Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous

16 Pages·2016·2.57 MB·English

viruses
Article
Human Cytomegalovirus Nuclear Egress Proteins
Ectopically Expressed in the Heterologous
Environment of Plant Cells are Strictly Targeted to the
Nuclear Envelope
ChristianE.Lamm1,KatrinLink1,SabrinaWagner2,JensMilbradt2,ManfredMarschall2and
UweSonnewald1,*
1 DivisionofBiochemistry,DepartmentofBiology,Friedrich-AlexanderUniversityErlangen-Nuremberg,
Staudtstrasse5,Erlangen91058,Germany;[email protected](C.L.);[email protected](K.L.)
2 InstituteforClinicalandMolecularVirology,Friedrich-AlexanderUniversityErlangen-Nuremberg,
Schloßgarten4,Erlangen91054,Germany;[email protected](S.W.);
[email protected](J.M.);[email protected](M.M.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:ThomasHohn
Received:22December2015;Accepted:29February2016;Published:10March2016
Abstract: Inalleukaryoticcells,thenucleusformsaprominentcellularcompartmentcontainingthe
cell’snucleargenome. Althoughstructurallysimilar,animalandplantnucleidiffersubstantially
indetailsoftheirarchitecture. Oneexampleisthenuclearlamina,alayeroftightlyinterconnected
filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous
lamingenescouldbedetectedinplantgenomesandputativelamin-likeproteinsareonlypoorly
described in plants. To probe for potentially conserved features of metazoan and plant nuclear
envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus
pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and
recruitsthenuclearlocalizedpUL53toit,formingheterodimers. Uponexpressioninplantcells,a
verysimilarlocalizationpatternofbothproteinscouldbedetermined. Notably,pUL50isspecifically
targetedtotheplantnuclearenvelopeinarim-likefashion,alocationtowhichcoexpressedpUL53
becomesstrictlycorecruitedfromitsinitialnucleoplasmicdistribution. UsingpUL50asbaitina
yeasttwo-hybridscreening,thecytoplasmicre-initiationsupportingproteinRISPcouldbeidentified.
InteractionofpUL50andRISPcouldbeconfirmedbycoexpressionandcoimmunoprecipitationin
mammaliancellsandbyconfocallaserscanningmicroscopyinplantcells,demonstratingpartial
pUL50-RISPcolocalizationinareasofthenuclearrimandotherintracellularcompartments. Thus,
ourstudyprovidesstrongevidenceforconservedstructuralfeaturesofplantandmetazoannuclear
envelopsandidentifiesRISPasapotentialpUL50-interactingplantprotein.
Keywords: human cytomegalovirus; pUL50; pUL53; nuclear envelope; plant cells; re-initiation
supportingproteinRISP
1. Introduction
Althoughalreadyobservedinthebeginningofthe18thcenturybytheDutchmicroscopistAntonie
vanLeeuwenhoek,thenatureoftheeukaryoticnucleusstillraisesnumerousquestionsespeciallyin
plantsandissubjecttoextensivestudies. Superficially,animalandplantnucleiaresimilarbutthey
differintheircompositionaldetails.Thiscanbeseenalreadyintheconstitutionofthenuclearenvelope
(NE):Inbothkingdoms,thestructureconsistsofadoublemembranelayerthatdelimitsthecytoplasm
andthenucleoplasm. Theoutermostofbothnuclearmembranesiscontinuouswiththeendoplasmic
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reticulum(ER).Inbothkingdomsthenuclearporecomplexes(NPCs)regulateamultifacetedmutual
exchangeofmetabolitesandmacromoleculesacrossthenuclearenvelope[1]. However,NPCsarenot
identicalstructuresinplantandanimalcells. SomeNPCproteinsareconservedattheaminoacid
sequencebutothersareonlyfunctionallyandstructurallyrelated[2–4].
These evolutionary differences in nuclear architecture are not restricted to the NPC, but are
also found in the protein composition of the nuclear envelope and its associated compartments,
with one of the best examples being the nuclear lamina. This structure directly underlying the
inner nuclear membrane (INM) literally is a meshwork of proteins, mainly comprised of lamins
andvarioustypesoflamin-associatedproteins[5–7]. Themanifoldfunctionsofthislatticeinclude
supportingandshapingofthenucleus[8–10],aswellastetheringofheterochromatintothenuclear
periphery[6,11,12].OtherfunctionalaspectsrangefromDNAdamagerepairandreplicationtocellular
signaltransduction[13–15]. Interestingly,onthebasisofprimarysequencesimilarity,noplantlamin
geneshavebeenidentifiedsofar. However,theexistenceofamesh-likestructuresimilartothenuclear
laminaofmetazoancellshasbeendemonstratedbyelectronmicroscopyintobacconuclei[16],anda
numberofnuclearproteinswereidentifiedanddiscussedasproteinswithsimilarsecondarystructure,
localization and molecular functions, but varying amino acid sequence as compared to metazoan
lamins. Inordertoemphasizebothsimilaritiesanddifferences,thepostulatedplantequivalenttothe
metazoanlaminahasbeenreferredtoasplamina[17–21].
Inthiscontext,thetransmembraneSad1/UNC-84proteins(SUN)areimportanttomentionsince
theseproteinsareamongthefewNEconstituentssharinganevolutionaryconserveddomainandcan
befoundlikewiseinmetazoans,plantsandfungi[22].Intheanimalkingdom,SUNdomain-containing
proteinswerefoundtolocalizeattheinnernuclearmembraneintrimericarrangementsandtointeract
withlaminsviatheiraminotermini.[23–27]. TheuseofFörsterresonanceenergytransfermicroscopy
(FRET)illustratedthatplantSUNproteinstargettheNEandcouldbeshowntoassociatewithaplant
lamin-likeprotein[28,29]. Inmetazoa,SUNproteinsareabletobindouternuclearmembraneproteins,
designatedKASH(Klarsicht,ANC-1andSYNEhomologyproteins),therebybuildingabridgeacross
the nuclear envelope, which enables interactions with actin filaments, microtubules, intermediate
filamentsormotorproteins[24,30,31]. Ofnote,noknownKASHhomologshavebeenidentifiedin
plantsbasedonprimarysequencecomparisons,butfunctionalanalogswerepostulatedadoptingsome
relatedfunctionalityofKASH-likeproteins. ArabidopsisthalianaWPPdomain-interactingproteins
(WIPs)couldbeshowntointeractwithbothArabidopsisthalianaSUN1andSUN2,aninteractionwhich
isnecessarytoretainWIPeffectivelyattheNE[32]. UnlikemetazoanKASHproteins,theseWIPs
arenotdirectlylinkedtothecytoskeleton,butseemtobedependentontheinteractionwithother
plantouternuclearmembraneproteins. PreviousanalysesidentifiedaWIPinteractionwithWPP
domain-interacting tail-anchored proteins (WITs), which themselves are able to bind myosin XI-I,
furtherstabilizingtheassociationwiththecytoskeleton[33].
