University of Colorado, Boulder
CU Scholar
Undergraduate Honors Theses
Honors Program
Spring 2017
Correlative Light and Electron Microscopy on
Mouse Polyomavirus-Infected Fibroblasts
Christina Henderson
[email protected]
Follow this and additional works at: http://scholar.colorado.edu/honr_theses
Part of the Cell Biology Commons, Developmental Biology Commons, and the Virology
Commons
Recommended Citation
Henderson, Christina, "Correlative Light and Electron Microscopy on Mouse Polyomavirus-Infected Fibroblasts" (2017).
Undergraduate Honors Theses. 1363.
http://scholar.colorado.edu/honr_theses/1363
This Thesis is brought to you for free and open access by Honors Program at CU Scholar. It has been accepted for inclusion in Undergraduate Honors
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April7
2017
CorrelativeLightandElectron
MicroscopyonMousePolyomavirusInfectedFibroblasts
By:ChristinaHenderson
TheGarceaLaboratory
DefenseCommittee:
Dr.RobertGarcea,ThesisAdvisor
Dr.JenniferMartin,HonorsCouncilRepresentative
Dr.MichaeleFerguson,OutofDepartmentMember
1. Abstract
Polyomavirusesaresmall,DNAtumorvirusesthatcaninfectmultiplespecies,including
humans.Inrecentyears,thenumberofidentifiedhumanpolyomaviruses(huPyV)has
grown.ToidentifypossibletherapeutictargetsforhuPyVs,abetterunderstandingofvirus
infectionatthestructurallevelisneeded.Confocalmicroscopyallowsforthevisualization
offluorescently-taggedproteinsbutwithoutseeingthecell’ssubcellularstructures,
functionalinformationcanbelost.Electronmicroscopyprovidesstructuralinsightbut
doesnotalwaysprovidebiologicalorbiochemicaldetails.Combiningthesetwoapproaches
canrelatebiologicalfunctiontostructuralinformationaboutvirusreplication.
Polyomaviruses(PyV)co-opthostproteinsthatfunctionduringnormalDNAdamage
repairtoaidinreplicationofviralgenomes.Herewedescribeamouseembryoniccellline
(MEF)thatstablyexpressesafluorescently-taggedcomponentofthereplicationprotein
complexA(RPA).RPAismadeupofthreeproteins(RPA70/RPA32/RPA14)andisan
importanthostcomponentforPyVvDNAreplication.MEFsstably-expressingGFP-human
RPA32orRPA70(GFP-huRPA32/70)wereinfectedwithmurinepolyomavirus(MuPyV)to
studyviralDNA(vDNA)replicationbycorrelativelightandelectronmicroscopy(CLEM).
LightmicroscopystudiesfoundthatGFP-huRPA32/70localizedtositesofvDNA
replicationalongwithotherhostDNAdamageresponse(DDR)proteins,whichhavebeen
previouslycharacterized.WeattemptedtocorrelatetheimmunofluorescenceofGFPhuRPAproteinsatviralreplicationcenterstotheultrastructureofvirusfactoriesthathave
beenobservedbyelectronmicroscopy(EM).
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2.Introduction
Polyomaviruses(PyVs)infectdifferenttypesofmammalsandbirds(reviewedin
Decaprio&Garcea,2013).Untilrecently,twohumanpolyomaviruses(HuPyVs)havebeen
studied:JCpolyomavirus(JCPyV)andBKpolyomavirus(BKPyV)(Gardner,etal.,1971).
SerologicalstudiesindicatethatBKPyVinfectsupto90%ofthegeneralpopulationandJC
infectsgreaterthan50%ofthegeneralpopulation(Hirschetal.,2003;Rinaldoetal.,
2013).BKPyVandJCPyVinfectiontypicallyoccursduringchildhoodandresultsina
lifelonginfectionthatisasymptomaticforhealthyindividuals(Maginnisetal.,2014;
Rinaldoetal.,2013).Severepathologicalmanifestationsappearinpatientswhoare
immunocompromised(Hirschetal.,2003).ThetumorigeniceffectsofPyVinfectionhave
beenstudiedforovertwentyyearsinmurinepolyomavirus(MuPyV)andhaveprovided
crucialinsightregardingthecellularandmolecularbiologyofPyVs(Chengetal.,2009;Guy
etal.,1992;Decaprio&Garcea,2013).
