2015 Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus

2015·1.94 MB·English

VirusResearch208(2015)136–145

ContentslistsavailableatScienceDirect

Virus Research

journal homepage: www.elsevier.com/locate/virusres

Functional characterization and proteomic analysis of the

nucleocapsid protein of porcine deltacoronavirus

SunheeLee,ChangheeLee∗

AnimalVirologyLaboratory,SchoolofLifeSciences,BK21plusKNUCreativeBioResearchGroup,KyungpookNationalUniversity,Daegu702-701,

SouthKorea

a r t i c l e i n f o a b s t r a c t

Articlehistory: Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes
Receiv ed1May2015 enteritis anddiarrheain piglets.L ike o therco ronaviruses ,PDCoVcom monly contains4m ajor struc-
AReccceepivteedd i1n 5 rJeuvni see2d0 f1o5rm 11 June 2015 tural pro teins : spike (S ), e nvelope (E), m embr ane (M), and nu cleocap sid (N) prot eins. Amo n g these , the N
proteinisknowntobethemostabundantandmultifunctionalviralcomponent.Therefore,asthefirststep
Availableonline20June2015
towardunderstandingthebiologyofPDCoV,thepresentstudyinvestigatedfunctionalcharacteristicsand

expressiondynamicsofhostproteinsinastableporcinecelllineconstitutivelyexpressingthePDCoVN
Keywords:
protein.SimilartoNproteinsofothercoronaviruses,thePDCoVNproteinwasfoundtointeractwithitself
PDCoV
toformnon-covalentlylinkedoligomersandwasmainlylocalizedtothenucleolus.Wethenassessed
Nucleocapsid
Oligomerization alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time
Nucleolarlocalization pointsbymeansofproteomicanalysis.Accordingtotheresultsofhigh-resolutiontwo-dimensionalgel
Proteomic s electro ph oresis, at otalof43 proteins potswere in itial lyfoun d tobedifferentia llyexpressedin PK-

Cellularproteinregulation PDCoV-NcellsincomparisonwithcontrolPKcells.Ofthesespots,10proteinspotsshowedastatistically

significantalteration,including8up-regulatedand2down-regulatedproteinspotsandwerepickedfor

subsequentproteinidentificationbypeptidemassfingerprintingfollowingmatrix-assistedlaserdes-

orption/ionizationtime-of-flightmassspectrometry.Theaffectedcellularproteinsthatweidentifiedin

thisstudywereclassifiedintothefunctionalgroupsinvolvedinvariouscellularprocessessuchascell

division,metabolism,thestressresponse,proteinbiosynthesisandtransport,cytoskeletonnetworks

andcellcommunication.Notably,twomembersoftheheatshockprotein70familywerefoundtobe

up-regulatedinPK-PDCoV-Ncells.Theseproteomicdatawillprovideinsightsintothespecificcellular

responsetotheNproteinduringPDCoVinfection.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction 2014a,b;Wangetal.,2014).Furthermore,recentindependentstud-

iesdemonstratedthatPDCoVcancausesevereentericlesionsand

Porcinedeltacoronavirus(PDCoV)isanemergingviralpathogen clinicaldiarrheaintheabsenceofotherpathogensinneonataland

that was first reported in 2012 in Hong Kong, China (Woo gnotobioticpigs(Jungetal.,2015).Shortlythereafter,PDCoVwas

et al., 2012). In February 2014, the detection of PDCoV was first identifiedinKoreain2fecalsamplesindependentlycollectedin

announcedinOhio,UnitedStates,inconjunctionwithoutbreaksof AprilandJune2014,andeachofwhichwerepositiveforarotavirus

diarrheawithoutotheretiologicagents.Sinceitsemergence,this andnegativeforotherentericviruses(LeeandLee,2014).Genetic

novelcoronavirushasbeendetectedin17USstates,andalmost and phylogenetic analyses showed that these Korean strains are

80%ofthetestedsamplescorrespondedtocasesofcoinfectionof morecloselyrelatedtotheUSstrainsthantotheHongKongHKU15

PDCoV with other enteric viral pathogens such as a rotavirus or strains(LeeandLee,2014).

porcine epidemic diarrhea virus (Li et al., 2014; Marthaler et al., PDCoVisalarge,envelopedviruspossessingasingle-stranded,
positive-se n se RNAg enomeofa ppro ximately25 .4 kbwitha5(cid:3)cap
and a 3(cid:3) polya deny lated tai l, b elonging to th e ge nus Delt ac oron-

aviruswithinthefamilyCoronaviridaeoftheorderNidovirales(de
∗ Corresponding author at: School of Life Sciences, College of Natu- Groot et al., 201 1; Woo et al., 2012) . T he PDCoV genome c on-
ral Sciences, Kyungpook National University, Daegu 702-701, South Korea.
tains six common coronaviral genes in the following conserved
Tel.:E +-8m2a 5il3a d9d5r0e s7s3:6c5h;a fnagxh: e+e8@2k 5n3u 9.a5c5.k 5r5(C2.2L.ee). order : 5 (cid:3) untransl ated region (UTR) -O RF1a -ORF-1b-S -E-M-N-3(cid:3)

http://dx.doi.org/10.1016/j.virusres.2015.06.013
0168-1702/©2015ElsevierB.V.Allrightsreserved.
S.Lee,C.Lee/VirusResearch208(2015)136–145 137

UTR. ORF1a/b encompasses two-thirds of the genome encoding 70-kDa protein (HSC70), (cid:3)-actin, (cid:4)-tubulin, and Sp1 were pur-

2 overlapping viral replicase polyproteins, 1a and 1ab, which chasedfromSantaCruzBiotechnology(SantaCruz,CA).

arethenproteolyticallyprocessedintomaturenonstructuralpro-

teins.Asinothercoronaviruses,productionofpolyproteins1aand 2.2. ConstructionofthePDCoVNplasmid
1abre qu ire sa− 1ribosomalfra meshiftdu rin gtranslation of the

genomicRNA.Thelastthirdofthegenomeencodesthe4structural DNA manipulation and cloning were performed according

proteins,spike(S),envelope(E),membrane(M),andnucleocapsid to standard procedures (Sambrook and Russell, 2001). The
(N),aswe llast woa ccessory gen es,nonstruc tura lgen e6(NS6)and Es cherichia c oli strain D H5(cid:4) (RBC Bios cience, T aiwan) was

NS7gene,betweenMandN,andwithinN,respectively(Laietal., used as the host for general cloning. The full-length N gene

2007; Lee and Lee, 2014; Li et al., 2014; Marthaler et al., 2014a; was amplified from the PDCoV KNU14-04 strain (Lee and