Theseexamplesillustratethatthenuclearenvelopeofplantandanimalcellsmightnotbedirectly
conservedonthelevelofprimaryaminoacidsequences,butthatfunctionalhomologsmayexistthat
expressfunctionalredundancyconferredbyevolutionarydivergentcomplexes.
To address the question whether functional similarities exist between metazoan and plant
nuclearenvelops,weemployedaheterologousproteinexpressionapproach,inwhichhumanviral
proteinswereusedasbaitstoidentifycandidateproteinsassociatedwiththenuclearenvelopeand
toinvestigatetheconservationofnuclearmolecularfunctions. Humancytomegalovirus(HCMV)is
adouble-strandedDNAvirusofthefamilyHerpesviridae,whichreplicatesinthehostcellnucleus.
Thisnuclearphaseincludestheformationofviralnucleocapsids,thepackagingwithviralgenomic
DNAandasubsequentnucleocytoplasmictransitionofparticlestoovercomethenuclearenvelope.
Severalstepsofsubsequentextranuclear,cytoplasmictraffickingarefollowingtofinallyresultinthe
cellularreleaseofinfectiousprogenyvirions. However,priortothebuddingthroughbothinnerand
outernuclearmembranes,viralparticleshavetoreorganizetheproteinaceousportionsofthenuclear
envelope[34,35]. Withthenuclearporesbeingfartoonarrowtoallowfordirectexportofthe130nm
capsids, HCMV has evolved a strategy of regulated nuclear capsid egress [36,37]. To achieve this
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task, HCMV assembles a protein complex consisting of both viral and cellular components at the
nuclearenvelope,thesocallednuclearegresscomplex(NEC).Inmammaliancells,themultimeric
NECisabletomediatelaminphosphorylation,whichinturnleadstodestabilizationanddisassembly
of the nuclear lamina. It has been suggested that the cellular peptidyl-prolyl cis/trans isomerase
Pin1inducesaconformationalchangeofnuclearlaminsthatpromotesthedisassemblyprocessof
thenuclearlaminainHCMV-infectedcells[38]. ThecoreNECisformedbythetwoconservedviral
proteinspUL50andpUL53,whichheterodimerizeandremainanchoredtotheinnernuclearmembrane
byacarboxy-terminaltransmembranedomainofpUL50[39–41]. Together,theseviraleffectorsare
thoughttoprovideabasisforfurtherrecruitmentofproteinfactorsnecessaryforthedestabilizationof
thelamina,suchasproteinkinases,bridgingfactorsandfunctionallyuncharacterizedcomponents.
AmongthesearethecellularkinasePKCα,theviralkinasepUL97,theinnernuclearmembraneprotein
emerinandthemultifunctionalcellularproteinp32/gC1qR[42–49]. Inthisstudy,weprovidefirst
evidencethatamolecularmechanismenablingINM-retentionisconservedacrosskingdoms,andthat
thenucleoplasmiclocalizationofpUL53aswellasitspUL50-mediatednuclearrimrecruitmentis
surprisinglysimilarinplantandhumancells. Furthermore,weidentifiedapUL50-interactingplant
protein,re-initiationsupportingprotein(RISP,locusidentifierAT5G61200),whichuponcoexpression
withpUL50associatestogranularstructuresattheplantnuclearenvelope. Theconclusionsdrawn
from the observed patterns of protein interaction and translocation in plant cells as well as their
possiblesignificanceforunderstandingnuclearplantarchitecturearediscussed.
2. MaterialsandMethods
2.1. PlantMaterials
Nicotianabenthamianaplantsweregrowninsoilinagreenhousemaintaininga16hlight/8h
darknessphotoperiodandtemperaturesof25and20˝C,respectively.
2.2. PlasmidConstructs
Forexpressioninplants—thecodingsequencesofpUL50andpUL53wereamplifiedbyPCR
from pcDNA-UL50-HA and pcDNA-UL53-FLAG [43], respectively, using specific oligonucleotide
primers(sequencesgiveninTable1). InbothcasestheGateway® Cloningsystem(ThermoFisher
Scientific, Waltham, MA, USA) was used: After subcloning of the PCR products into the vector
pENTR/D-TOPO®,thefinalconstructsweregeneratedusingtheLR-Clonase®EnzymeMixandthe
destinationvectorspK7WGF2[50]andpGWB660[51]forpUL50andpUL53respectively,leadingto
eGFP::pUL50andpUL53::tagRFPfusionproteins.
TogenerateaRFP-destinationvector,thesequenceoftagRFPwasamplifiedfrompGWB660[51]
usingappropriateoligopeptideprimers(sequencesgiveninTable1). Theresultingampliconwas
subcloned into the entry vector pCR™-Blunt (Thermo Fisher Scientific), excised by the restriction
enzymesSalIandPstIandinsertedintothevectorpRB35S[52],givingrisetothevector35S-X:RFP.
The RISP coding sequence was amplified from Arabidopsis thaliana cDNA and cloned in the same
manner (restriction enzymes BamHI and SalI) and inserted into the latter vector, resulting in a
RISP::tagRFPconstruct.
The expression of all constructs was driven by the constitutively active cauliflower mosaic
virus(CaMV)35Spromoter, whereasterminationoftranscriptionwasachievedbytheCaMV35S
terminator,thenopalinesynthaseterminatorandtheoctopinesynthaseterminatorforeGFP::pUL50,
pUL53::tagRFPandRISP::tagRFP,respectively.
Forexpressioninyeast—aplasmidcodingforpUL50infusionwiththeGAL4DNA-binding
domain(GAL4BD-UL50)wasdescribedpreviously[43].
For expression in human cells—a plasmid coding for RISP with a C-terminal FLAG-tag was
generatedbyPCRamplificationoftheRISPopenreadingframe. StandardPCRamplificationwas
performed using pENTR-RISP as template with oligonucleotide primers purchased from Biomers
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(Ulm, Germany); sequences of oligonucleotides are given in Table 1. After cleavage with EcoRV
and NotI, the PCR product was inserted into the mammalian expression vector pcDNA3.1(+)
(Thermo Fisher Scientific). The mammalian expression constructs pcDNA-UL50(1-358)-HA,
pcDNA-UL53-HA,pcDNA-UL53-FLAGandpcDNA-UL97-HAhavebeendescribedpreviously[43,53].
