Interferon escape of respiratory syncytial virus [Elektronische Ressource] : functional analysis of nonstructural proteins NS1 and NS2 / by Sabrina Marozin

Sabrina Marozin

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Poids de l'ouvrage: 2 Mo

Extrait

Max-vonPettenkofer-InstituteandGeneCenter,
LudwigMaximiliansUniversity,Munich
Prof. Dr.Karl-KlausConzelmann
Instituteof MolecularAnimalBreedingandBiotechnology, GeneCenter
Facultyof VeterinaryMedicineoftheLudwigMaximilianUniversity,Munich
Prof.Dr.EckhardWolf
InterferonEscapeofRespiratorySyncytialVirus:
FunctionalAnalysis
oftheNonstructuralProteinsNS1andNS2
Thesisfor theattainmentofthetitleDoctorinVeterinaryMedicine
fromtheFacultyof VeterinaryMedicineoftheLudwigMaximilianUniversity,Munich
by
SabrinaMarozin
fromMilan,Italy
Munich, 2005Ausder
AbteilungfürVirologiedesMax-vonPettenkofer-Institutes,
Genzentrum, München
ArbeitsgruppeProf. Dr.Karl-KlausConzelmann
Leiter Prof. Dr. UlrichKoszinowski
Vorgelegt über
dasInstitutfürTierzuchtderTierärztlichenFakultät
der Ludwig-Maximilians-UniversitätMünchen
Lehrstuhl für MolekulareTierzucht undBiotechnologie, Genzentrum
Univ.-Prof.Dr. EckhardWolf
Unterdrückung derInterferon-vermittelten Immunantwort
durchdasRespiratorischeSynzitialVirus:
FunktionelleAnalyse derNicht-Strukturproteine
NS1undNS2
Inaugural-Dissertation
zur ErlangungdertiermedizinischenDoktorwürde
der TierärztlichenFakultät
der Ludwig-Maximilians-UniversitätMünchen
von
SabrinaMarozin
ausMailand, Italien
München2005GedrucktmitGenehmigungderTierärztlichenFakultätder
Ludwig-Maximilians-UniversitätMünchen
Dekan: Univ.-Prof.Dr. E.P.Märtlbauer
Referent: Univ.-Prof.Dr.E.Wolf
Korreferent: Priv.-Doz.Dr.H.-Ch.Siebert
TagderPromotion:10.Februar 2006Part ofthis workis published:
Bossert B., MarozinS., ConzelmannK.K., 2003, Non structural proteins NS1 and NS2 of bovine
respiratory syncytial virus block activation of interferon regulatory factor 3, J. Virol., 77: 8661-
8668
SchlenderJ., HornungV.,FinkeS., Gunthner-BillerM., MarozinS., BrzozkaK., MoghimS.,
Endres S., Hartmann G. and Conzelmann K.K. , 2005, Inhibition of toll-like receptor 7- and 9-
mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory
syncytial virus and measles virus, J. Virol., 79: 5507-5519
Other publications:
Gregory V., Lim W., Cameron K., Bennett M., Marozin S., Klimov A., Hall H., Cox N., Hay
A. and Lin Y.P., 2001, Infection of a child in Hong Kong by an influenza A H3N2 virus closely
related to viruses circulating inEuropean pigs, J.Gen. Virol., 82: 1397-1406
Marozin S., Gregory V., Vallette M., Aymard M., Barigazzi G., Y. P. Lin and Hay A., 2002,
Recent evolution of influenza A H1N1 and H1N2 subtypes in European pigs, J. Gen. Virol., 83:
735-745
MarozinS.,PrankU.andSodeikB., 2004, Herpes simplex type I infection of polarized epithelial
cells requires microtubules and access to receptorspresent at cell-cell contact sites, J. Gen. Virol.,
85: 775- 786If a man will begin with certainties, he shall end in doubts,
but if he will content to begin with doubts, he shall end in certainties.