Wooetal.,2012). Lee, 2014) with the following primer pair: KNU14-04-N-Fwd
Am o ng thestructuralproteinsofcoronaviruses,theNprotein (5(cid:3)-G CCGGT CGAC ATG GCTGCACC AGTAG- 3(cid:3))and KNU14-04-N-Rev
is abundan tly produced in infecte d cells and has m ult ip le func- (5(cid:3)-CGGCTCTAGACGCTGCTGATTCCTGC-3(cid:3)),w her eunderlinesindi-

tionsinviralreplicationandpathogenesis(McBrideetal.,2014). cate the SalI and XbaI restriction enzyme sites, respectively. The

Asthesolestructuralcomponentoftheviralcapsid,theNprotein PCR amplicon was initially inserted into the pBudCE4.1 vector

ofcoronavirusesinteractswiththenucleicacidanditselfforself- (Invitrogen)thatcontainsamycepitopeand6repetitivehistidine

associationtoprotecttheviralgenomefromextracellularagents, codons,andtheresultingplasmidpBud-PDCoV-Nwasverifiedby

servingasthecriticalbasisforribonucleoprotein(RNP)complexes nucleotidesequencing.BecausethePDCoVgenomecontainsone

duringvirusassembly(McBrideetal.,2014).Theentirelifecycle accessoryNS7genewithintheNgeneinadifferentreadingframe,

ofcoronavirusestakesplaceinthecytoplasmofinfectedcells,and thepresenceofNS7couldaffectourfunctionalstudiesofthePDCoV

accordingly, the N protein is distributed mainly in the cytoplas- Nprotein.Topreventanysuchsideeffects,thetranslationiniti-

mic compartments. In addition to their cytoplasmic localization, ation codon and the fifth codon of the NS7 ORF were modified

coronaviral N proteins are commonly localized to the nucleolus, to disrupt its expression by pBud-PDCoV-N. To accomplish this,

suggesting their non-structural functions in ensuring successful overlappingPCRwasconductedtosimultaneouslychangetheATG

virusinfection(Hiscoxetal.,2001;McBrideetal.,2014).Althougha startcodonandthefifthcodonoftheNS7genetoTTAandTAAat

varietyofstudieshaveconfirmedthatNpossessesmultifunctional genomicnucleotidepositions24,096–24,098and24,108–24,110,

significanceincoronavirology(McBrideetal.,2014),detailedchar- respectively, using pBud-PDCoV-N as a template with the fol-

acteristicsofthisproteinanditsroleinthereplicationofPDCoV lowingprimersfortheT24097C/T24109Amutation:NS7-KO-Fwd
remainun kn own. (5(cid:3)-GGC AAcGGA GT TCC GCTaAACTCCGCCAT C-3(cid:3)) and NS7-KO-Rev
In th e present study, alterations in cellular gene expression (5(cid:3)-GATGGCGGAGTTtAGCGGAACTCCgTTGCC-3(cid:3)) ,whe relowercase

that are caused by the N protein were evaluated as a first step letters indicate the mutated nucleotides. Both mutations were

towardunderstandingthebiologicalroleoftheNproteininPDCoV translationallysilentwithrespecttotheORFencodingtheNpro-
replicat ion.Toaccomp lish thistask,s tabl ep orc in e-origin ce lllines tein,andthere sultin gpla smidpB ud -PD CoV- Nw/oNS7w asv er ified
constitutive lye xpressingt heP DCoV Nprot einweregener ated and bynu cleo tide sequenci ng.Afrag mentofPDCoVNw/oNS7cD NAthat

characterizedinthisstudy.Changesinexpressionpatternsofvar- was prepared from pBud-PDCoV-N was then subcloned into the

iouscellularproteinsintheNprotein-expressingporcinecellsin pFB-Neoretroviralvector(Stratagene,LaJolla,CA)usingtheSalI

comparisonwithcontrolcellswereexaminedbyproteomicanal- andEcoRIrestrictionsitestoconstructthePDCoVNgeneexpression

ysisatdifferenttimepoints.Ourproteomicdataareexpectedto plasmidpFB-Neo-PDCoV-N-myc/HisthatproducesrecombinantN

providenovelinformationforbetterknowledgeoftheproperties protein.

andfunctionsoftheNproteinduringPDCoVinfection.

2.3. GenerationofstablePK-15celllinesexpressingthePDCoVN

protein
2. Materialsandmethods

The retrovirus gene transfer system (Stratagene) was used

2.1. Cellsandantibodies to generate cell lines constitutively expressing the recombinant

PDCoVNgeneoranemptyvectoronlyasdescribedelsewhere(Lee

HEK-293T cells (CRL-1573) were purchased from the Ameri- et al., 2010; Nam and Lee, 2010; Oh and Lee, 2012). Antibiotic-

can Type Culture Collection (ATCC, Manassas, VA) and cultured resistant continuous cell clones were analyzed by RT-PCR to

in Dulbecco’s modified Eagle medium (DMEM) with high glu- determinethepresenceofthefull-lengthNgene,andthepositive

cose(Invitrogen,Carlsbad,CA)with10%fetalbovineserum(FBS; clones(PK-PDCoV-NandPK-Neo)wereamplifiedforsubsequent
Invit rogen) and antibiotic– ant imyco tic s oluti ons (10 0×; In vitro- experim ents.

gen).PK-15cellsweregrowninRPMI1640medium(Invitrogen)

supplementedwith10%FBSandantibiotic–antimycoticsolutions 2.4. Immunofluorescenceassay(IFA)
(100×). The c ells w ere ma intai ned at 37◦C in an atm osphere

of humidified air containing 5% CO . PAM-pCD163-N cells that PK-PDCoV-Ncellsweregrownonmicroscopecoverslipsplaced
2

stably express the N protein of porcine reproductive and respi- in6-welltissuecultureplates.At48hpost-seeding,thecellswere

ratory syndrome virus (PRRSV; Sagong and Lee, 2010) were fixedwith4%paraformaldehydefor10minatroomtemperature

cultured in RPMI 1640 medium (Invitrogen) supplemented with (RT) and permeabilized with 0.2% Triton X-100 in PBS at RT for
10% FBS, an tibiot ic–ant imycotic solutions (1 00×), 10mM H EPES 10m in. T he cells were block ed us ing 1% bovine se rum al bum in

(Invitrogen), 1mM sodium pyruvate (Invitrogen), and nonessen- (BSA)inPBSfor30minatRTandthenincubatedwithananti-Histag
tial amino ac id s (1 00×; Inv itrogen) in the presen ce o f 50(cid:2)g/ml orant i-m yc ant ibo dyfo r 2h .Aft erwa shing5tim esin P BS,thec ells
Zeo cin (Inv itroge n) and 200(cid:2)g/ml G4 18 ( Invitrogen ). The anti- we reincuba tedfor1h at R Tw itha goatanti -m ouse se cond ary anti-