Construct pDsRed1-N1, expressing the red fluorescent protein (RFP), was purchased from BD
Biosciences Clontech (Heidelberg, Germany) and was used as a positive control for transfection
experimentsinmammaliancells.
Table1.Sequencesofoligonucleotideprimersusedforcloning.
PrimerName Sequence(5’to3’)
PrimersforRFPVectorConstruction
RFPfwSalI GTCGACGGATCTGGTGTGTCTAAGGGCGAAGAGCTG
RFPrevPstI CTGCAGCTATCAATTAAGTTTGTGCCCCAGTTTG
PrimersforLocalizationStudies
inPlants
pUL50fw CACCATGGAGATGAACAAGGTTCTCCATC
pUL50rev TCAGTCGCGGTGTGCGGAG
pUL53fw CACCATGTCTAGCGTGAGCGGC
pUL53rev AGGCGCACGAATGCTGTTGAG
RISPfw GGATCCAACAATGTCGAGTAATTGGGGAAGTAGCTCG
RISPrev GTCGACTACAGCAGGAAGAGGAACTAAGCAAGTTG
inHumanCells
5-RISP_EcoRV-Acc65I TCAGGTACCGATATCATGAACGACCTGAGTGAACATGTAC
TCAGGGCCCGCGGCCGCTTACTTGTCGTCATCGTCTTTGTAGT
3-RISP-FLAG_NotI-ApaI
CTACAGCAGGAAGAGGAACTAAGC
Expression of both RISP-FLAG and pUL50(1-358)-HA was driven by the HCMV immediate
earlypromoter/enhancer(PCMV),whileterminationwasachievedbythebovinegrowthhormone
polyadenylationsignal(BGHpA).
2.3. TransientExpressioninNicotianabenthamianaandConfocalLaserScanningMicroscopy
Transient expression was achieved through pressure infiltration of Agrobacterium tumefaciens
culturesasdescribed. IncaseofRISP::tagRFPexpressions,atomatobushystuntvirusp19-construct,
whichwaskindlyprovidedbyKathrinPaulus,Friedrich-AlexanderUniversityErlangen-Nürnberg,
wascoinfiltratedtominimizeposttranscriptionalgenesilencing[54].
Forconfocalmicroscopy,leafsegementsofN.benthamianawerecutandtheabaxialsideswere
observed. Pictures were obtained using a Leica TCS SP5 II confocal laser scanning microscope,
fluorescent dyes and proteins were excited using lasers with wavelengths of 405 nm, 488 nm and
561 nm for DAPI, eGFP and tagRFP, respectively. To prevent fluorescence bleed through during
expressionofmultiplefluorescentproteins,sequentialscanswereconducted.
Life cell DAPI-stainings were achieved by leaf infiltration of a 1 mg/mL DAPI
(41,6-diamidino-2-phenylindole)solution. After10min,leaveswereflushedbyinfiltrationofwater
andsubsequentlyobservedunderthemicroscope. Whenstatisticalanalysiswereemployed,theLAS
AFsoftware(LeicaMicrosystems,Wetzlar,Germanywasused. Scalebarsforthefiguresdepicting
theregionsofinterestusedforcolocalizationinthesupplementarymaterialswereintroducedwith
FIJI[55].
2.4. TransientExpressioninHumanEpithelialHelaCells,IndirectImmunofluorescenceAssayandConfocal
Laser-ScanningMicroscopy
Humanepithelialcells(HeLa)wereseededoncoverslipsfortransienttransfectionexperiments.
CellsweretransfectedusingLipofectamine2000(ThermoFisherScientific)witheukaryoticexpression
Viruses2016,8,73 5of16
plasmidscodingforhemagglutinin(HA)-taggedpUL50(pcDNA-UL50-HA)andFLAG-taggedpUL53
(pcDNA-UL53-FLAG)accordingtothemanufacturer’sprotocols. At2dayspost-transfection,cells
were fixed and permeabilized followed by indirect immunofluorescence staining. Stainings were
performed as described previously [43,48] using primary antibodies rabbit polyclonal antibody
(pAb), anti-HA (HA.11; Eurogentec Deutschland GmbH, Cologne, Germany) and Alexa Fluor
488-/555-conjugatedsecondaryantibodies(MolecularProbes,Eugene,OR,USA).Cellsweremounted
usingDAPI-containingVectashieldmountingmedium(VectorLaboratories,Burlingame,CA,USA).
ImageswereacquiredbysequentialscanningusingaTCSSP5confocallaser-scanningmicroscope
(LeicaMicrosystems)andanalyzedusingLASAFsoftware(LeicaMicrosystems).
2.5. PlantProteinPAGEandWesternBlotAnalysis
To obtain a total protein extract, two leaf discs (Diameter appr. 9 mm) were frozen in liquid
nitrogen,groundin70µLextractionbuffercontainingurea(225mMTrisHClpH6.8,50%glycerol,
5%SDS,250mMDTT,8Murea,0.02%bromophenolblue)andcentrifugedfor3minat15,900ˆg.
SupernatantwasloadedonaBis/Trisgelcontaining12%acrylamideforelectrophoresis. Proteinswere
transferredontoanitrocellulosemembrane,whichafterwardswasblockedforatleast1hinblocking
buffer(20mMTrisHCl,500mMNaCl,0.1%(v/v)Tween20,5%(w/v)milkpowder). Detectionwas
carried out using suitable primary antibodies and horseradish peroxidase-conjugated secondary
antibodies,followedbyenhancedchemiluminescencedetection. Nitrocellulosemembranestainings
wereconductedusingasolutionof0.5%(w/v)PonceauSin1%aceticacid.Membraneswereincubated
for2mininthestainingsolutionandsubsequentlydestainedindistilledwater.
2.6. YeastTwo-HybridScreening
Foryeasttwo-hybridscreening,theGAL4-based[56]ClontechMatchmaker3system(Clontech,
Heidelberg,Germany)wasused. Inshort,thevectorpGBT9containingaGAL4DNA-bindingdomain
inframewithpUL50waschosentotransformtheyeaststrainY187[57]usingthepreviouslydescribed
PEG/Lithium acetate/ssDNA method [58]. As a cDNA library, an Arabidopsis thaliana library in
AH109byeastcellsobtainedfromtair[59]wasused. Aftermatingbothstrainsandplatingonminimal
medium,yeastcolonieswereobtainedafter5–7days. X-Galfilterstainingassayswereconductedto
verifypositiveinteractions[60]. Theidentityofpositivecloneswasrevealedbyplasmidpreparation
andfollowingsequencing.
