UNDERSTANDINGFACTORSCONTROLLINGGAGANDGAG-POL POLYPROTEINPRECURSORPROCESSINGINHIV-1 By GREGORYBLOOM ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFUFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA . Dedicatedtomywifeandfamilyforalltheirloveandsupport. ACKNOWLEDGEMENTS Iamextremelygratefulfortheloveandsupportofmyfamilywhohave encouragedmeinmypursuitofhighereducation. Myparents,BernardandSue,have alwaysbelievedthatknowledgeenricheslifeandhavestrovetoinstillthatprincipleinall theirchildren.IgreatlyappreciatemywifeAnnaforherinvaluablesupportand J generosityallthroughoutmycareer. v Iamdeeplyindebtedtomymentor,ProfessorBenDunn,forprovidingmeagreat opportunitytodevelopmyskillsasascientist. Hehasgivenmeinvaluableadviceand guidancethroughoutmygraduatework. Iwouldalsoliketothankthemembersofmy supervisorycommitteeDr.MaureenGoodenow,Dr.HarryNick,DrNancyDenslow,and Dr.CharlesAllen,fortheirexpertiseandsuggestionsthathelpedmetocompletethe workpresented. TheDunnlaboratoryhasbeenawonderfulenvironmentforlearningaswellas research.IthankBillFarmariewhohasalwaysbeenaninspiration. Iamgratefulforthe supportandfriendshipofthemembersoftheDunnlab. IamindebtedtoJen,Jeff, Deepa,Brian,Marina,Chetana,Kevin,Su,Becky,Patty,Constanza,Jay,JoeandMike formakingmytimeinthelabaveryenjoyableone. iii TABLEOFCONTENTS page ACKNOWLEDGEMENTS iii LISTOFTABLES vii LISTOFFIGURES viii ABBREVL\TIONS x ABSTRACT xii CHAPTERS BACKGROUNDANDINTRODUCTION 1 1 Classification 1 OriginsandDiscovery 2 CurrentInfectionStatistics 4 ViralStructure 8 GenomicStructure 10 ViralLifecycle 14 AttachmentandCoreEntry 14 ReverseTranscription 15 Integration 17 TranscriptionandExpressionofViralProteins 18 ViralAssemblyandMaturation 20 Protease 22 Structure 22 Function 24 InhibitionofProteaseFunction 27 Gag-Pol 38 2 MATERIALSANDMETHODS 46 Gag-Pol 46 iv ConstructionofGag-PolPrecursorExpressionSystem 46 PatientSamples 48 Cloningof1.7kbRegionofGag-PolintoExpressionSystem 49 ConstructionofChimericGag-PolPrecursors 52 FrameshiftingProcedure 52 SequencingoftheGag-PolPrecursor 54 ExpressionoftheGag-PolPrecursor 54 AnalysisofPrecursorExpression 55 Protease 56 CloningofProteaseRegionfromGag-Pol 56 Expression 57 Purification 58 KineticAnalysis 59 3 EXPRESSIONANDANALYSISOFORIGINALVARIANTS 60 Rationale 60 Results 62 Discussion 63 4 KINETICANAYSISOFTHEPROTEASEREGIONFROMORIGINAL VARIANTS 72 Rationale 72 Results 73 Discussion 73 5 CHIMERICCONSTRUCTS 77 Rationale 77 Results 78 Discussion 85 6 GAG-POLMUTAGENESIS 89 Rationale 89 Results 90 Discussion 96 7SUMMARYANDFUTUREDIRECTIONS 101 Summary 101 FutureDirections 108 V LISTOFREFERENCES 112 BIOGRAPHICALSKETCH 124 1 vi LISTOFTABLES Table page 1-L ListingofthenumberofpersonsinfectedwithHIV-1asof1996foreight countries 5 1-2. CurrentlistingofmutationsinHFV-lproteaseassociatedwithdrug resistance 31 3-1. AminoacidsequenceoftheregiondefiningtheCcleavagesiteforeachof theeightvariantsusedinthisexperiment 69 4-1. Kineticanalysisofgag-polHXB2andthreevariantsofHIV-1proteaseforthree substrates 74 5-1. Summarylistingofeachofthechimericconstructsanalyzedinthissection, showingthegagandpolregionsusedintheirrespectiveconstruction 84 vii . LISTOFFIGURES Figure page 1-1. Leadingcausesofdeathamongpersons25to44yearsoldintheUnited Statesfrom1982to1992 7 1-2. StructureoftheHIV-1virionwithstructuralandaccessoryproteins identified 9 u.- 1-3. IllustrationofthegenomicmapoftheHIV-1andHIV-2viruses 11 1-4. Illustrationofthemajorstepsinthelife-cycleoftheHTV-lvirus 16 1-5. CrystalstructureofHIV-1proteaseshowingactivesiteresiduesand majorstructuralregions 23 1-6. Structureofthegag-polprecursorshowingtheeightcleavagesitesand theirsequences 40 2-1. Procedureusedtomovethe1500bpfragmentisolatedfrominfectedpatients intothepGAG-POLexpressionplasmid 51 2-2. Diagramofthegenerationofchimericconstructsillustratingtheregion ofgag-polexchangebetweenvariants 53 3-1. Westernblotofthreevariants:Dl.lO,D2.21,andMD.03 64 3-2. Diagramoftheoriginalgag-polprecursorbeinganalyzedinFigure3-1 65 3-3. Aminoacidsequenceoftheproteaseregionforeachoftheeightvariants usedinthisexperiment 67 5-1. WesternblotanalysisofchimerasCl.O,C2.0,andC3.0 79 5-2. Westernblotanalysisofpl10gag-pol processingforchimerasC4.0 andC5.0 81 5-3. Westernblotanalysisofpi10gag-polprecursorprocessingforchimeras C6.0andC7.0 83 viii 6-1. Theaminoacidsequenceofthegagregionencompassedby theSpeIandBglIIrestrictionsitesisshown 92 6-2. AnalysisandcomparisonofvariantMD.03,andanalteredMD.03 variantcontainingamutatedCsite 93 6-3. AnalysisandcomparisonofvariantMD.03,andanalteredMD.03 variantcontainingamutatedCsiteandKtoRmutation 94 6-4. AnalysisandcomparisonoftheoriginalvariantMD.03tothatofa MD.03variantinwhichmutationshavebeenperformedtochangeitsgag regiontothatofthegagregionsseenforvariantsD2.21andD2.22 95 U.J ^ ix ABBREVIATIONS (x)g timesgravitationalconstant amp ampicillin ben N-a-benzoyl bp basepairs d deoxy DMSO dimethysulfoxide E.coli Escherichiacoli EDTA ethylenediaminotetraaceticacid FPLC fastproteinliquidchromatography gag groupspecificantigen IPTG isopropylthiogalactopyranoside kana kanamycin kcat turnovernumber kcat/Km specificityconstant Kj inhibitionconstant Km Michaelis-Mentenconstant LB LuriaBroth MES 2-(4-morpholino)-ethanesulfonicacid mg milligram X