PRRSVnucleocapsid(N)monoclonalantibody(MAb)andanti-myc bodyconjugatedwithAlexaFluor488(MolecularProbes,Carlsbad,
MAbw erepurchased fro mMEDIAND iagnostic s(Chun che on,South CA).T hecellswe refin allyc ounte rsta inedwith4 (cid:3),6-diam idino-2-
Korea ) and Invitroge n, re spectivel y. Antibodie s to the 6× histi- phen ylin dole (DAPI; Sigma ,St.Louis,MO),a ndth ecellstainingwas

dinetag,glucose-regulatedprotein78(GRP78),heatshockcognate visualizedbythefluorescenceLeicaDMILLEDmicroscope(Leica,
138 S.Lee,C.Lee/VirusResearch208(2015)136–145

Wetzlar,Germany)oraConfocalLaserScanningmicroscope(Carl 48h.Forcross-linkingstudies,thecellswerewashedtwicewith

Zeiss,Gattingen,Germany). coldPBSandthenincubatedatRTfor30minwitha2mMsolution
of a mem bra ne p ermeable a nd th iol -cle ava ble cr o ss -link er, 3,3(cid:3)-

2.5. Fluorescence-activatedcellsorting(FACS)analysis dithiobis(succinimidylpropionate)(DSP;Pierce),dissolvedin10%

dimethylsulfoxide(v/vinPBS).Thereactionwasquenchedwith

The N expression in PK-PDCoV-N cells was analyzed by flow 50mMTris–HCl[pH7.5]andincubatedforanadditional15min.

cytometry. The cells were trypsinized at 48h post-seeding and Thecellswerelysedwithlysisbuffer,andtheresultantcelllysates
centrifuged at 2 50× g (Han il Centrifug e F LET A 5) for 5min. The wer ethe nsub jected toa West ernblo tass aya sdescribe da bove.

cell pellet was washed with cold washing buffer (1% BSA and
0.1% sodiu maz ideinPB S),an d106 cellswer eresus pen dedi n1%
form aldehyd esolu tio ninco ldw ashbuffe rforfi xationat4◦C in the 2.9. Preparationofproteinsamplesforproteomicanalysis

darkfor30minfollowedbycentrifugationandincubationofthe
pelle tin 0.2 %Tr itonX-10 0i nPBSat37◦C for1 5minforp erm e- Pellets of cultured cells were washed twice with ice-cold

abilization.Aftercentrifugation,thecellpelletwasresuspendedin PBS, placed in sample lysis solution consisting of 7M urea,

asolutionoftheprimaryanti-Histagantibodyornormalmouse 2M thiourea, 4% 3-[(3-cholamidopropyl) dimethyammonio]-1-

IgG1(SantaCruzBiotechnology)andthemixturewasincubatedat propanesulfonate(CHAPS),1%dithiothreitol(DTT),2%pharmalyte
4◦Cf or30m in.T hecellswerewa shed an dallowe dtor eactwitha n (pH3.5–10,Amer shamBio scie nces),and1m Mben za midine,and

AlexaFluor488-conjugatedanti-mouseIgGsecondaryantibodyat were then sonicated for 10s using a Sonoplus device (Bandelin
4◦Cfo r30m ininthedark.Th estainedce llsw erewashe dagainan d Electr onic, Germany) . Th e sa m ples we re subseq uently incubated
anal yze do nth e BD FACSA ria IIIflow cyto meter (BDBio scien ces, for 1h at 4◦C. After c entr ifugation at 1 5,000×g for 1 h at 4◦C,

Belford,MA). insoluble cellular debris were discarded, and the supernatant
was colle cted and stored at − 80◦C until use. The protein con-

2.6. Cellproliferationassay centrations were measured by the Bradford assay as described

previously(Bradford,1976).

ThegrowthpropertiesofcellsexpressingPDCoVNproteinand

controlcellsweredeterminedbya3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) detecting 2.10. Two-dimensionalgelelectrophoresis(2DE)andimage

cellviabilityasdescribedpreviously(KimandLee,2013).AllMTT analysis

assayswereperformedintriplicate.

Proteinsampleswereanalyzedby2DEusingcommercialIPG

2.7. CellfractionationandWesternblotanalysis drystrips(pH4–10,24cm;GenomineInc.,Pohang,SouthKorea)

forthefirst-dimensionalseparation(isoelectricfocusing;IEF)and

WholecelllysateswerepreparedfromPK-PDCoV-Ncellsgrown sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-
at 5×105 cell s/well in 6-w ell tissu e cul ture plates a t ind icated PAGE)f orthese conddimension.TheIP Gd rystripswerel oaded
tim e p oints using ly sis buffer as desc ribed p revious ly (Nam and indepe nde ntly with20 0(cid:2)gofeach pro tein sam plean drehy drated

Lee, 2010). For cell fractionation, PK-PDCoV-N cells were frac- overnightinrehydrationbuffer(7Murea,2Mthiourea,2%CHAPS,
tion ated us ing the N uclear/Cytoso l Fractionatio n Kit (BioV ision, 1% DTT, a nd 1% pharma lyte). IEF w as pe r for med at 2 0◦ C using

Mountain View, CA) according to the manufacturer’s manuals. a Multiphor II electrophoresis unit and an EPS 3500 XL power

The protein concentrations of the cell lysates were determined supply (Amersham Biosciences) with the following parameters:

usingtheBCAproteinassay(Pierce,Rockford,IL).Thecelllysates gradientincreasefrom150to3500Vfor3hand3500Vforatotal
were mix ed w ith 4× NuPA GE sam ple buffe r (I nvit roge n) and of96,000 Vh.Foll owin gIEF se parat io n,t h e IPG strips w ere i ncu-
boiled at 70 ◦C for 10 min. Equa l amoun ts of t otal protein w ere ba tedfor 10 m inatRTw ith gentleshak ingi neq uilibra tionb uffer

separated in a NuPAGE 4–12% Gradient Bis-Tris Gel (Invitrogen) (50mMTris–ClpH6.8,containing6Murea,2%SDS,and30%glyc-

undernon-reducingorreducingconditions,andelectrotransferred erol)containing1%DTTandweresubsequentlyincubatedinthe

onto an Immobilon-P membrane (Millipore, Billerica, MA). The equilibrationbuffercontaining2.5%iodoacetamideunderthesame

membranes were blocked with 3% powdered skim milk (BD conditions.Eachequilibratedstripwastheninsertedinto10–16%
Biosciences) inTBS (10mM Tris–H Cl[ pH8.0],15 0mM NaCl) with polyacrylam ideg els(20cm× 24cm )an dSDS -PAGEw asru nusing
0.05%Tween -2 0(TB ST) at4 ◦Cfor2h and react edw itht hepri mary a Hoefer DALT 2D s yst em (A m ersh am Biosciences ) at 20◦ C for
antibo dy agains t the 6 × His -tag , G RP7 8, HSC7 0, or (cid:3)- actin at 1 700Vh accord ing tothem anufacturer’ sinstructions .Th e2Dg els
4◦Cover night.Th ebl otsw erethen incuba tedwith ah orserad ish were st ai nedbythe si lver stainingmetho dasdescribed pre vio usly

peroxidase(HRP)-labeledgoatanti-mouseIgGorgoatanti-rabbit withsomemodifications(Oakleyetal.,1980).Tocompensatefor