PyVsarenon-enveloped,dsDNAtumorvirusesthatreplicateinthehostcell’s
nucleus.The5-kbcircular,DNAgenomecanbedividedintotworegions,earlyandlate,
whichencodeuptosixgenes,dependingonthestrain.TheearlyregionencodestheT
antigens,whichareresponsibleforprimingthecellforvDNAreplication(Myersetal.,
1981).MuPyVencodesthreeTantigenproteins,small,middle,andlargeTantigen(ST,MT,
LT),whicharisefromsplicingvariationsduringtranscription.Thelateregionencodesthe
viralcapsidproteins(VP1,VP2,andVP3)(Yanetal.,1996;Klein,1980).VP1isthemajor
viralcapsidprotein,whereasVP2andVP3arethesmaller,minorcapsidproteins(Yanet
al.,1996;Klein,1980).MuPyVisa45nmicosahedralviralcapsid(Yanetal.,1996;Klein,
3
1980).Theviralcapsidiscomposedof72capsomeres,formedfromonemoleculeofVP2or
VP3associatedtoaVP1pentamer(Fig1)(Barouch&Harrison,1994).
A
B
Adaptedfrom(Chenetal.,1998)
Figure1.(A)Cryo-EMoftheMuPyVcapsidcomposedof72capsomeres(VP1pentamersassociated
toamoleculeofVP2orVP3).(B)AstructuralmodelofanSV40VP1-VP2complex
PyVsinfectcellsbybindingsialicacidsresiduesonthehost’scellsurfaceviaVP1
(Tsaietal.,2003).PyVisinternalizedandtraffickedtotheendoplasmicreticulumina
microtubule-dependentmanner(Norkinetal.,2002;Gilbertetal.,2003).Inorderforthe
virustotraffictothecytosolandeventuallythenucleus,itco-optsendoplasmicreticulumassociateddegradation(ERAD)translocationcomplexproteins(Goodwinetal.,2011;
Bennettetal.,2013).Thevirusgainsentryintothenucleususinganuclearlocalization
signalonaviralcapsidprotein(Neuetal.,2009;Nakanishietal.,2007).Onceinthe
nucleus,theearlygenesareexpressed,andLTdrivesthecellintoSphase.Studiesusing
simianvirus40(SV40)havedemonstratedLT’sabilitytosequesterthetumorsuppressor,
p53,andcompromisecheckpointcontrolofG1toSphase(Damaniaetal.,2009).In
addition,LTbindstheretinoblastomatumorsuppressor(Rb)proteinfamily,leadingtothe
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activationofdownstreamgenesessentialforentryintoSphase(Damaniaetal.,2009).
OncethecellhasenteredintoSphase,MuPyVusesthecell’smachinerytopromoteviral
DNA(vDNA)replication.AfterLTunwindsdouble-strandedDNA(dsDNA)usingits
helicasedomain,itrecruitsthehost’spolymerase/primasecomplextotheviraloriginof
replicationandinitiatessynthesis(Damaniaetal.,2009).FollowingDNAreplication,the
viralcapsidproteinsareexpressed(Kawanoetal.,2006).Theviralcapsidproteinsform
capsomeresinthecytosol,aretransportedintothenucleus,andbyunknownmechanisms,
packagevDNA(Kawanoetal.,2006).Thevirusspreadsbylysingthehostcell,thereby
releasingthevirions(Damaniaetal.,2009).
Inadditiontousingthehost’sreplicationmachinery,LTusesthehost’sDNA
damageresponseproteins(DDR)topromotevDNAreplication.Oneoftheearliest
complexesrecruitedtositesofDNAdamageistheMRNcomplex(D’amoursetal.,2002).
TheMRNcomplexisatrimericcomplexmadeupofthreeproteins,Mre11,Rad50,and
Nbs1(D’amoursetal.,2002).TheMRNcomplexbindsdouble-strandedDNA(dsDNA)
breaksandrecruitsataxia-telangiectasiamutated(ATM)kinasetositesofDNAdamage
(Zhaoetal.,2008).TheMRNcomplexisphosphorylatedbyATMthenactivatesATMto
phosphorylatedownstreamsubstrateproteins(Zhaoetal.,2008).PhosphorylatedATM
(pATM)kinaseinitiatesasignalingcascadethattriggersp53-mediatedG1toSphase
checkpointarresttopromoteDNArepair(Kastanetal.,2004;Shiloh2003).DuringSV40
infection,theMRNcomplexmayactinviralreplicationcenterstomaintainATMactivation
(Zhaoetal.,2008).InhibitionofATMduringSV40replicationresultsinalossofviral
replicationcenters,indicatingthattheactivationofATM,maintainedbyMRN,maybe
necessaryfortheinfectedcelltoremaininSphase(Zhaoetal.,2008).
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TheDDRprotein,γH2AX,isimportantformaintainingrepairenzymesaroundDNA
breaksiteswhilepreventingprogressionofthecellcycle(Bassingetal.,2002;Celesteetal.,
2003;Matsuokaetal.,2007).Uponinfection,H2AXisphosphorylatedatserine139by
pATM(Kastanetal.,2004).AlthoughγH2AXisnotessentialforMuPyVgenomereplication
andvirusassembly,itplaysanimportantroleinrecruitingDDRproteinsandpromoting
theiractivation(Heiseretal.,2016).