2.7. CoimmunoprecipitationAssay
Humanembryonickidneyepithelialcells(293T)werecultivatedandcotransfectedwithexpression
plasmidscodingforHA-orFLAG-taggedcytomegaloviralproteinswithFLAG-taggedRISPbytheuse
ofpolyethlyeniminine-DNAcomplexesasdescribedpreviously[48,61]. Threedayspost-transfection,
cellswereusedforprotein-proteininteractionexperimentsutilizingcoimmunoprecipitation(CoIP).
Immunoprecipitationwasperformedunderpreviouslydescribedconditionsusing2µLofmouse
monoclonal antibody (mAb) anti-HA (12CA5; Roche Applied Science, Risch, Switzerland) [43].
CoIPsamplesandexpressioncontrols(input)takenpriortotheadditionoftheCoIPantibodywere
subjectedtostandardWesternblotanalysisusingmousemAbanti-FLAG(M2;Sigma,St. Louis,MO,
USA)andrabbitpolyclonalantiserum(pAb)anti-HA(HA.11;CovanceInc.,Princeton,NJ,USA).
3. Results
3.1. HCMVpUL50LocalizestothePlantNuclearEnvelope
Inordertoinvestigatethearchitectureofplantnuclei,theherpesviralproteinpUL50waschosen
forheterologousexpressionexperiments,astheproteinislocalizedtotheinnernuclearmembranein
humancells[43,48]. Forlocalizationstudies,eGFPwasfusedtotheN-terminusofpUL50(FigureS1a).
The construct was transiently expressed in leaves of Nicotiana benthamiana using Agrobacterium
Viruses 2016, 8, x 6 of 16
3. Results
3.1. HCMV pUL50 Localizes to the Plant Nuclear Envelope
Viruses2016,8,73 6of16
In order to investigate the architecture of plant nuclei, the herpesviral protein pUL50 was
chosen for heterologous expression experiments, as the protein is localized to the inner nuclear
tumefamcieemnbsr-amnee dinia theudmtarna ncsefllosr [m43a,4ti8o].n F.oTrh leocianltizeagtrioitny sotufdeiGesF, Pe:G:pFUP Lw5a0s cfousueldd tboe tchoe nNfi-rtemrmedinbuys oWf estern
blotanpaUlyLs5i0s .(FUigsuinreg Sm1ao).u Tsheem coonnstorculcot nwaalsa tnratnibsioedntyly( mexAprbes)sdedir ienc tleeadveasg oafi nNsitcoptiUanLa 5b0en(tmhaAmiban-Ua uLs5in0g; kindly
Agrobacterium tumefaciens-mediated transformation. The integrity of eGFP::pUL50 could be
provided by Stipan Jonjic and Tihana Lenac, University of Rijeka, Rijeka, Croatia), a band at the
confirmed by Western blot analysis. Using mouse monoclonal antibody (mAb) directed against
corresponding size of ca. 70 kDa was detected, verifying proper protein expression (Figure S1b).
pUL50 (mAb-UL50; kindly provided by Stipan Jonjic and Tihana Lenac, University of Rijeka, Rijeka,
Whenobservedunderaconfocallaser-scanningmicroscopetwodaysafterAgrobacteriuminfiltration,
Croatia), a band at the corresponding size of ca. 70 kDa was detected, verifying proper protein
eGFP::epxUprLe5ss0iosnh o(Fwigeudrea Sc1lbe)a. rWahnedn ocbosnetrivneudo uunsdleor caa clioznafoticoanl ltaosetrh-secannuncinlega mriecnrovseclooppee t,wino ddiasytsin agftueri shable
fromthAegprohbyasctieorliougmic ianlfirlitrmatiloonc,a elGizFaPti::opnUoLf50t hsehonwaetidv ae cpleraort eainnd( cFoingtuinreuo1ubs, lpoacanleizlasti1o–n4 t)o. AtheD nAuPclIeasrt aining
servedenavsealopme,a irnkdeirsticnognufiisrhmabilne gfrothme thloec pahliyzsaiotlioognicaalt rtihme loncualcilzeaatironp oefr itphhe enraytivien pprolatenint c(Feilglusr(eF 1ibg,u re 1a,
panelsp1a–n2e)ls. 1N–4o)t. eAw DoArtPhI ys,taeinGinFgP s::eprUveLd5 a0s aa lmsoarlkoecr acloinzfeidrmtiongt hthee eloncdaolizpaltaiosmn aitc threet nicuuclleuamr p,ewriphhicehryc anbe
in plant cells (Figure 1a, panels 1–2). Noteworthy, eGFP::pUL50 also localized to the endoplasmic
seeninamaximumprojectionofaz-stackimageseries(Figure1a,panels3,4). Interestingly,aninitial
reticulum, which can be seen in a maximum projection of a z-stack image series (Figure 1a, panels
localizationofpUL50totheendoplasmicreticulumwasalsodescribedinHCMV-infectedhuman
3,4). Interestingly, an initial localization of pUL50 to the endoplasmic reticulum was also described
cells,whichis,duringthecourseofviralreplication,abolishedbypUL53-mediatedINM-retentionof
in HCMV-infected human cells, which is, during the course of viral replication, abolished by
pUL50p[U62L]5.3-mediated INM-retention of pUL50 [62].
Figure 1. Expression and localization of pUL50 (a) Transient expression of eGFP::pUL50 in Nicotiana
Figure1.ExpressionandlocalizationofpUL50(a)TransientexpressionofeGFP::pUL50inNicotiana
benthamiana: panel 1, DAPI-staining was performed to identify nuclei (blue), the GFP-fusion
benthamiana:panel1,DAPI-stainingwasperformedtoidentifynuclei(blue),theGFP-fusionconstruct
construct (green) was localized to the nuclear envelope; chloroplast autofluorescence is shown in
(green)waslocalizedtothenuclearenvelope;chloroplastautofluorescenceisshowninyellow,the
yellow, the dotted box indicates the section shown in panel 2; panel 2, magnified section from panel
dottedboxindicatesthesectionshowninpanel2;panel2,magnifiedsectionfrompanel1;panel3,

pUL50alsolocalizedtotheendoplasmicreticuluminNicotianabenthamiana,maximumprojectionofa
z-stackimage,thedottedboxindicatesthesectionshowninpanel4;panel4,magnifiedsectionfrom
panel3revealsthelocalizationofeGFP::pUL50tothenet-likestructureoftheendoplasmicreticulum;
(b)ImmunolocalizationofpUL50-HA(green)andpUL53-FLAG(red)inHeLacells:Panels1-4,single
transfectionofpUL50-HAinDAPI(blue)stainedcellsrevealedthelocalizationtothenuclearenvelope;
panels5-8,singletransfectionofpUL53-FLAGinDAPIstainedcellsshowednucleoplasmiclocalization;
panels9-12,cotransfectionofpUL50-HAandpUL53-FLAGdemonstratingthepotentialofpUL50to
recruitpUL53tothenuclearrim.Whitebarsrepresent10µm.