IgGantibody(SantaCruzBiotechnology)atthedilutionof1:2000 thevariabilityof2DE,threeindependentexperimentswerecon-
for 2h at 4◦C . Final ly, th e proteins were v isua lized by en hanced duc tedforfurt he rstat istical analysis.

chemiluminescence (ECL) Reagents (Amersham Biosciences, Pis- Thestainedgelswereimagedusingahigh-resolution2DgelCCD

cataway,NJ)accordingtotheinstructionsofthemanufacturer.To imageanalyzer,Dyversity(Syngene,Frederick,MD),andquantita-

analyzetheexpressionkineticsofcellularproteinsinPK-PDCoV-N tiveanalysisofthedigitizedgelimageswascarriedoutusingthe

cells,thebanddensityofeachproteinwasquantifiedrelativeto PDQuestsoftware(version7.0,Bio-Rad,Hercules,CA)accordingto
(cid:3)-act inu singd ensitom etr ywit htheW righ tCellImag ingFacil ity theproto colsprov idedby the manufac turer.Dat are presenting 3

(WCIF) version of the ImageJ software package (http://www. independent2DEexperimentsforeachsamplewerestatistically

uhnresearch.ca/facilities/wcif/imagej/). analyzedusingStudent’st-test,andp-valuesoflessthan0.05were

consideredtobestatisticallysignificant.Thequantitativedatafrom

2.8. Chemicalcross-linking eachproteinspotwerenormalizedbasedonthetotalvalidspot

intensityforeachgel.Onlyproteinspotsthatshowedsignificant
A chemical cross-linking assay was performed as described differenti ale xpres sion (p<0 .05)wit ha±2 -fold consist entchange

previously(LeeandYoo,2006).Briefly,PK-PDCoV-NcellsandPAM- intheexpressionlevelincomparisonwiththecontrolsamplewere

pCD163-N cells were grown in a 6-well tissue culture plate for selectedformassspectrometry(MS)analysis.
S.Lee,C.Lee/VirusResearch208(2015)136–145 139

2.11. Proteinidentificationbypeptidemassfingerprinting(PMF) (Woo et al., 2012). PDCoV is the fifth coronavirus infecting pigs

in addition to transmissible gastroenteritis virus, porcine respi-

Eachselectedproteinspotwasexcisedmanuallyfromthesilver- ratory virus, porcine hemagglutinating encephalomyelitis virus,

stainedgelsandwasenzymaticallydigestedin-gelaspreviously andporcineepidemicdiarrheavirus(PEDV).Todate,interactions

described (Shevchenko et al., 1996) and using modified porcine between this novel virus and the host have not been studied

trypsin(Sequencinggrade;Promega,Madison,WI).Thegelpieces yet. Furthermore, alterations of cellular protein expression in

werewashedwith50%acetonitrile(ACN),air-driedthoroughlyat responsetoPDCoVoreachviralproteinuponinfectioncurrently
RT,an dthenr ehydr ated for8–10ha t37◦C withmod ifiedsequen c- remain u nd etermin ed . In t he cu rrent s tudy, the PDCo V N pro-
ing grad epor cinetrypsin (8– 10ng / (cid:2)l ).The prote olyticrea ctionwas tein wa s chosen in the cu rren t study for the pro teomic a na lysis
term inate d by ad dition of 5(cid:2) l of 0.5 % tri fluoroaceti c acid (T FA). to e valua te spec ific ho st cellul ar res pon ses, which is n ot only

The tryptic peptides were recovered by combining the aqueous the most abundant viral component but also performs several

phase from several extractions of gel pieces with 50% aqueous biological functions in completing viral replication. For this pur-

ACN.Afterconcentration,thepeptidemixturewasdesaltedusing pose, sublines of PK-15 cells were established to stably express
C Zi pTips (Millipore),an dth epeptid eswere elut edin1–5 (cid:2)lof recom binant P DC oV N unde r the control of a r etrovir al long-
18

50%ACN.Analiquotofthissolutionwasmixedwithanequalvol- terminal-repeat (LTR) promoter. Ten generated cell clones were
ume of a sat urated sol utio n of (cid:4)-cy ano -4-hyd roxyc inn amic acid initially collecte d and subjected to RT -PCR and Wes tern blo tting
(CHC A; Si gma)in50 %ACN/0 .1% TFA,and1(cid:2)lofthismixture was to confi rm the N gen e expressi on at mRN A an d protei n levels,

immediatelyspottedontothetargetplate.MALDI-TOFanalysiswas respectively(datanotshown).AccordingtotheresultsoftheWest-

performedonaMicroflexLRF20instrument(BrukerDaltonics,Bil- ern blot analysis, one PK-PDCoV-N cell clone that constitutively

lerica, MA) as described by Fernandez et al. (1998). The spectra expressed the highest levels of N was selected for subsequent

were collected from 300 shots per spectrum over the m/z range studies.

600–3000 and calibrated by two point internal calibration using To characterize PK-PDCoV-N cells, we examined intracellular

trypsinauto-digestionpeaks(m/z842.5099,2211.1046).Thepeak expressionlevelsofNbyIFA,FACSanalysisandWesternblotting.As

list was generated using Flex Analysis 3.0. We used the follow- showninFig.1A,thespecificcellstainingwasclearlyevidentwhen

ing threshold for peak-picking: 500 for minimum resolution of PK-PDCoV-N cells were reacted with the anti-His tag antibody,

monoisotopicmass,5forS/N.ThesearchprogramMASCOTfrom confirming the constant high expression level of the N protein.

MatrixScience(http://www.matrixscience.com/)wasusedforpro- Furthermore,themajorityofthecellsconsistentlyexhibitedspe-

tein identification by PMF. The following parameters were used cific fluorescent signals, indicating a homogenous population of

forthedatabasesearch:trypsinasthecleavingenzyme,amaxi- cellsintermsofNexpression(Fig.1B).Time-courseWesternblot

mumofonemissedcleavage,iodoacetamide(Cys)asacomplete analysis revealed that the PK-PDCoV-N cells stably express and
modifi ca tion ,oxidat ion(Met) asapartialmo dificat ion , monoiso- accumul ate robus t lev els of a ∼45kDa reco mbinan t N pro tein,
topicmasses, andamas stolera nc e of±0.1 Da.ThePMFa cceptance largerthani tspredi ctedm ole cu larw eigh tofapproxima tel y38kDa

criterionwasprobabilityscoring. possiblyduetopost-translationalmodificationsandthepresence

of C-terminal myc and histidine tags (Fig. 1C). In addition, the

2.12. Quantitativereal-timeRT-PCR overallgrowthkineticsofPDCoVNgene-expressingPKcellswas

found to be similar to that of the parental PK-Neo cells, indicat-

TotalRNAwasextractedfromthelysatesofPK-PDCoV-Ncells ingthatthePDCoVNexpressionhasnoeffectoncellproliferation

at24and48hpost-seedingbytheTRIzolReagent(Invitrogen)and (Fig.1D).