ADDRproteinrequiredforthereplicationofchromosomalDNAandSV40
replicationisreplicationproteinA(RPA)(Kennyetal.,1990).RPAisaheterotrimeric
single-strandedDNA(ssDNA)bindingproteinessentialforDNAreplicationandrepair
pathwayssuchashomologousrecombination(Fanning,2006).TheRPAheterotrimeris
comprisedofa70-kDsubunit(RPA70),a32-kDsubunit(RPA32),anda14-kDsubunit
(RPA14)(Hanetal.,1999).ThecomplexhasfourssDNAbindingsites,onewithinRPA32
andthreewithinRPA70(Fanning,2006).RPA32isphosphorylatedatserine23(RPA32pSer23)inacell-cycledependentmannerandinresponsetoDNAdamage(Fotedar&
Roberts,1992).AlthoughitisunclearifRPA32-pSer23isrequiredforMuPyVreplication,
activationofRPA32uponDNAdamageallowsfortherecruitmentofadditionalDDR
proteins(Hendricksenetal.,1996;Justiceetal.,2015).IthasbeenshownthatLT’sorigin
bindingdomain(OBD)associateswithRPA32andRPA70andweakensRPA’sbindingto
ssDNA(Fig2)(Fanning,2006).RPA’sloweredaffinityforssDNAresultsinastrandofDNA
beingreleased;LTusesthecell’smachinerytoreplicatetheviralgenome(Fanning,2006).
6
Adaptedfrom(Arunkumaretal.,2005;Fanningetal.,2006)
Figure2.SV40’sLTassociateswithRPA32andRPA70subunitsandutilizesthehost’smachineryto
replicateitsgenome:greenshapesdepictLT’shelicaseandorigin-bindingdomains(OBD);orangeshapes
depictthehost’spolymerase-primasebindingtotheDNA.
Inaddition,MuPyVutilizesataxia-telangiectasiaandRad-3related(ATR)kinase,a
DDRprotein,toreplicateitsgenome(Cicciaetal.,2010).ATRisrecruitedbyRPAupon
DNAdamageandcantargetthecheckpointprotein,Chk1(Cicciaetal.,2010).Inturn,Chk1
phosphorylateschromatin-boundproteinstopromotestabilityofthereplicationfork
(Cicciaetal.,2010).Duringinfection,activatedChk1(pChk1),mayplayaroleinSphase
cellcyclearrest(Justiceetal.,2015;Liuetal.,2000).pChk1’sroleinMuPyVisstillbe
investigatedasrecentdatahasshownthatChk1isnotrequiredforMuPyVreplicationor
assembly(Heiseretal.,2016).
IthasbeenshownpreviouslythatMuPyVactivatesandreorganizeshostDDR
proteinsinthenucleus.ActivatedRPA32localizestovDNAreplicationcentersalongwith
DDRsignalingproteinspATM,pChk1,andγH2AX(Heiseretal.,2016).Byelectron
microscopy,weobservedtubularstructuresadjacenttoprogenyvirionsininfectedmurine
embryonicfibroblasts(MEFs).Thetubularstructuresarecomposedofthecapsidprotein,
VP1,andarethoughttobeanassemblyintermediateduringencapsidationoftheviral
genome.Thesetubularstructures,togetherwithprogenyvirions,arecalled“virus
factories”(Ericksonetal.,2012).ItisunknownhowvDNAreplicationcentersandDDR
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proteinlocalizationobservedbyimmunofluorescenceanalysis(IFA)correlatetovirus
factoriesobservedbyEM.
Correlativelightandelectronmicroscopy(CLEM)isanimagingtechniquethat
complementsthecapabilitiesoftheelectronmicroscope(EM)andthelightmicroscope
(LM)(Perkovicetal.,2014).CLEMcombinestheabilityofLMtoviewfluorescentlytagged
proteinswithEM’sabilitytoprovidehigh-resolutionimagesofthemorphologyand
ultrastructureofthelocationwheretheproteinresides(Perkovicetal.,2014;Hodgsonet
al.,2014).CLEMhasthreeessentialphasesafterLMimaging:findasuitableprobe,process
thesample,andanalyzethecellusingEM(Hodgsonetal.,2014).Wehavetakenadvantage
ofRPA’slocalizationtoviralreplicationcentersinMEFs.Weanalyzedtherecruitmentof
GFP-humanRPA32/70andDDRproteinstonuclearviralreplicationcentersinMEFs.
CLEMcanprovideinsightaboutMuPyVreplicationcenterswhenimmunofluorescence
analysis(IFA)ofstablecelllinesexpressingGFP-huRPA32/70arecorrelatedtoEMstudies
ofvirusfactories.