Viruses 2016, 8, x 7 of 16
1; panel 3, pUL50 also localized to the endoplasmic reticulum in Nicotiana benthamiana, maximum
projection of a z-stack image, the dotted box indicates the section shown in panel 4; panel 4,
magnified section from panel 3 reveals the localization of eGFP::pUL50 to the net-like structure of the
endoplasmic reticulum; (b) Immunolocalization of pUL50-HA (green) and pUL53-FLAG (red) in
HeLa cells: Panels 1-4, single transfection of pUL50-HA in DAPI (blue) stained cells revealed the
localization to the nuclear envelope; panels 5-8, single transfection of pUL53-FLAG in DAPI stained
Viruses2016c,e8l,l7s 3showed nucleoplasmic localization; panels 9-12, cotransfection of pUL50-HA and pUL53-FLAG 7of16
demonstrating the potential of pUL50 to recruit pUL53 to the nuclear rim. White bars represent 10
µm.
3.2. pUL50isAbletoRecruitHCMVpUL53totheNuclearPeripheryinNicotianabenthamiana
3.2. pUL50 is Able to Recruit HCMV pUL53 to the Nuclear Periphery in Nicotiana benthamiana
InordertoinvestigatepUL50-pUL53interactionincoexpressingplantcells,afusionproteinfor
In order to investigate pUL50-pUL53 interaction in coexpressing plant cells, a fusion protein for
pUL53wasgeneratedcarryingaC-terminallytaggedRFP(FigureS2a).AutofluorescentpUL53::tagRFP
pUL53 was generated carrying a C-terminally tagged RFP (Figure S2a). Autofluorescent
wastransientlyexpressedinNicotianabenthamianaleavestodetermineitslocalization. Thecorrect
pUL53::tagRFP was transiently expressed in Nicotiana benthamiana leaves to determine its
expressionoftheconstructwasverifiedbyWesternblotanalysis,showingabandatthecorresponding
localization. The correct expression of the construct was verified by Western blot analysis, showing a
sizeofappr. 70kDa(FigureS2b). ConfocallaserscanningmicroscopyrevealedthatpUL53::tagRFP
band at the corresponding size of appr. 70 kDa (Figure S2b). Confocal laser scanning microscopy
localirzeevdeaaletdv athraiot upsUiLn5t3r:a:tnaugRclFePa rlosciateliszeadl saot ivnacrliuodusin ignttrhaneunculecalre osiltuess (aFlsiog uinreclu2dai–ndg) .thFeo nruccolmeopluasr ison
with(tFhieguhrue m2aa–ndc).e lFlourl acrolmocpaalriizsoanti owni,thp UthLe5 0huamndanp UceLll5u3larw leorcealtirzaantisofne,c tpeUdLi5n0 HanedL apcUeLll5s3i nwperaer allel.
Durintrgansisnfegclteedtr iann HsfeeLcati ocenl,lsp iUn Lp5a3rawlleal.s Dtaurrginegte sdintgoleth treannusfcelcetuiosn(, FpiUguL5re3 1wba,sp taanrgeeltse5d– t8o) tbhue tn,uinclecuosn trast
topla(Fnitgcuerell s1,b,p pUaLne5l3s 5d–i8d) bnuott, ienx choinbtirtaast ntou cplleaontl acrelalsc,c puUmLu53la dtiido nnoatn edxhwibaits aa nlsuoclenooltara ascdcuismpuelrasteiodn asin
Nicotaiannda wbeanst haalsmo iannoat .aUs pdoisnpecroseexdp arse sisni oNni,cobtoiatnha flbuenotrheasmcieannat.p Uroptoenin csosetxrpicretlsysiocno,l obcoatlhiz feludoirnesNceincot tiana
proteins strictly colocalized in Nicotiana benthamiana. Importantly, eGFP::pUL50 was able to recruit
benthamiana. Importantly, eGFP::pUL50 was able to recruit pUL53::tagRFP from its even nuclear
pUL53::tagRFP from its even nuclear localization to the nuclear rim (Figure 2e–h). This is in
localizationtothenuclearrim(Figure2e–h). ThisisinaccordancewiththesituationinHeLacells,
accordance with the situation in HeLa cells, where a cotransfection of both viral constructs led to a
whereacotransfectionofbothviralconstructsledtoaverysimilarresult(Figure1b,panels9–12).
very similar result (Figure 1b, panels 9–12). Noteworthy, in Nicotiana benthamiana, both signals
Noteworthy,inNicotianabenthamiana,bothsignalswerenotevenlydistributedacrossthenuclear
were not evenly distributed across the nuclear envelope, but localized to a patch-like pattern at the
envelnoupcel,eabru rtimlo.c Talhizise pdattotearnp watacsh p-lairkteicpulaatrtleyr ndiasttinthcte wnuhecnle darepriicmte.dT thhirsopugahtt ethrne mwaaxsimpaurmtic purloajrelcytidonis tinct
whenodf ae pzi-csttaecdk tihmraogueg shertihees (mFiagxuirme 2uim–k)p. rojectionofaz-stackimageseries(Figure2i–k).

Figure 2. Expression of pUL53::tagRFP (depicted in magenta) and colocalization with eGFP::pUL50
Figure2.ExpressionofpUL53::tagRFP(depictedinmagenta)andcolocalizationwitheGFP::pUL50
(depicted in green). (a)–(d) Transient expression of pUL53::tagRFP in Nicotiana benthamiana: No
(depicted in green). (a)–(d) Transient expression of pUL53::tagRFP in Nicotiana benthamiana: No
fluorescence in the GFP-channel, tagRFP-signal corresponding to pUL53 was restricted to the
fluorescence in the GFP-channel, tagRFP-signal corresponding to pUL53 was restricted to the

nucleoplasm; (e)–(h) Coexpression of eGFP::pUL50 and pUL53::tagRFP in Nicotiana benthamiana:
ColocalizationofbothconstructssuggestedthatpUL50wasabletorecruitpUL53tothenuclearrim;
(i)–(k)CoexpressionofeGFP::pUL50andpUL53::tagRFPinNicotianabenthamiana,z-stackmaximum
projection:Constructscolocalizedinpatch-likestructuresatthenuclearperiphery.Whitebarsrepresent
10µm.