wastreatedwithDNaseI(TaKaRa,Otsu,Japan)accordingtothe Self-association of the N protein has been observed in many

manufacturer’sprotocols.TheconcentrationsoftheextractedRNA viruses,includingcoronaviruses,andisessentialforassemblyofthe

weremeasuredusingaNanoVuespectrophotometer(GEHealth- viralcoreconstructingthebasicarchitectureofviruses.Sequence

care,Piscataway,NJ).Quantitativereal-timeRT-PCRwasperformed analysisofthePDCoVNproteinindicatedthatitiscomposedof

usingaThermalCyclerDiceRealTimeSystem(TaKaRa)withgene- 342 amino acids residues and contains no cysteine residues. As

specificprimersetsasdescribedpreviously(SagongandLee,2011). expected, no band corresponding to a disulfide-linked N dimer

The primer sequences are available upon request. The RNA lev- wasdetectedbySDS-PAGEundernon-reducingconditions(Fig.2A,
els o f cellul ar genes w ere normalize d to p orcine (cid:3) -act in mR NA, lane 4), indic ati ng that th e PDC oV N protein does not u nde rgo

and relative quantities (RQ) of mRNA accumulation were calcu- cysteine-linkedhomodimerization.Asapositivecontrol,thePRRSV
lated usingt he2−(cid:2)(cid:2)Ct meth od (Livak andSchmittge n,200 1).To Nproteininthe N-expressingstable PA M cellsw asclear lyd emon-

detect alterations of cellular mRNA levels in the presence of the strated to form 35-kDa N-N dimers under the same conditions

PDCoVNprotein,therelativefoldchangeofeachcellulargenewas (Sagong and Lee, 2010; Fig. 2A, lane 2). The ability of N to form

calculatedinaccordancewiththecomparisonofPK-PDCoV-Nand non-covalentdimerswasfurtherinvestigatedinachemicalcross-

controlPK-Neocells. linkingexperiment.AsshowninFig.2B,thePRRSVNproteinin

PAM-pCD163-Ncellsformedanumberofhigher-orderoligomers

2.13. Statisticalanalysis (lane 1) as reported previously (Wootton and Yoo, 2003). When

the N protein in PK-PDCoV cells was subjected to cross-linking,

We used the Student’s t-test for all statistical analyses, and numerousmultimericformsoftheNproteinwereidentified(lane

differenceswithp-values<0.05wereconsideredstatisticallysig- 2),indicatingthattheNproteinofPDCoVexistsintheformofnon-

nificant. covalently linked oligomers that are used for assembly the viral

capsid.
3. Results We next determined whether the recombinant N protein

expressedinPK-PDCoV-Ncellsissubjecttonucleolarlocalization

3.1. Generationandcharacterizationofstableporcine-origincell thatisknowntobeacommonfeatureofcoronaviralNproteins.The

linesexpressingthePDCoVNprotein stainingpatterninPK-PDCoVcellswasfoundtobepredominantly

cytoplasmicandnucleolic;thispatternpersistedforupto60hafter

Asetofsevennovelmammalianandaviancoronaviruseswas seeding(Fig.3A).NearlyallcellsexpressingPDCoVNshoweddis-

recently discovered, including one porcine coronavirus, PDCoV tinctfluorescentsignalsinthenucleolusat48hpost-seeding,and
140 S.Lee,C.Lee/VirusResearch208(2015)136–145

Fig.1. ConstitutiveexpressionoftheNproteininPK-PDCoV-Ncells.(A)ImmunofluorescenceassayofthePDCoVNprotein.PK-PDCoV-Ncellsgrownina6-welltissueculture

platewerefixedwith4%formaldehydeatindicatedtimepointsandincubatedwiththeHistag-specificantibodyfollowedbygoatanti-mousesecondaryantibodyconjugated
with Alexa green (up per panels).Thec el lswereth enco unters tain edwithDA PI(m idd lep anels)and examined usinga flu oresc entmicrosc opeat400 ×magnifi cation.(B)

IntracellularexpressionofPDCoVN.Onemillioncellswereharvestedat48hpost-seedingandincubatedwithanti-Histagantibody(grayhistogram)oranisotypecontrol
(whitehisto gram)anda na lyzedb y flow cytome try.( C)Im munoblot an aly si softheNpr otein .PK-PDCo V-N cellswer egr ownina 6-we lltissuecu ltu rep lateat 4×105

cells/wellfor6,12,24,36,and48h.CelllysateswerepreparedattheindicatedtimepointsandsubjectedtoWesternblotanalysiswithanti-Histagantibodytodetermine
theexpre ssio nl eve lof the Np rote in (up perpan el).T heblotwa s also reactedw ithm ouseM Ab against(cid:3)- act intocon firm equalpr otein loading (low erpanel) .(D ).Growth

kineticsofthestablePDCoVNproteinexpressingcells.CellproliferationwasmeasuredattheindicatedtimesbytheMTTassay.Valuesarerepresentativeofthemeanfrom

threeindependentexperimentsanderrorbarsdenotestandarddeviations.

thereafter,thePDCoVNproteinwaslocalizedmainlytothecyto- 3.2. 2DEanalysisofporcinecellsconstitutivelyexpressingthe

plasmat72hpost-seeding.ThenucleolarlocalizationofPDCoVN PDCoVNprotein

wasconfirmedbytransienttransfectionofBHK-21orSTcellswith

theplasmidpBud-PDCoV-N(Fig.3B).Thecellfractionationassay Cellular proteins in the parental PK-Neo cells and PK-PDCoV-

alsorevealedthepresenceoftheNproteininboththecytoplasmic Ncellswereextractedandsubjectedto2DEanalysistocompare

and nucleic fractions (Fig. 3C). These observations demonstrated the host protein expression profiles. To reduce the variability of

that, like N proteins of other coronaviruses, PDCoV N protein is gel electrophoresis, three independent 2DE analyses of cellular

mostlydistributedinthenucleolusalongwiththecytoplasmand extracts from control or N gene-expressing PK cells were per-

hasaconservedsubcellularlocalizationproperty. formed,andspotintensitydatafromtriplicategelswereselected
forstati stica lana lysis.On avera ge,1 090±63p rote inspo tswere

resolved by 2DE within a pH gradient 4–10 and were visualized

using silver staining; the molecular weights of the spots ranged

from 10 to 200kDa. These spots were used for the comparative

analysis.Fig.4Ashowsrepresentativeimagesof2DEgelsfromcon-

trolPKcells(upperpanel)andNgene-expressingPKcells(middle

andlowerpanels).Atotalof43proteinspotswereinitiallyfound

tobedifferentiallyexpressedinPK-PDCoV-Ncellswhencompared

withcontrolPK-Neocells.Onthebasisofthestatisticalcomparison,

onlythosespotsthatconsistentlyshowedalterationinexpression

levelsbetweenPK-PDCoV-Ncellsandthecontrolcellswerechosen

forfurtherproteinidentification.