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2.MaterialsandMethods
CloningGFP-TaggedRPA32/RPA70IntoaPiggyBacTMPlasmid
IndividuallyGFP-taggedRPAhumangeneswereobtainedasplasmidsfromMarc
WoldandclonedintothePiggyBacplasmid(pLP28)containinganampicillinresistance
gene(giftofAmyPalmer).Theplasmids(pLP28,GFP-huRPA32,andGFP-huRPA70)were
digestedwithNheIandBamHI.Thedigestswereresolvedbyelectrophoresisandthe
appropriatebandswereexcisedandpurifiedusingtheQIAquickgelextractionkit.
Thevector(pLP28)andinserts(GFP-huRPA32orGFP-huRPA70)wereligated.Each
ligationwastransformedintoDH5αE.coliandplatedonLuriaBroth(LB)agarplates
containingampicillin(Amp).Positivecloneswerescreenedbyrestrictiondigestionwith
NheIandBamHI.Positiveclones(pLP28-huRPA32.1,pLP28-huRPA32.2,pLP28-huRPA70.9,
andpLP28-huRPA70.12)werecarriedforwardtogeneratestablecelllines.
GenerationofstablecelllinesexpressingGFP-taggedRPAproteins
C57MEFsweremaintainedinDMEM,supplementedwith10%fetalbovineserum
(FBS),penicillin/streptomycin/antimycotic(SigmaA5955)and55μMofβmercaptoethanol(βME)inahumidenvironmentat37°Cand5%CO2.Fortransfection,
MEFswereseededonasix-welldishandallowedtoadhereovernight.Thefollowingday,
eachwellwastransfectedwithpLP10(PiggyBacTransposaseplasmid)plusoneofthe
followingplasmids:pUC18,pLP28,pLP28-huRPA32.1,pLP28-huRPA32.2,pLP28huRPA70.9,orpLP28-huRPA70.12usingLipofectamine2000inordertogeneratestable
celllines.(Theexpressionplasmidscontainingahygromycinresistancegenewerediluted
to100ng/uLandthepLP10PiggyBacTMTransposaseplasmidwasdilutedto40ng/uL).
9
ThepUC18plasmidwasincludedinthenegativecontrolwellinordertobringthetotal
amountoftransfectedDNAequaltotheotherwells.TheDNA/Lipofectaminemixwas
addedtogrowthmedia,andthecellswereincubatedat37°Cforthreedaysbeforethe
mediawasreplacedwithfreshgrowthmediacontaining50ug/mLofhygromycin.Thecells
wereincubatedandmonitoreddailyforcelldeathandsubsequentgrowthofhygromycinresistantcellsasstatedabove.
InducingDNAdamageinstablecelllinesexpressingGFP-taggedRPAproteins
Stably-transfectedC57cellsthatexpressedeitherGFP-RPA70orGFP-RPA32were
grownonimagingdishes(MatTek)ThegrowthmediawasreplacedwithFluorobrite
DMEMcontainingHoechstdyeandimagedonthelaserscanninglightmicroscopeusingthe
environmentalchamberat37°C,70%humidity,and5%CO2.Weoutlinedaregion-ofinterest(ROI)withtheimagingsoftware(Nikon)andinducedDNAdamageusingthe405
laserat50%for1minwithintheROI.Thecellswereimagedat30-secondintervalsfora
maximumof40min.
VirusInfection
Cellswereplatedandgrownovernightasdescribedabove.Thenextday,thegrowth
mediawasreplacedwithstarvemedia(DMEM/0.5%FBS/antibiotics/βME),and
incubationwascontinuedforanother16-18hrs.MuPyV(NG59RA)waspreparedfor
infectionbysonication,heat,andcentrifugation.Briefly,theviruswassonicatedfor1
minuteat75%amplitudeandplacedat45°Cfor20min.Thecelldebriswaspelletedby
centrifugationat13,000rpmfor3minutes.Thevirussupernatantwasdiluted1:25with
adsorptionbuffer(Hank’sbuffer/FBS/10mMHepes,pH5.6).Themediawasaspirated
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fromthecellsandthedilutedviruswasaddedtoeachwellandincubatedat37°C.After1hourincubation,viruswasremoved,andnormalgrowthmediawasaddedtothewells.The
cellswereincubatedat37°Cforthetimeindicatedforeachexperiment.
ImmunofluorescenceAnalysis
Cellswereplatedonacid-etchedcoverslipsandinfectedasdescribedabove.