3.3. pUL50InteractswithArabidopsisthalianare-InitiationSupportingProtein(RISP)
GiventhenuclearenvelopelocalizationofeGFP::pUL50inNicotianabenthamiana,candidateplant
proteinspossiblyinteractingwithpUL50wereinvestigated. Tothisend,ayeasttwo-hybridscreening
was performed using an Arabidopsis thaliana cDNA library fused to the GAL4 activation domain,
Viruses 2016, 8, x 8 of 16
nucleoplasm; (e)–(h) Coexpression of eGFP::pUL50 and pUL53::tagRFP in Nicotiana benthamiana:
Colocalization of both constructs suggested that pUL50 was able to recruit pUL53 to the nuclear rim;
(i)–(k) Coexpression of eGFP::pUL50 and pUL53::tagRFP in Nicotiana benthamiana, z-stack maximum
projection: Constructs colocalized in patch-like structures at the nuclear periphery. White bars
represent 10 µm.
3.3. pUL50 Interacts with Arabidopsis thaliana re-Initiation Supporting Protein (RISP)
Given the nuclear envelope localization of eGFP::pUL50 in Nicotiana benthamiana, candidate
Viruses2016,8,73 8of16
plant proteins possibly interacting with pUL50 were investigated. To this end, a yeast two-hybrid
screening was performed using an Arabidopsis thaliana cDNA library fused to the GAL4 activation
edmomplaoiyni,n gemaptrluoynicnagte da vterursniocanteodf pvUeLrs5i0on(a mofi npoUaLci5d0s 1(a–m35i8n,ol aacckiidnsg i1t–s3C5-8t,e rlmaciknianlgt raitns smC-etmerbmrainnael
dtroamnsamine)mfubsreadnet odtohmeaGinA)L f4uDseNd Ato-b tihned iGngAdLo4m DaNinAa-sbianbdainitgp drootmeianin(F iagsu ar eb3aai)t .pArfotteerinm (aFtiingugroef 3tha)e.
yAefatsetr cmloantiensge iotfh ethret ryaenassfto crmlonedesw eiitthhetrh etrcaDnsNfoArmlibedra wryitohr tthhee cbDaiNtAco nlisbtrraurcyt ,otrh ethcee lblasiwt ceorensptlrautcetd, tohne
sceellelsc twioenrem pedlaituemd toond seetleercmtioinne mtheedeifufimci etnoc dyeotferthmeinmea tthineg epffrioccieenssc.yI noft othtael ,mapaptirnogx ipmroatceelsys.3 .I4n ˆto1ta0l6,
mapaptirnogxiemvaetnetlsy w3.e4r e× o1b0s6e mrvaetdin.gY eeavsetnctesl lwseexrep roebssseinrvgetdh.e Yireraespt ocretlelrs geexnperses(sHinIgS3 thaneidr lraecpZo)rtaenrd gtehnuess
c(HonItSa3i nainndg plaoctZe)n tainaldly thinutse rcaocntitnagincionngs ptroutcetnstwiaellrye isnetleercatecdtinbgy cthonesutrsuecotfs mwienriem saellemceteddiu bmy athned uXs-eG aolf
fimltienrimstaali nminegdiausmsa yasn.dH Xe-rGebayl ,fwilteeri dsetanitnifiinegd assisxaiynsd. iHviedruebalyy, ewaes tidcleonnteifsi,ewd hsiixc hinwdeivreidfuuartl hyeeraasnt aclloynzeeds,
bwyhpiclahs mwiedrep rfeuprtahrearti oannaalnydzesdeq buye npcliansgm.iAdl lpsriexpcaloranteiosnh aarnbdo rseedqtuheenicdienngt.i cAalllp sriexy cplolansems ihda,rcboonrteadin itnhge
aidfernagtimcaeln tporefyth eplcaosdminidg, secqonuteanicneinogf Aar afbriadgompseisntth aolifa nthaer e-cionditiinagti osnesquupepnocert ionfg pArroatbeiidnop(RsiIsS Pth,alolicaunsa
irdee-inntiitfiiaetrioAnT 5sGup61p2o0r0ti)n.gIn pprroetveiionu s(RwISoPrk, s,lothcuesp liadnetn-stpifeiecri ficART5ISGP61w2a0s0)d. esIncr ibperdevtioouins tewraocrtkws,i ththae
cpaluanlitfl-ospweecrifmic oRsIaSicPv wirauss dpersoctreiibne,dtr aton sianctetirvaactto wrvitihro ap lcaasumliifnlo(wTAerV m),oinsaoircd veirrtuos spurpopteoirnt, tthreantrsaancstilvataitoonr
rvei-rionpitliaastmionino (fTpAolVy)c,i sitnr oonridcemr RtoN Asu.pApsoartp tohsetu tlraatnedslpatairotno freth-ientitriaantisolant ioonf pmoalcyhciinsterroyn,iRcI SmPRwNaAs.f oAusn da
tpoobsetuplaretesedn ptainrtd ooft -tlhikee traagngsrleagtiaotens minatchheinceyrtyop, RlaIsSmP [w63a]s. fToouvnedr itfoy bthee prreessuelnttf rionm dtohte-liykeea satgtgwreog-haytebsr iidn
sthcree ecny,totwploasomf t[h6e3]s.i xTore svceuriefdy pthlaes mreisduslt( Ffriogmur eth3eb )yweaesrte tcwootr-ahnysbfroidrm secdreweni,t htwpoG AofL t4hBeD s:i:xp UreLs5c0ueind
ypelaassmt cidelsl s(FaingdurXe- G3ba)l fiwleterre sctoatirnainnsgfoarsmsaeyds wwietrhe ppGerAfoLr4mBeDd::.pUALs5n0e gina tyiveeascto cnetlrlosl sa,ntdh eXr-eGsaple cfitlitveer
esmtaipntiyngv eacstsoaryssw wereereu speedr,fowrmheerde.a sAtsh eneSgVa4t0ivlea rcgoenTtroalnst, igtheen rinesppGecAtiDve4 2e4mapntdy pv5e3ctionrsp GwBeTre9 uwseerde,
cwohtrearnesafso trhmee SdVa4s0p loasrigtiev Te caonntitgroeln( bino tphGsAupDp4l2ie4d abnyd tph5e3m inan puGfaBcTtu9r ewre).reW choitlreatnhsefocromtreadn safso rpmosaittiiovne
wcointhtrtohl e(bnoetgha stiuvpepcloiendt rboyls thdeid mnaontulfeaacdtutroers)t.a Winihnigle otfheth ceoytreaansstfocormloantiieosnu wpiotnh Xth-eG naelgtraetaivtme ceonnt,trtohles
idnitde rnaoctt iloenado ftoS sVta4i0nTinagn otfig tehne yaneadstp c5o3lorensiueslt uedpoinn Xan-Geaxlp trreesastimonenotf, tthhee irnetpeorartcetriogne onfe SsVan40d Tth aunstitgoena
balnude pst5a3i nriensgulotefdth ien caonlo enxipesre.sInsiocans oefo tfhpeU reLp5o0ratenrd gRenISePs, aanlldc othlounsi etos oa fbtlhuee tsetsatiendindgu opfl itchaete csoeloxnhiibesit.e Idn
acasstero nofg pblUuLe5s0ta iannindg R,iInSdPi,c aatliln gcoalosnoileids ionft etrhaec titoensteodf bdouthpplicroatteeisn sexinhiybeitaesdt, cao nstfirromngin gbltuhee pstraeivniionugs,
riensduiclta(tFiniggu ar eso3lci)d. interaction of both proteins in yeast, confirming the previous result (Figure 3c).