3.3. Identificationofthedifferentiallyexpressedproteins

To identify the differentially expressed cellular protein spots

in PK-PDCoV-N cells at different time points, 10 protein spots

withastatisticallysignificantalteration,including8up-regulated

and 2 down-regulated protein spots (Fig. 4B), were selected
Fig.2. Homo-oligomerizationofthePDCoVNprotein.(A)Absenceofthedisulfide-
and manually excised from the stained gels. Subsequently, the
linkedNhomodimers.CelllysateswerepreparedfromPAM-pCD163-NandPK-
PDCoV -N cellsgrownfo r48 handsu bjecte dtoWest ernblo tanalysisinthe prese nce trypsin-digestedspotsweresubjectedtoMALDI-TOFanalysis.With
(lanes1a nd3) orabs enc e( la nes2 and4)of (cid:3) -mercapt oeth anol((cid:3)M E ).M onomers combinedPMFa ndda tabas esearchin g, identityofa ll10pro teins
(N)an d dimm e rs (2N)ofth ePRR SV Np ro tei nareindicatedaspo sitivec ontrols.(B) wassucce ssfull yde termined .Informatio nonall th ese pr oteinsin
Np rote inoligom eriza tio nby DSPc ro ss-linkin g.E achcellli ne express ingPRRSV N
PK-PDCoV-Ncells,withtheirproteinscoresandsequencecoverage,
(lane1)orPDCoVN(lane2)wasindependentlycross-linkedwith2mMDSPfor
issummarizedinTable1.Tobetterunderstandtheimplications
n30o nm-rined. uCceilnl glycsoantedsi twioenrse. Mpruelptiamreedri canfodr msusbojfeNctepdro ttoe iWnseosftebront hbvloirtu asneaslayrseiss huonwdenr. of the cellular re spons es to the PD CoV N pro tein , we further
S.Lee,C.Lee/VirusResearch208(2015)136–145 141

Fig.3. NucleolarlocalizationofthePDCoVNproteinexpressedinPK-PDCoV-Ncells.(A)SubcellularlocalizationofNincellsstablyexpressing(A)andtransientlyexpressing

(B)PDCoVN.Cellswerefixedatindicatedtimepointspost-seedingorpost-transfectionandincubatedwithanti-Hisoranti-myctagMAbfollowedbyAlexagreen-conjugated
goa tanti-m o usese cond aryan tib ody.The cellsw erevi sualizedusing a fluorescentmicro scop eat400×m agn ification .Nu cleus(Nu )a ndnu cleolus(N o) arein dicatedbyarrows.

(C)NuclearandcytoplasmicfractionationofPKcellsexpressingPDCoVN.EachnuclearandcytosolicfractionwaspreparedfromPK-PDCoV-Ncellsatindicatedtimepoints
pos t-seedin gan dsubjected toWesternb lot an alysis withthea ntibody s pecifi cforHis -tag (toppan el),Sp1 asa nuclearp rotein marker(sec ondp an el),or(cid:4)- tubul inasa
cytosolicprot einm arker(th ird panel).A llblo tswere alsor eact edwith(cid:3) -actinan tib odytov erify equalp rote in lo ading(b ottomp anel).

categorizedtheidentifiedproteinsbybiologicalprocessesaccord- We were able to detect mRNAs corresponding to nearly all pro-

ingtotheGeneOntologydatabaseasdescribedpreviously(Zhang teins identified by proteomics, except for histone H4. Although

et al., 2009). These proteins showing altered expression were the altered expression levels of the remaining 9 proteins were

associated with various cellular functions including intracellu- consistent with the real-time RT-PCR results, we observed only

lar transport, metabolic processes, gene regulation, the stress modestincreaseorreductioninmRNAlevels.Theseresultscould

response,proteinsynthesis,cytoskeletonnetworks,andcelldivi- beexplainedthecasethecorrelationbetweenmRNAandprotein

sion.Sincechangesincellularproteinexpressionmaybeattributed abundancecouldbeinsufficienttopredictproteinexpressionlev-

to alterations in the corresponding mRNA levels, transcriptional elsfromquantitativemRNAdata(Gygietal.,1999),suggestingthat

changes for all the identified proteins were tested by real-time thedifferencesinproteinlevelsmightbeduetopost-translational

RT-PCR to confirm the results of the proteomic analysis (Fig. 5). modificationsorproteinstabilityratherthanmRNAlevels.

Table1

ListofdifferentiallyexpressedcellularproteinsinPDCoVN-expressingPKcells.

Spotno. Proteinname Accessionno. MW(kDa) pI Protein Sequence Function

score coverage(%)

Down-regulatedproteins
3006 Tran slocon-associatedprotein gi|545887405 19.017 5.49 107 38 ProteintransporttotheER

subunitdeltaisoform
4009 Ferritin gi|346421372 20.165 5.75 141 65 Metabolicprocess

Up-regulatedproteins
0019 H istoneH4 gi|354480096 15.032 11.02 177 53 Generegulation
1627 78kDag luc ose-regulated gi|466006301 72.449 5.06 163 35 Stress response

protein
1636 Heatshock70kDaprotein8 gi|345441750 71.050 5.37 281 36 Stressresponse
5735 Ezrin gi|744614044 67.571 6.79 186 44 Linksa ctintotheplasmamembrane
6004 Perox iredoxin-5, gi|47523086 17.484 5.71 126 42 Metab olicp ro cess

mitochondrial
6108 Phosphoglyceratemutase1 gi|4505753 28.9 6.67 222 62 Metabolicprocess
6310 Septin2 gi|34509096 9 41.8 13 6.19 215 51 Cytokinesi s
7733 Elonga ti onfactor2 gi|335282386 96.262 6.41 351 47 Proteinsynthesis
142 S.Lee,C.Lee/VirusResearch208(2015)136–145

Fig.4. Representativegelimagesshowingspotsthatdisplayedsignificantlydifferentialexpression.(A)Themagnifiedpicturesrepresenttheproteomicanalysisofcontrol

PK-Neo(upperpanel)andPK-PDCoV-Ncells(middleandlowerpanels)andexhibiteddifferentiallyregulatedproteinsidentifiedinNgene-expressingcells.Four-digit

numbersonthetoprepresentindividualspotidentification.(B)Thequantityofeachspotwasnormalizedbasedonthetotalvalidspotintensityforeachgelandtherelative

foldchangeofeachspotwasthencalculatedbetweencontrolPK-NeoandPK-PDCoV-Ncells.Datarepresentingthreeindependent2DEexperimentsforeachsamplewere

statisticallyanalyzedanderrorbarsrepresentstandarddeviations.*P=0.001–0.05.