Coverslipswerewashedtwicewithcoldphosphatebufferedsaline(PBS)andincubatedon
icefor5minutesinCSKbuffer(10mMPIPES,pH6.8/100mMNaCl/300mMsucrose/3
mMMgCl2/1mMEGTA/0.5%TritonX-100)withproteaseinhibitor(Roche,Complete
Mini).CoverslipswerewashedtwicewithcoldPBSandfixedin4%paraformaldehyde
(PFA)for15minatroomtemperature(RT).Followinganovernightblockin2%bovinecalf
serum(BCS)inPBS(blockingbuffer)at4°C,coverslipswereincubatedwithprimary
antibodyat37°Cfor1hrandwashedthreetimeswithblockingbuffer.Thecoverslipswere
incubatedwithsecondaryantibodyunderthesameconditionsandwashedthreetimes
withblockingbufferandthenmountedusingDAPIProlongAnti-FadeGold(Invitrogen)
andallowedtocurefor24hrsatRT.CellswereimagedontheNikonA1Rlaser-scanning
lightmicroscopeusing100Xobjective.Z-stacksthroughthenucleuswerecollected;images
shownrepresentasinglez-planethroughthecenterofthenucleus.ImageJsoftwarewas
usedtoanalyzetheimages.
ImmunofluorescenceAntibodies
Primaryantibodiesusedforimmunostainingwereanti-LT(E1,rat),1:2000;anti-
RPA32(3A2,rat;giftofH.Nasheuer),1:5;anti-RPA32-pSer23(8H3,rat;giftofH.
Nahseuer),1:20;anti-pATM(Millipore,mouse),1:1000;andanti-γH2AX(AbCam,rabbit),
11
1:1000.ForRPA32co-stainingwithanti-LTantibody,theanti-RPA32antibodywasdirectly
conjugatedwithAlexaFluor546(Invitrogen)andusedata1:5dilutionaftertheLT
primaryandsecondaryantibodyincubations.Thesecondaryantibodiesusedwere
conjugatedwithAlexaFluor647(Invitrogen)andusedata1:2000dilution.Allprimaryand
secondaryantibodiesweredilutedin2%blockingbuffer.
FluorescentInSituHybridization(FISH)ofMuPyVDNA
TheMuPyVgenome(NG59RA)wasclonedintopUC18andaFISHprobewas
generatedbynicktranslationofpUC18-MuPyVplasmidusingPromofluor-550NTLabeling
Kit.Cellsweregrownoncoverslipsandinfected,fixed,andimmunostainedforviral
and/orhostproteinsasdescribedaboveexceptaftersecondaryantibodystaining,thecells
werewashedthreetimeswithPBS,fixedwith4%PFAinPBS,andwashedthreetimeswith
2%BCSinPBSfollowedbytwowashedin2XSSC.Thecellsweretreatedwith0.2mg/ml
RNaseTypeIII(Sigma)in2XSSCat37°Cfor15minandwashedthreetimesin2xSSC.The
FISHprobewasdilutedincDenHyb(InSitus)andhybridizedwithsamplesfor3minutesat
90°C,followedby2minutesateachtemperature80°C,70°C,60°C,50°C,45°C,andfinally
overnightat37°Cinahumidenvironment.Coverslipswerewashedfor5minuteseachat
45°Cwith1.5xSSC,1.5xSSC/50%formamide,and1.5xSSCandafinalwashinPBS.Cells
weremountedontoglassslidesusingDAPIProlongAnti-FadeGold(Invitrogen)and
allowedtocurefor24hrsatRT.
High-PressureFreezing,FreezeSubstitution,andEmbedding
StablecelllinesexpressingGFP-RPA70orGFP-RPA32wereculturedonT75flasks
andinfectedwithMuPyV(strainNG59RA).Thecellsweretrypsinizedandcollectedby
12
centrifugation.Thecellpelletwasresuspendedinasmallvolumecryoprotectant(either
20%dextran/5mMsucroseor150mMMannitol)tomakeacellslurry.Theslurrywas
placedintoaluminumplanchettes(0.1µmdepth)andfrozenbyhighpressureona
WohlwendCompact02high-pressurefreezer.Afterfreezing,thehatscontainingthe
sampleswerereleasedimmediatelyintoaliquidnitrogenbathandplacedincryotubes
containingfreeze-substitutionmedia.
Threefreeze-substitution(FS)mediasinacetoneweretested:0.1%uranylacetate
(UA),0.025%UA,oracetonealone.Cryotubescontainingfrozensamplesweretransferred
toafreeze-substitution/low-temperatureembeddingapparatus(AFS,LeicaMicrosystems)
toallowforuser-controlledtemperaturechanges.Thesampleswerewarmedto-85°Cfor
12hrs.Thetemperaturewasthenraisedto-35°C.Thesampleswerewashedwithcold
acetone,andinfiltratedwithincreasingconcentrationsofHM20.Afterthreechangesin
100%HM20,samplesweredistributedbetweenfourBEEMcapsulesandtheresinwas
polymerizedat-35°Cfor24hrsunderUVlightandsubsequentlywarmedtoRT.
