Understanding factors controlling gag and gag-pol polyprotein precursor processing in HIV

139 Pages·1998·4.9 MB·English

by  Bloom, Gregory Charles

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