Francis BaconI
CONTENTS
Page
1 INTRODUCTIONANDOBJECTIVES 1
2REVIEWOFTHELITERATURE 3
2.1Structureandgenome 3
2.1.1Virionstructure 3
2.1.2Genomeorganization 4
2.1.3Viral proteins 5
2.2Replicativecycle 7
2.3EpidemiologyandPathogenesis 10
2.4Immunity 12
2.4.1Humoral immunity 12
2.4.2Cell-mediatedimmunity 13
2.4.3Innateimmunity 14
2.4.4TheInterferon / system 15
2.5ViralantagonistsofIFNtypeIresponse 18
2.6TheRSVnonstructuralproteinsNS1andNS2 19
3MATERIALSANDMETHODS 21
3.1Cellcultureandviruses 21
3.1.1Cells 21
3.1.2RSVpropagationandtiterdetermination 21
3.1.3Rabiesvirus(RV) stocksandtiter determination 22
3.2Generalcloningprocedures 22
3.2.1Restrictionenzymedigest 22
3.2.2ExtractionofDNAfragmentsfromagarosegel 23
3.2.3DNAligation 23
3.2.4Transformationintocompetent bacteria 24
3.2.5Preparationof DNAminiprepsandmidipreps 25
3.3ReverseTranscriptionandPCRconditions 26
3.3.1ExtractionofRNAfromcellsandreversetranscription 26
3.3.2PCRconditions 27
II
3.4GenerationofrecombinantBRSVvirusesexpressing
HRSVNS1andNS2proteins 29
3.4.1ConstructionofrBRSV virusesexpressingNS proteinsofHRSV
strainLong(rBRSVh1/2) 29
3.4.2ConstructionofrBRSVexpressingamutatedHRSVNS2protein
T26I(rBRSVh1/2*)29
3.4.2.1 Quick site-directed mutagenesisof HRSVLong NS2gene 2
T26I3.4.2.2GenerationofrBRSVh1/2* virus 30
3.5Recoveryof recombinantBRSVviruses 31
3.6GenerationofrecombinantSADeGFP-Pvirusesexpressing
HRSVNSproteins 31
3.6.1.GenerationofrecominantSADeGFP-P viruses 31
3.6.2Rescueof recombinant eGFP-P virusesexpressing RSVNS proteins 32
3.7GrowthcharacteristicsofrecombinantBRSVandeGFP-Pviruses 32
3.7.1rBRSVvirusesexpressingHRSVNS proteins 32
3.7.2Recombinant rabieseGFP-P virusesexpressing RSVNS proteins 33
3.8IFNtreatmentofrBRSVviruses 33
3.9CloningofthehumanTANK-bindingkinase1(TBK1)and
TRAF-bindingprotein(TANK) 33
3.10GeneReporterAssay 35
3.10.1ModifiedGeneReporterAssayinVero-p125Luccell line 35
3.10.2Dual luciferaseassayandtransfectionofreporter plasmids 35
3.10.3TranscriptionfactorsactivationandTBK1inhibitionbyRSVinfection 36
3.11Proteinexpressionanalysis 36
3.11.1Extractionofproteinsfromcells 36
3.11.2SDS-polyacrylamidegel electrophoresis(SDS-PAGE) 37
3.11.3Electroblotting 38
3.11.4Westernblotting 39
3.12Immunofluorescence 40
3.13ImmunoprecipitationofIgTANKandIgTBK1 40
3.14Materialsandequipment 41
3.14.1Serologicalreagents 41
3.14.2Chemicals 42
3.14.3Enzymes 43III
3.14.4Kits 43
3.14.5Miscellaneous 44
3.14.6Tissueculturereagents 45
3.14.7Equipment 45
3.14.8Bacteriaand plasmids 46
4RESULTS 49
4.1Nonstructuralproteins(NS)ofBRSVblockactivationof
interferonregulatoryfactor3 49
4.2HRSVprototypes,A2andLong,differintheirability
toinducetypeIIFN 50
4.2.1DetectionofIFN- induction 50
4.2.2Analysisof activationof IFN- transcriptionfactors
AP-1,NF-kBandIRF3 51
4.3SequencecomparisonofRSVNS1andNS2proteins 52
4.3.1Sequenceanalysisof HRSVA2 andHRSVLongnonstructuralproteins 52