Sectioning,Staining,andImagingofEmbeddedSamples
70nmsectionswerecollectedusingan(Leica)ultramicrotomeequippedwitha
diamondknifeandmountedonnickelandcoppergrids.Thesectionswereimagedusing
theNikonA1Rlaser-scanninglightmicroscopeusing100xobjectivelensanda488laser
line,toidentifyGFP-positivecells.Thesectionswerethenpost-stainedwithmethanolic1%
UAfollowedbyleadcitrateandimagedontheFEITwinSpiritT12electronmicroscope
operativeat100kV.
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3.Results
GenerationofMEFsexpressingGFP-taggedhumanRPA32orRPA70
ThehostRPAcomplexisassDNAbindingproteinthatisrecruitedtositesofvDNA
replication.WeobtainedplasmidscontainingGFP-taggedhumanRPA70orRPA32from
MarcWold.WeclonedtheGFP-taggedgenesintothePiggyBacTransposasesystemin
ordertogeneratemousefibroblastsstablyexpressinghumanGFP-RPA32orGFP-RPA70.
WedigestedthepLP28PiggyBacplasmidandtheGFP-huRPA32/70plasmidswithNheI
andBamHI.RestrictiondigestsweregelpurifiedusingtheQIAquickgelextractionkitand
theGFP-huRPAgeneswereligatedintopLP28usingT4DNAligase(Fig3A).Theligations
weretransformedintoDH5αE.coliandplatedontoLuria-Brothagaroseplatescontaining
ampicillin.Colonieswerescreenedwithamini-plasmidprepanddigestedwithNheIand
BamHI.Positiveclones(pLP28-huRPA32/70)werecarriedforwardtogeneratestablecell
lines(Fig3B).
Figure3.(A)DiagramofthedigestionandligationofpLP28andGFP-huRPA:blackbarsindicatesites
ofdigestionwithNheIorBamHI;grayarrowsindicatesitesofinsertionoftheplasmidencodingGFPhuRPA into the pLP28 plasmid. (B) Diagram of the resulting pLP28-huRPA plasmid after digestion
andligation.
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StablecelllinesweregeneratedbytransfectingMEFswitheitherpLP28-GFPhuRPA32orpLP28-GFP-huRPA70togetherwithpLP10,theplasmidcontainingthe
transposase.CellsthathadstableintegrationoftheGFP-huRPAplasmidswereselectedin
growthmediacontaininghygromycinandscreenedforexpressionofGFP-RPA32/70.
InordertodeterminewhetherthestablecelllinesexpressedGFP-huRPA32/70,we
lookedatthecellsbylightmicroscopyforGFPexpression.SinceRPAisrecruitedtositesof
DNAdamageanditwastaggedwithGFP,weexpectedtoseeGFPexpressionwhenDNA
damagewasinduced.Therefore,weinducedDNAdamagebyirradiatingspecificsites
withinthecellsandcharacterizingsitesofGFP-huRPA32/70localization,denotedbyan
increasedintensityofGFP.WefoundthatstablecelllinesexpressingGFP-huRPA32(Fig4)
andGFP-huRPA70(datanotshown)displayedanincreasedintensityofGFPatthesiteof
DNAdamage,butnotoutsideoftheROI,by6minutesafterirradiation.Ourresults
demonstratethatGFP-huRPA32andGFF-huRPA70arerecruitedtositesofinducedDNA
damageinMEFs.
Figure4.GFP-huRPA32andGFP-huRPA70arerecruitedtositesofDNAdamageinstablecelllines.
MEFs stably-expressing GFP-huRPA32were grown on imaging dishes and the nuclei were stained with
Hoechst dye. Cells were maintained in an environmental chamber at 37°C, 70% humidity and 5% CO2
during imaging. Region of interests (ROI) were selected using the Nikon software and are denoted by
white outlines.DNA damage was induced withinthe ROI using the 405laserat 50% for1 minute.Live
cellswereimagedbeforeandafterirradiationat1-minuteintervalsontheNikonA1Rlaser-scanninglight
microscope. Image above shows GFP-huRPA32 before irradiation (t=0 min) and after irradiation (t=6
min).
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RPA32wasfoundpreviouslytoco-localizetositesofvDNAreplicationcentersand
LTsitesduringMuPyVinfectionofMEFs(Heiseretal.,2016).Wedeterminedwhether
stablecelllinesexpressingGFP-huRPA70andGFP-huRPA32couldbeinfectedwithMuPyV
andifso,wouldGFP-huRPA32/70localizetositesofvDNAreplicationandLT.Wealso
determinedwhetherstablecelllinesexpressingGFP-huRPA70localizedtothesamesites
asendogenousmouseRPA32(mu-RPA32).WeinfectedthestablecelllinewithMuPyVand
harvestedthemat28hrspostinfection.Thecellswerethenfixed,permeabilizedand
stainedforvDNA,LT,andmu-RPA32.WegeneratedafishprobebycloningtheMuPyV
genomeintoapUC18plasmid,followedbynicktranslation(Erickson,2012).TheFISH
probewashybridizedtothesamplesandthecellswereimagedontheNikonA1Rlaserscanninglightmicroscope.