3.4. Alteredexpressionoftheheatshockprotein70(HSP70) expressionofthesestress-responseproteinsundertheinfluenceof

familyinresponsetothePDCoVNprotein PDCoVN,weperformedtime-courseWesternblotanalysis.Total

cellularlysateswerepreparedfromPK-PDCoV-Ncellsatdifferent

TwomembersoftheHSP70family,glucose-regulatedprotein timepoints,andtheexpressionkineticsoftheGRP78andHSC70

78(GRP78)andheatshockcognate70-kDaprotein(HSC70),were wascomparedbetweencontrolandNprotein-containingextracts.

foundtobeup-regulatedinthePDCoVN-expressingcellsbythe HigherexpressionofbothproteinswasfirstevidentinPK-PDCoV-

proteomic analysis. To further verify the dynamic alterations in Ncellsasearlyasat12hpost-seedingincomparisonwithcontrol

PK-Neocells,andtheproductionofbothproteinswaspersistently

highduringthelatertimepoints,suggestingthattheirenhanced

expression is dependent on the N protein (Fig. 6). These results

wereconsistentwiththeproteomicanalysisof2DEgelsandreal-

timeRT-PCR,demonstratingthatthesynthesisofGRP78andHSC70

isindeedup-regulatedinresponsetoexpressionoftheNprotein

ofPDCoV.

4. Discussion

Sincevirusesareobligateintracellularparasites,theymustuti-

lize the cellular machinery and biosynthetic components of the

hostcellfortheirownreplication.Afterviralinfection,theinfected

cellslaunchanumberofhostantiviraldefensiveresponses,which

are turned on to eliminate the invading viruses. On the other

hand, viruses employ their own evasion strategies to complete

their replication and to successfully spread to neighboring cells.

This series of interactions between the virus and host results in

compensatorymodificationsincellulargeneproduction.Accord-

ingly,numerousstudieshavebeenextensivelyconductedbymeans

of various molecular and genomic methods to understand how

Fig.5. TranscriptionalalterationofidentifiedcellulargenesinPK-PDCoV-Ncells. the altered host gene expression affects viral infection and the
The mR NAlevelofeach genewasa sse ssedbyqu antitativ ereal- tim eRT-PCRand nor- asso ciatedp atho genic processes.T heprot eomic analysis coup ling
mal izedto that of porc ine(cid:3) -acti n.Relativ eq uantities(RQ )ofmRN Aaccum ula tion high-resolu tion2DEan dMALDI-T OF/M Sisatool thatcan generate
wereeva lu ated by 2−(cid:3)(cid:3)Ctm ethoda ndthere lativefoldc hang eo feach genewasthen
ampledataoncellularproteinprofilesmodifiedbyviralreplication.
calculatedbetweencontrolPK-NeoandPK-PDCoV-Ncells.Resultsareexpressedas
Withtheproteomictechniques,itisnowpossibletoidentifyrel-
rthepe rmeseeannt svtaalnudesa rfdrodmev tihatrieoen isn.d*Pep=e0n.0d0e1n–t 0e.x0p5e;r†iPm<e0n.0ts0 i1n. duplicate and error bars ative cha ngesinpro teinabunda nc ef orev aluation of hostcell ular
S.Lee,C.Lee/VirusResearch208(2015)136–145 143

wasfoundtobepredominantlypresentinboththecytoplasmand

nucleolusofPK-PDCoV-Ncells.ThelocalizationofnidovirusNpro-

teinstothenucleolusmaybenecessaryforcontrolofRNAsynthesis

orribosomebiogenesisviaassociationwithribosomalsubunitsand

interaction with nucleolar proteins (Chen et al., 2002; Lee et al.,

2006;Wurmetal.,2001;Yooetal.,2003;).Similarly,thePDCoV

N protein may participate in such a viral strategy to favor viral

replication and pathogenesis. In addition, the nucleolar localiza-

tionsequencedetectorprogram(http://www.compbio.dundee.ac.

uk/www-nod/)predictedthatPDCoVNharborsaputativenucleo-

larlocalizationsignal(NoLS)consistingofastretchofbasicamino

acids.Therefore,furtherresearchisneededtoidentifyafunctional

NoLStoconfirmtheintracellularlocalizationofthePDCoVNpro-

tein. These biological characteristics of N gene-expressing cells

haveyettobeconfirmedusinganauthenticNproteininPDCoV-

infectedcells,butaccordingtoourresults,PDCoVNappearstoact

asamultifunctionalproteinplayingstructuralandnon-structural

rolescontributingtocompletionofviralreplication.Ourproteomic

datarevealedthat10differentiallyexpressedcellularproteinsare

identifiableinPDCoVNgene-expressingporcinecells.Theproteins

thatweidentifiedinthisstudyareinvolvedindiversecellularpro-

cesses:metabolism,thestressresponse,proteinbiosynthesisand

transport,cytoskeletonnetworksandcellcommunication,andcell

division.Thesignificanceofthefunctionalrolesoftheselectedhost

proteinsaffectedbytheinteractionofthePDCoVNproteinwiththe

hostcellisdiscussedbelow.

The most interesting finding in the present study is that two

cellular chaperone proteins belonging to the HSP70 family are

up-regulatedconcomitantlybytheNproteinofPDCoV.Thefirst

up-regulatedproteinisGRP78incellsexpressingthePDCoVNpro-

tein,whichisassociatedwithendoplasmicreticulum(ER)stress.In

eukaryoticcells,theERisthemajorsiteforsynthesisandfolding

oftransmembraneandsecretedproteins,andaccordingly,animal

viruses also use the ER as a site of synthesis and processing of

theirownproteins.TheamountofproteinenteringtheERcandif-

fer under physiological and environmental conditions. If protein

synthesis exceeds the folding capacity of the ER, unfolded pro-

teinsaccumulatethere,resultinginERstress.TomaintaintheER

homeostasis,cellshavedevelopedasignalingpathwayknownas

theunfoldedproteinresponse(UPR)thattransmitssignalsacross

the ER membrane to the cytosol and the nucleus and ultimately

Fig.6. DifferentialexpressionoftheHSP70familyinPK-PDCoV-Ncells.Celllysates reduces protein translation and enhances the ER folding capac-
wer ep reparedfrom PDCoVNg en e-e xpress ingPKc el lsatindicated time poi ntsand itybyu p-regula tingchaper onep roteins(R ona nd Walter, 2007).