4.4 ChimericBRSVvirusesexpressing HRSVNSgenes 54
4.4.1Constructionofrecombinant BRSVvirusharbouringLong-derivedNS1
andamutatedformofNS2 54
4.4.2HRSVNS proteinexpression andcDNArestrictionanalysis 55
4.4.3Growthkineticsof recombinantBRSVviruses 56
4.4.4Sequencecomparison ofHRSVNS genes 58
T26I4.4.5rBRSVh1/2* butnotrBRSVh1/2inhibitsIFN- induction 60
T26I4.4.6Recombinant BRSVh1/2* selectively reducesactivation of
transcriptionalfactorIRF3 62
4.4.7Residue26of NS2proteindoesnot influenceinterferonresistanceof
recombinant BRSV viruses 63
4.5IkappaBkinase-relatedcomplex:TBK1,IKK- andTANK 64
4.5.1CloningofTANK-BindingKinase1(TBK1)andTRAFfamily
member-associated NF-kappaBactivator (TANK) 64
4.5.2TBK1 activatesIFN- production 66
4.5.3HRSVstrainA2inhibitsTBK1activation 68
T26I4.5.4rBRSVh1/2* inhibitsTBK1activationsimilarlytoHRSVA2 69
IV
4.6RecombinantRVexpressingRSVnonstructuralproteins 72
4.6.1Constructionofrecombinant SADeGFP-Pvirusesharbouring
RSVNSproteins 72
5DISCUSSION 75
5.1 Humanrespiratorysyncytialvirus:strainsA2andLong 75
5.1.1SuppressionofIFN- inductiondiffersinHRSVA2andLong 75
5.1.2ActivationofIFN- transcriptionfactors 76
5.1.3Sequenceanalysisof nonstructural NS proteinsinHRSV 77
5.2AmutatedLong-derivedNS2proteinpreventsinductionofIFN- 78
5.2.1rBRSVexpressingHRSVnonstructural protein 78
T26I5.2.2rBRSVh1/2* infectioninhibitsIFN- inductionbyblocking
IRF3activation 79
5.3ThenovelIkB-relatedkinases(IKK):IKK- andTBK1 80
5.3.1CloningTANK-bindingkinase1(TBK1)andTRAFfamilymember–
associated NF-kBactivator (TANK) 80
5.3.2TBK1-dependent IFN- induction 82
5.4InhibitionofTBK1byRSV 82
5.4.1HRSVA2 blocksTBK1-induced IFN- expression 82
5.4.2Residue26of NS2proteinisimportantforTBK1inhibition 83
5.5RecombinantRabiesVirusesexpressingRSVnonstructuralproteins 86
5.5.1GenerationofSADeGFP-P virusesharbouringRSVNS1and
NS2proteins 86
5.5.2Recombinant eGFP-PNS2/NS1virusesdonot counteract IFNproduction 86
5.6Finalconsiderations 87
6SUMMARY 89
7ZUSAMMENFASSUNG 91
8BIBLIOGRAPHY 93
V
Abbreviations
AAmper
APS ammoniumpersulfate
ATP adenosintriphosphate
b bovine
BRSV Bovinespiratorysyncytialvirus
bp base pair
BSA bovine serumalbumin
cDNA complementaryDNA
cm centimeters
CMV cytomegalovirus
CoA coenzymeA
CPE cytopathiceffect
CS newborncalfserum
C-terminus carboxyterminus
DEPC diethylpyrocarbotate
DL Dualluciferaseassay
DMEM Dulbecco’s ModifiedEagleMedium
DNA desoxyribonucleicacid
dNTP desoxyribonucleosidetriphosphates
DTT dithiothreitol
EDTA ethylenediaminetetraaceticacid
e.g examplegiven
Etal. Etalii
FCS fetalcalfserum
FITC fluoresceinisothiocyanate
FFU focus formingunits
ggram
GFP green fluorescent protein
h human
h, hrs hour(s)
HRSV Humanrespiratorysyncytialvirus
IFN interferon
Ig immunoglobulin
IRF Interferonregulatoryfactor
IU internationalunits
kb kilobase
kDa kilodalton
l liter
LB Luria Broth
LPS lipopolysaccharides
Luc luciferase
M molar, marker