WefoundthatGFP-huRPA70localizedtoviralreplicationcenters(Fig5A)aswellas
sitesofLT(Fig5Aand5B)andmu-RPA32(Fig5B).WealsoobservedGFP-huRPA32’s
localizationtovDNAreplicationcenters,indicatingthatthesecelllinesbehavedsimilarlyto
theGFP-huRPA70celllines(datanotshown).TheseresultssuggestthatGFP-huRPA70is
beingrecruitedtothesamesitesasendogenousmu-RPA32andmaybefacilitatingvDNA
replicationbyassociatingwithLT.
16
2
Figure5.GFP-huRPA32andGFP-huRPA70arerecruitedtositesofvDNAreplication.GFP-huRPA70
isalsorecruitedtositesofmu-RPA32andLTwithinthenucleus.StablecelllinesexpressingGFP-huRPA70
andGFP-huRPA32wereinfected withMuPyV (NG59RA), fixed, permeabilized, and immunostained at 28
hours post infection. Images were taken on the A1R light microscope; cells were stained for (A) MuPyV
DNA(FISH)andLT(B)Mu-RPA32andLT
MuPyVrecruitsandactivateshostDDRproteinstositesofvDNAreplication(Heiser
etal.,2016).WethereforestudiedtherecruitmentandactivationofhostDDRproteinsto
viralreplicationcentersinstablecelllinesexpressingGFP-huRPA32/70.Weinfectedthe
stablecelllineswithMuPyVandharvestedthemat28hrspostinfection.Thecellswere
thenfixed,permeabilizedandimmunostainedforpATM,RPA32-pSer23,andγH2AX.FISH
wasperformedonthecellstoanalyzetheco-localizationofGFP-huRPA32/70andhost
DDRproteinswithsitesofvDNAreplication.LMrevealedthatpATMandRPA32-pSer23
wererecruitedtositesofGFP-huRPA70duringinfection(Fig6Aand6B).Additionally,the
hostDDRsignalingprotein,γH2AX,localizedtositesofGFP-huRPA32duringinfection(Fig
6C).ThelocalizationofRPA32-pSer23tositesofGFP-huRPA70indicatesthatactivated
RPA32wasbeingrecruitedtothesesites.ThelocalizationofhostDDRproteinstositesof
GFP-huRPA32andGFP-huRPA70indicatethatthehumanRPAproteinmaybefacilitating
17
therecruitmentofactivatedendogenousRPA32(RPA32-pSer23)aswellaspATMandγ
H2AX.
Figure 6. DDR proteins are recruited to sites of vDNA replication in stable cell lines expressing
GFP-huRPA32 and GFP-huRPA70. Stable cell lines expressing GFP-huRPA32 or GFP-huRPA70 were
infected with MuPyV (NG59RA), fixed, permeabilized, and immunostained at 28 hours post infected.
ImagesweretakenontheA1Rlight microscope;cellswere stainedfor(A)pATMinGFP-huRPA70cells
(B)RPA32-pSer23inGFP-huRPA70cells(C)γH2AXinGFP-huRPA32cells.
CLEMcanbeusedtovisualizethesubcellularlocalizationoffluorescently-tagged
proteins.WeanalyzedthelocalizationofGFP-huRPA32/70tositesofvDNAreplicationand
DDRproteinsviaLMandattemptedtostudyGFP-huRPA32/70’slocalizationtosubcellular
structuresusingthehigh-resolutionpowerofEM.TodetermineifGFPisretainedandvirus
18
factoryformationispresentinstablecelllinesexpressingGFP-huRPA32/70afterCLEM
processingweinfectedthestablecelllineswithMuPyVandperformedhigh-pressure
freezing.Thesampleswereplacedinafreezesubstitutionmediaandinfiltratedwithresin
thatwaspolymerizedunderUVlight.Sectionsweregeneratedontheultramicrotomeand
imagedontheLMtoanalyzetheretentionoffluorescence.Followingthis,thesections
werepost-stainedandimagedontheEMtoanalyzevirusfactoryformation.LMofthin
sectionsrevealedthatGFPfluorescencewasnotretainedafterprocessing.EMofthin
sectionsrevealedvirionsinthenucleusofthesecellsbutsincewecouldnotcorrelate
fluorescencewithelectronmicrographs,wewereunabletodetermineifthevirionswere
formedinacellexpressingGFP-huRPA.
