfectedcell,andinallofthecellsprogenyIntegrase:viralnucleasethatclips2nucleotidesfromthe3endofthe2LTRs.InaconcertedreactionitthenmakesastaggeredcutinhostchromosomalDNA,andligatestheclipped3endsofviralDNAtohostDNA.Ri.mov,HostCellRNAPII,TranscriptionoftheHIVgenomicDNA,Provirus,PrimaryRNAtranscript,Ret.mov,HIV-1U3EnhancerSites,U3ofthe5-LTRoftheproviruscontainsthesingleviralpromoterwhichcontainsbindingsitesforfactorsfoundinTcellsandmacrophages.,FatesoftheprimaryRNAtranscript,Daughtergenome:tRNAadded;dimerof1xTranslatedtogagpolyproteinTranslatedwithaframeshifttogag-polSplicedtomRNAforenvSplicedtov-oncmRNAinvirusesthathavev-oncgene,HIV-1OpenReadingFrames,TheParadoxofHIV-1Tat,Trans-actingtranscriptionalactivator,Bindsstem-loopstructure(TAR)innascentHIV-1transcript,HowdoesTatstimulatetranscriptionoftheRNAtowhichitbinds?,TatEnhancesProcessivityofRNAPolymeraseII,RNApolII,CDK9,CyclinT,Tat,RNApolII,CTD,HowdoesHIV-1express9ORFsfrom1mRNA?,HIV-1expressesmorethan30mRNAs,Acceptor,Donor,UseofPolyproteinsIncreasesRetroviralCodingCapacity,RetroviralProteaseActivation,ViralProtease,NotrequiredforvirionassemblyRequiredforvirionmaturationandinfectivityInhibitorsofthisenzymearenowastandardpartoftheanti-HIVdrugregimenCombinationtherapynecessitatedbyresistance,REinE.coliandMyxococcusxanthus,TheseRT-genesarefoundinanoperonwithtwootherregions,msrandmsd.TheRTdomainistranslated,andtheresultingenzymereversetranscribestheremainingtranscriptthathasenteredastem-loopstructure.Reversetranscriptionstopsattheboundarybetweenmsrandmsd,resultinginachimericalRNA/DNAstructure.,Tissuee.g.brain,LysecellsandPurifymRNAs,mRNA,OligodT,RT,cDNA,TreatwithRNaseHoralkali,DNAPI,S1nuclease,Ds-cDNA,