FFiigguurree 33.. YYeeaasstt ttwwoo--hhyybbrriidd aassssaayy wwiitthh ppUULL5500 aass bbaaiitt.. ((aa)) BBaaiitt ccoonnssttrruucctt yyiieellddiinngg aa GGAALL44 bbiinnddiinngg
ddoommaaiinn--ppUULL5500f ufsuiosniopnr otperino,teuisne,d fuosresdc refeonri ngscarnedenciontgra nasfnodrm acotitorna.nPsAfoDrmHa,tailocno.h oPldAeDhHyd, roaglecnoahsoel
1dephryodmrootgeern;aTsAe D1 Hpr1o,maloctoehr;o TlAdeDhHyd1,r oalgceonhaosle d1ehteyrdmroingaetnoars;er 1es tterrimctiionnatsoirt;e rsewstreircetiuonse sditeass winedriec autseedd;
vaas luinedsiocvaeterds;c hveamlueess inodviecra tsechsiezmeeosf tihnedriceasptee cstiizvee ofrfa gthmee rnetsipnebctaisveep afrirasg;m(be)nPt rieny cboansestprauicrts;i d(ebn) tiPfireedy
dcounrisntrguscct riedeennintigfieadn dduursiendg fsocrreceontrinang safonrdm uasteiodn f,otrh ecoGtrAaLns4foarcmtivaatitoionn, tdhoem GaAinL4fu ascetdivtaotiaonfr adgommeanint
ofArabidopsisthalianaRISP;(c)X-GalfilterstainingassayconfirmsinteractionofpUL50andRISPin
yeastcells.
Toverifytheresultsobtainedintheyeasttwo-hybridsystem,weanalyzedtheputativeinteraction
between RISP and HCMV pUL50 by coexpression and coimmunoprecipitation (CoIP) in human
embryonickidney293Tcells. Therefore,wefirstgeneratedaconstructencodingFLAG-taggedRISPfor
expressioninmammaliancells(FigureS3). Mammalianexpressionconstructsforatruncatedversion
of pUL50 (i.e., amino acids 1–358; lacking its C-terminal transmembrane domain) (Figure S3) and
furtherHCMVcontrolproteinsweregeneratedpreviously[43]. TheHCMVproteinspUL50(1–358),
Viruses 2016, 8, x 9 of 16
fused to a fragment of Arabidopsis thaliana RISP; (c) X-Gal filter staining assay confirms interaction of
pUL50 and RISP in yeast cells.
To verify the results obtained in the yeast two-hybrid system, we analyzed the putative
interaction between RISP and HCMV pUL50 by coexpression and coimmunoprecipitation (CoIP) in
human embryonic kidney 293T cells. Therefore, we first generated a construct encoding
FLAG-tagged RISP for expression in mammalian cells (Figure S3). Mammalian expression
Viruses2016,8,73 9of16
constructs for a truncated version of pUL50 (i.e., amino acids 1–358; lacking its C-terminal
transmembrane domain) (Figure S3) and further HCMV control proteins were generated previously
[43]. The HCMV proteins pUL50(1–358), pUL53 or pUL97 were coexpressed with RISP in 293T cells.
pUL53orpUL97werecoexpressedwithRISPin293Tcells. Forprotein-proteininteractionanalysis,
For protein-protein interaction analysis, hemagglutinin (HA)-tagged viral proteins were
hemagglutinin (HA)-tagged viral proteins were immunoprecipitated with a tag-specific antibody.
immunoprecipitated with a tag-specific antibody. CoIP samples and lysate controls were analyzed
CoIPsamplesandlysatecontrolswereanalyzedbyWesternblotting(Figure4a,b). Importantly,RISP
by Western blotting (Figure 4a,b). Importantly, RISP was specifically coimmunoprecipitated along
wasspecificallycoimmunoprecipitatedalongwithpUL50(Figure4a,lanes3–4),whereasCoIPofRISP
with pUL50 (Figure 4a, lanes 3–4), whereas CoIP of RISP by other HCMV control proteins was
byothneegraHtiCveM (FVigcuorne t4rao,l lapnreost e5i–n6s).w Toagsenthegera,t tihveese(F riegsuurltes 4coan,lfairnmeesd5 t–h6e) .inTtoegraectthioenr, otfh ResISePr ewsiuthlt sHcCoMnfiVr med
theinptUerLa5c0t.i onofRISPwithHCMVpUL50.