19
4.Discussion
IthasbeenshownpreviouslybyLMthatendogenousRPA32localizestositesof
vDNAreplicationandreorganizeshostDDRproteinsinthenucleus(Heiseretal.,2016).In
addition,ithasbeenshownbyEMthatMEFsinfectedwithMuPyVdemonstratevirus
factoryformationinthenucleus(Ericksonetal.,2012).InordertoutilizeGFPhuRPA32/70forCLEM,weanalyzeditslocalizationwhenexpressedinMEFs.First,we
analyzedGFP-huRPA32/70’srecruitmenttositesofinducedDNAdamagewithintheROI
andfoundthatthehumanRPAswereappropriatelyrecruited.Followingthis,weanalyzed
GFP-huRPA32/70’slocalizationtositesofvDNAreplicationbyinfectingthestablecelllines
withMuPyV.WefoundthatGFP-huRPA32/70wasrecruitedtositesofvDNAreplicationas
wellasendogenousRPA32andLT.Ourresultsindicatethatstablecelllinesexpressing
GFP-huRPA32/70canbeinfectedwithMuPyVandLTmayutilizeGFP-huRPA32/70to
facilitatevDNAreplication.MuPyVinteractswithotherhostDDRproteins,suchaspATM,
γH2AX,andRPA32-pSer23,todrivethecellintoSphaseandpromoteviralreplication.In
ordertostudytherecruitmentofadditionalhostDDRproteinsinstablecelllines
expressingGFP-huRPA32/70westainedthestablecelllinesforhostDDRproteinsand
vDNAandimagedthemontheLM.OurresultsindicatethathostDDRproteins(pATM,
γH2AX,andRPA32-pSer23)wereappropriatelyrecruitedtositesofGFP-huRPA32/70
duringinfection.AlthoughitishasbeenshownthatGFP-huRPAproteinsarelocalizingto
theappropriatesites,furtherstudiesarenecessarytodetermineiftheGFP-huRPAproteins
areactivated.ImmunostainingforactivatedGFP-huRPA32withaspecificantibodyagainst
humanRPA32-pSer23mayprovidethisinsight.Furthermore,toinvestigatewhether
humanRPAproteinsaresufficientinfacilitatingvDNAreplicationindependentof
20
endogenousRPAs,smallinterferingRNA(siRNAs)maybeusedtosuppressthegene
expressionofhostRPAs.Followingendogenousgenesuppression,thestablecelllines
expressingGFP-huRPA32/70canbestudiedforMuPyVinfection,localizationtoviral
replicationcenters,andrecruitmentofhostDDRproteins.Thesecelllinescanalsobe
studiedbyEMtorevealthecharacteristicsofvirusfactoryformation.
AfterstablecelllinesexpressingGFP-huRPA32/70werehighpressurefrozenand
infiltratedwithresin,LMimagesdisplayedalackoffluorescence.Inaddition,EMimages
displayingvirionswereinconclusiveduetotheinabilitytocorrelatevirionsto
fluorescently-taggedRPAs.Ithasbeenreportedpreviouslythatfluorescenceisretained
afterprocessingusingaquickhigh-pressurefreezingprotocol(Peddieetal.,2014).
Therefore,decreasingtheamountoftimeoursamplesunderwenthigh-pressurefreezing
maypreserveGFPfluorescence.IthasalsobeenshownthatCLEMissuccessfuliflivecell
imagingontheLMisperformedpriortoprocessingthecellsforEM(Rijnsoeveretal.,
2008).Therefore,livecellimagingthestablecelllinesexpressingGFP-huRPAproteinson
griddedcoverslipsmayprovidepointsoforientationthatcanbeco-localizedtosubcellular
structuresseenbyEM.OurresultsdemonstratethatstablecelllinesexpressingGFPhuRPA32/70aresuitableforoptimizingCLEMtechniques.Furtherstudiestoimprove
processingandtechniquesforCLEMwillneedtobeexplored.
21
Acknowledgements
IwouldliketothankMarcWoldfortheplasmidsencodingGFP-taggedRPA32and
RPA70proteinsandAmyPalmerforsharingthePiggyBacTMplasmids.Iwouldliketothank
theJoeDragavonandtheBioFrontiersAdvanceLightMicroscopyCoreFacilityfor
microscopytraininganduse;CourtneyOzzello,GarryMorganandtheCU-BoulderElectron
MicroscopyFacilityforsamplepreparationandmicroscopeuse.Lastly,Iwouldliketo
thankKimberlyEricksonfortheoverwhelmingsupportandtimecommitmenttothis
project,Dr.RobertGarceaandtheGarcealabfortheirsupportandcontributiontothis
thesis.
22
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