immunoblottedtodeterminetheexpressionprofileofeachproteinwithantibodies
CoronavirusinfectionofculturedcellsisknowntocauseERstress
specificforGRP78andHSC70(toptosecondpanels).Theblotwasalsoreactedwith
anti-His tag (third pane l)and anti- (cid:3)- actin(b ottompa nel )ant ibod iesto confirm the andtoinducetheUPR,whichthencrosstalkswithvariouscellu-
statusof Ne xpress ionan deq ualproteinlo ading,re spectiv ely.Eachc ell ularprot ein lars ign alingpa thw ays,i ncludin gmi togen-activ ated proteink inase

expressionwasquantitativelyanalyzedbydensitometryintermsoftherelative cascades, autophagy, apoptosis, and innate immune responses,
density val ue to the (cid:3)-actin ge ne and PK -PD CoV-N sample re sults w er e co mpared indicating theinvolve mentofUP Rac tivation invirus– hostinter-
toPK-controlresults.Valuesarerepresentativeofthemeanfromthreeindependent
ex periments a nd erro r bars d en ote standard de vi atio ns. *P = 0.00 1–0.0 5;†P < 0.001. 2ac0t1io4n).sM aonrde ivnirtaelr epsatitnhgolgye,ngelosbisa l(Fpurontge oemt aicl.a, n2d01m4i;c rFouanrgra aynadn aLliyu-,

seshaveshownthattheexpressionofchaperonproteins,suchas

responsestoviralinfectionorviralproteinexpressionandtogain GRP78andGRP94,isup-regulatedincellsinfectedwithahuman

specific insights into the cellular mechanisms involved in viral coronavirusorincellsexpressingtheS2subunit(Jiangetal.,2005;

pathogenesis (Alfonso et al., 2004; Brasier et al., 2004; Neuman Yeungetal.,2008).Furthermore,theNproteinofPEDV,another

etal.,2008;OhandLee,2012;Ringroseetal.,2008;Sagongand porcinecoronavirus,overexpressionwasshowntotriggerERstress

Lee, 2010; Zhang et al., 2008). In the present work, a proteomic (Xuetal.,2013).Thus,itappearsthatPDCoVinfectionmayinduce

approachwasappliedforprofilingtheglobalproteinexpression ER stress and the UPR, leading to the up-regulation of GRP78 to

changesinhostcellsinresponsetotheNproteinofPDCoV,which counteract the ER stress; hence, the N protein may be responsi-

isamajorconstituentofthevirionandinfectedcells.Therefore,we bleforthisstressresponsepathway.ThesecondidentifiedHSP70

initiallyestablishedaporcinecelllinestablyexpressingthePDCoV family protein is HSC70, also known as heat shock 70-kDa pro-

NproteinandthencharacterizedtheNproteinproducedinthose tein 8 (HSPA8). HSC70 is a molecular chaperone with multiple

cells. Due to the absence of cysteine residues, a disulfide-linked functionsinproteinfoldingandtraffickinginalleukaryoticcells

homodimericNproteinofPDCoVwasnotdetectedinPK-PDCoV-N and in protecting cells from apoptosis or a wide array of stress-

cells.Rather,thePDCoVNproteinwasshowntobeself-assembled orssuchasheatandinfection(Morano,2007;Powersetal.,2008;

vianon-covalentoligomerizationasastructuralcomponentforfor- Takayama et al., 1999). Accumulating evidence has shown that

mation of the viral capsid. Interestingly, the N protein of PDCoV HSC70playsimportantrolesincertainprocessesinvolvedinviral
144 S.Lee,C.Lee/VirusResearch208(2015)136–145

infectionbymodulatingcellentry,viriondisassemblyandassem- In conclusion, to our knowledge, this is the first report of a

bly,andthecellularantiviralresponseandapoptosis(Chuangetal., proteomicanalysisofcellularresponsestothePDCoVNprotein.

2015;Gutiérrezetal.,2010;Ivanovicetal.,2007;Liuetal.,2013; Althoughdefinitefunctionsoftheproteinsthatweidentifiedhere

Radhakrishnanetal.,2010;Yanetal.,2010).Apoptosisisconsid- werenotdetermined,itislikelythatalterationsintheirexpression

eredaninnatedefensemechanismthatlimitspropagationofavirus areinvolvedinvirus–hostinteractions.Toresolvethesequestions

byeliminatinginfectedcells(EverettandMcFadden,1999).There- aswellasperformingvariousPDCoVresearch,obtainingaKorean

fore,manyviruseshaveevolvedtoemployvariousstrategiesthat PDCoVisolatethatcangrowincellcultureisnecessary;wearecur-

inhibitapoptosisinordertopreventprematurecelldeath,thereby rentlyworkingonthistask.Infuturestudies,weareplanningto

securingsufficienttimeforprogenyproduction.Thus,aconceivable assesscellularresponsestoPDCoVbyproteomicanalysistovali-

explanationisthatPDCoVmaytaketheadvantageofsuppressing dateourpresentdata;theproteinsthataredifferentiallyexpressed

apoptosisbyup-regulatingHSC70intheearlyphaseoftheinfec- inthecellsoverexpressingPDCoVNorinthecellsinfectedwith

tion,andtheNproteinseemstobeinvolvedinthismechanism. PDCoVcanbeanalyzedindetailinordertoidentifytheirprecise

In addition, HSC70 mediates the nuclear export of the influenza functioninthereplicationofPDCoV.Ontheotherhand,onelimita-

virusribonucleoproteincomplexviainteractionwithviralproteins tionoftheproteomicmethodologyusedinthisstudyistheinability

M1andNS2(Watanabeetal.,2006,2014).Inthepresentstudy, to reliably detect low-molecular-weight or low-abundance pro-

we found that PDCoV N can be localized to the nucleolus in N- teins such as cytokines, which are involved in the host immune

expressingcells.Ontheotherhand,Nproteinsmustbetrafficked response.Therefore,morecomprehensiveworksarealsoneededto

fromthenucleolustothecytoplasmtoaccomplishnucleocapsid expandourpresentfindingsandtoexploreotherproteinsthatmay

assemblyaswellasviralRNAsynthesisandvirus–hostinteractions. playaroleinhostresponsestothisprotein.Nevertheless,thedata

Accordingtoourresultsandexistingdata,HSC70mayfacilitatethe presentedhereareexpectedtoadvancetheknowledgeaboutthe

export of PDCoV N from the nucleolus to complete the viral life molecular mechanisms associated with PDCoV–host interactions

cycle. andviralpathogenesis.

Like many other viruses, coronaviruses turn off host protein

translation, while continuing to the synthesis of their own gene
Acknowledgment
productstofinishviralreplication.Oneofthemechanismsbehind

thistranslationalsuppressionisthroughinteractionoftheNpro-
tein with elongat ion factor 1(cid:4) ( EF1(cid:4)), a major tran sla tion fa ctor This research was supported by Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
inmammaliancells(Zhouetal.,2008).Inthepresentstudy,the
funded by the Ministry of Science, ICT & Future Planning
amount of another translational factor, EF2, was affected by the
(2013R1A2A2A01004355).
hostresponsetothePDCoVNprotein.Althoughwedonotknow

whethertheincreasedexpressionofEF2isrelatedtoitsbindingto

PDCoVN,suchdifferentialexpressionofatranslationfactormaybe References

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