Figure 4. Interaction between RISP with HCMV pUL50 in human cells. (a,b) Coimmunoprecipitation
Figure4.InteractionbetweenRISPwithHCMVpUL50inhumancells.(a,b)Coimmunoprecipitation
(CoIP) assay. Human 293T cells were transiently cotransfected with constructs coding for
(CoIP)assay.Human293TcellsweretransientlycotransfectedwithconstructscodingforFLAG-tagged
FLAG-tagged RISP and HA-tagged HCMV proteins pUL50(1–358) (ratio 1:1 and 1:3, lanes 3 and 4,
RISPandHA-taggedHCMVproteinspUL50(1–358)(ratio1:1and1:3,lanes3and4,respectively),
respectively), pUL97 (lane 5) or pUL53 (lane 6), or with an empty vector (pcDNA3.1, lane 2) as
pUL97(lane5)orpUL53(lane6),orwithanemptyvector(pcDNA3.1,lane2)ascontrol.Coexpression
control. Coexpression of HA-tagged pUL50(1–358) and FLAG-tagged pUL53 served as CoIP control
ofHA(l-atnaeg g7)e; dRFpPU eLx5p0r(e1s–si3o5n8 s)earnveddF aLs AtrGan-stafegcgtieodn pcoUnLtr5o3l (sleanrvee 1d). aAst C2 odIaPysc opnostrt-otrla(nlasnfeect7io);nR, cFePllse xwperrees sion
serveldysaesd,t raannds feHctAio-tnagcgoendt rporlo(tleainnes 1w).eAret 2imdmauynsopporsetc-itpriatantsefde cutisoinng, cmelolsuswe ermeolnyosceldon,aaln danHtibAo-dtayg ged
proteminAsbw-HeAre. iCmoImP usnamopprleesc i(pa)i taantedd lyussaitne gcomntoruolsse (imnpounto) c(blo) ntaakleann tpirbioord tyo mthAe bad-HdiAtio.nC oofI Pthsea CmopIPle s(a)
andlaynstaibteodcyo nwterroel ssu(binjepctuetd) t(ob s)tatnadkaernd pWrieostrertno bthloet aandadlyitsiios nusoinfgt htaeg-CspoeIcPifiacn atnibtioboddyiews aesr einsduicbajteecdt.e dto
standIagr-dHCW, ecrsotessr-nrebalcotitvaen baalnyds ifsour siminmgutnaogg-slopbeucliifinc haenavtiyb cohdaiiens. asindicated.Ig-HC,cross-reactiveband
forimmunoglobulinheavychain.
3.4. pUL50 Colocalizes with Arabidopsis thaliana Re-Initiation Supporting Protein (RISP)
3.4. pUL50ToC ionlovceasltiizgeastew witehthAerra tbhideo spusbiscetlhlaulliaarn laocRael-izInatiitoiant ioofn RSIuSpPp iosr itnin aggPrereomteeinnt( RwIiSthP )the interaction
with the herpesviral protein when expressed in plants, we generated a fusion protein consisting of
ToinvestigatewetherthesubcellularlocalizationofRISPisinagreementwiththeinteractionwith
RISP and tagRFP (Figure S4). Subsequently, the RISP::tagRFP fusion protein was transiently
thehecropexepsvreirsaseldp rwotiethin ewGhFPen::peUxLp5r0e sisne dNiincoptilaannat sb,enwtheagmeinanear.a tTeod oavfeurscioomnep prootset intracnosncsriispttiinognaolf gReInSeP and
tagRFsiPle(nFciignug roefS t4h)e. SRuISbPs ecqounestnrutlcyt,, tthhee RtoISmPa:t:ota bguRsFhPy fsutusinot nvpirruost esiilnenwciansgt rsaunpspireensstloyr cpo1e9x wpraess saelsdo with
eGFP::pUL50inNicotianabenthamiana. ToovercomeposttranscriptionalgenesilencingoftheRISP

construct,thetomatobushystuntvirussilencingsuppressorp19wasalsocoexpressed[54]. Aftertwo
days,therespectivefluorescencesignalsweremonitoredbyconfocallaserscanningmicroscopy.
InaccordancewithobservationsofThiébeauldetal.[63],RISP::tagRFPwasfoundtolocalizein
cytoplasmicaggregates(Figure5b). Confirmingtheprecedingresultsfromtheyeasttwo-hybridscreen
aswellasthecoimmunoprecipitation,eGFP::pUL50couldbefoundincytoplasmicdot-likestructures
(Figure5a),ofwhichmostwerecolocalizingwiththeRISP-aggregates(Figure5d,e). WeusedPearson’s
correlationcoefficientasastatisticaltoolforquantifyingcolocalization. Therefore,aregionofinterest
(ROI)wasdefinedenclosingthewholecelloftheinsetshowninFigure5e(seeFigureS5),sincethis
cellcoexpressedbotheGFP::pUL50andRISP::tagRFP.APearson’scorrelationcoefficientof0.53ata
colocalizationrateof60.4%demonstratedastrongcolocalizationbetweenpUL50andRISPaccording
totheclassificationbyZinchuketal.[64].
Viruses 2016, 8, x 10 of 16
coexpressed [54]. After two days, the respective fluorescence signals were monitored by confocal
laser scanning microscopy.
In accordance with observations of Thiébeauld et al. [63], RISP::tagRFP was found to localize in
cytoplasmic aggregates (Figure 5b). Confirming the preceding results from the yeast two-hybrid
screen as well as the coimmunoprecipitation, eGFP::pUL50 could be found in cytoplasmic dot-like
structures (Figure 5a), of which most were colocalizing with the RISP-aggregates (Figure 5d,e). We
used Pearson’s correlation coefficient as a statistical tool for quantifying colocalization. Therefore, a
region of interest (ROI) was defined enclosing the whole cell of the inset shown in Figure 5e (see
Figure S5), since this cell coexpressed both eGFP::pUL50 and RISP::tagRFP. A Pearson’s correlation
Viruses2016,8,73 10of16
coefficient of 0.53 at a colocalization rate of 60.4% demonstrated a strong colocalization between
pUL50 and RISP according to the classification by Zinchuk et al. [64].

Figure5. ColocalizationstudiesofeGFP::pUL50(depictedingreen)andRISP::tagRFP(depictedin
magenta).(a)Notethatuponcoexpression,eGFP::pUL50wasfoundretainedincytoplasmicaggregates;

(b)Asreportedearlier[63], RISP::tagRFPwasmainlylocalizedtocytoplasmicdot-likestructures;
(d) The merged image demonstrates colocalization of eGFP::pUL50 and RISP::tagRFP. The dotted
boxindicatesthesectionmagnifiedin(e);(e)Magnifiedsectionfrom(d),arrowheadspointtowards
someofthecytoplasmicaggregatesthatincorporatebotheGFP-andtagRFP-fluorescencesignals;
(f)DuringcoexpressionwithRISP::tagRFP,eGFP::pUL50stilllocalizedtothenuclearrim, inpart
alsoinnucleus-associatedaggregates;(g)RISP::tagRFPlocalizedtoaggregatesinthevicinityofthe
nucleus;(i)BotheGFP::pUL50andRISP::tagRFPsignalsoverlappedtoasubstantialextentatnuclear
sites.Thedottedboxrepresentstheareamagnifiedin(j),inwhichthecolocalizationbecomesobvious,
arrowheadsindicateaggregatesofeGFP-andtagRFP-signalsthatmergeinassociationwiththenuclear
envelope;whitebarsrepresent10µm.