2015 Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats

2015·1.56 MB·English

VirusResearch202(2015)160–167

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Virus Research

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

Characterisation of different forms of the accessory gp3 canine

coronavirus type I protein identified in cats

Anne-LaurePham-Hungd’Alexandryd’Orengiania,b,c,LidiaDuartea,b,c,NicolePavioa,b,c,
SophieLePo dera,b,c,∗

aUniversitéParis-Est,EcoleNationaleVétérinaired’Alfort,UMR1161Virology,Maisons-Alfort,France
bINRA,UMR 1161Vir ology, Maisons-A lfort,Franc e
cANSES ,Anim alH ealthLabo ratory,UMR11 61Virology,Maisons-Alfort,France

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

Articlehistory: ORF3isasupplementalopenreadingframecodingforanaccessoryglycoproteingp3ofunknownfunc-
Availab leonline7February2015 tion,o n ly presentinge notyp eIcanin estra in(CCo V-I )an dsomea typicalfeline FCo Vs trains.In these

latterhosts,theORF3genesystematicallydisplaysoneortwoidenticaldeletionsleadingtothesynthesis
Keywords: oftru ncated pr oteins gp3- (cid:2)1andgp3-(cid:2) 2.Asde letio ns inC oVacces soryprot einshav e alre adybeen
Coronavirus in volvedint issueorh ostswit ch,s tudiesof the sedifferen tg p3p roteinswe recondu cted incanin eand
Accessory protein felinecel l.A llpro tei nsoli gomeris ethrou gh coval entbond s,are N-glyco sylate dandare ma intaine din
Feline
theERinnon-infectedbutalsoinCCoV-IIinfectedcells,withoutanyspecificretentionsignal.However,
Canine
deletionsinfluencetheirlevelofexpression.Incaninecells,allproteinsareexpressedwithsimilarlevel
GSppe3cies barrier whereas i n feline ce lls, th e exp re ssion of gp3 -(cid:2) 1 is hig her t han the two oth er forms o f gp3 . None o f the
gp3proteinsmodulatetheviralreplicationcycleofheterologousgenotypeIICCoVincaninecellline,

leadingtotheconclusionthatthegp3proteinsareprobablyadvantageousonlyforCCoV-Iandatypical

FCoVstrains.
©2015ElsevierB.V.Allrightsreserved.

1. Introduction diseases(Pedersen,2009;DecaroandBuonavoglia,2008).CCoVs

areseparatedintotwogeneticclusters,CCoV-IandCCoV-II,accord-

The genome of the Coronaviridae (CoV) family is a large ing to the S gene sequence. One accessory gene, named ORF3

single-strandedandpositive-senseRNAmoleculeofabout30kDa is a singularity of the CCoV genotype I (CCoV-I). Its sequence

(Gorbalenya et al., 2006). For all CoVs, the first two thirds of is highly conserved among CCoV-I, suggesting its importance in

the genome encode non-structural proteins which are essen- some aspects of the viral infection. It is the only CCoV acces-

tiallyinvolvedintheCoVreplicationandtranscriptionmachinery sorygenewhichhasbeeninvestigatedsofar.Invitrotranslation

(Bredenbeeketal.,1990).Therestofthegenomecomprisesgenes assaysdemonstratedthatORF3encodesanN-glycosylatedprotein

ofthestructuralproteinsS(spike),E(envelope),M(membrane), of28kDa,namedgp3(Lorussoetal.,2008).Still,thefunctionof

N (nucleocapsid) and eventually HE (hemagglutinin–esterase) gp3remainsunknown.Ourrecentphylogeneticstudiesconducted

(De Groot, 2006). Additional genes named accessory genes are on the characterisation of CoV strains infecting cats lead to the

interspacedbetweengenesencodingthestructuralproteins.The discoveryofatypicalFCoVstrainsharbouringafelineSgeneand

numberandpositionofthesegenesvaryfromoneCoVtoanother. downstreamgenesgeneticallyrelatedtoCCoV-I.Sequencesofthe

Witheightaccessorygenes,SARS-CoVpossessesthehighestnum- ORF3genewerealsopresentinthesestrainsbutdisplayeddele-

berofthem,whereashCoV-229Econtainsonlyone(Dijkmanetal., tionsneverdescribedsofar.AllORF3sequencesfromatypicalFCoV

2006). strainssharedadeletionof29-nt,whichintroducedastopcodon

Feline CoV (FCoV) and canine CoV (CCoV) are common andanotherfrequentlyobserveddeletionof27-ntdeletedthepre-

pathogensofcatanddogpopulations,sometimesleadingtofatal dicted protein of nine additional a.a. (Le Poder et al., 2013). The

completegp3proteinofCCoV-Iisof207a.a.inlength,whereasthe

deletedformspresentinatypicalFCoVstrainsarepredictedtopro-
duceeit herad eletedp ro tein,nam edg p3-(cid:2)1 of1 58a.a.ora no ther
∗ Corresponding author at: Université Paris-Est, Ecole Nationale Vétérinaire form ,name d gp3-(cid:2)2 ,of149 a.a.Inc omparis on with gp 3, gp3-(cid:2)1
d’Alfort, UMR 1161 Virology, 94704 Maisons-Alfort, France. andg p3-(cid:2)2 losttheC -te rmi nus pa rtfromthec ystei nere siduein
Tel.:E +-3m3a 1il a4d3d 9r6es 7s:3s 2le5p; ofadxe:r @+3v3e t1-a 4lf3o 9rt6.f r73(S 9.6Le. Poder). posi tion157 onw ard .Gp3-(cid:2)2isf urthe rtrun cat edof9a.a .betwe en

http://dx.doi.org/10.1016/j.virusres.2015.01.027
0168-1702/©2015ElsevierB.V.Allrightsreserved.
A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167 161

Lys88 andPhe96.Allproteinsmaintainapredictedsignalpeptide during1hat37◦Ctoeliminatetheparentalplasmid.LibraryEffi-
attheN-t erminu se xtremity andaput at iveglycos ylation siteon ciency D H 5(cid:3) bact eri a (Invitro gen) were tr ansforme d with the
As n116 .Nootherp articularfu nctio n aldomai nwaspredicte dby in digesti onprod uctsacco rdingtothe manuf acturer’sreco mmen da-

silicoanalysis. tions.

Coronavirusesarewellknownforcrossingthespeciesbarrier,

sometimes leading to the emergence of new pathogens, like the 2.3. Immunoblotting

SARS-CoV or the MERS-CoV. Adaptation to a new host is often

accompaniedbymodificationsofsomeaccessoryproteins,among At 24h post-transfection, cells were lysed with RIPA extrac-

others(McBrideandFielding,2012).Withtheaimofunderstand- tionbuffer(20mMTris–HCl,pH7.5,0.2%NonidetP-40,150mM

ingtheroleofthegp3deletionsintheadaptationofatypicalFCoVs NaCl, 0.5mM EGTA, 1.5mM MgCl2, 10% Glycerol) supplemented

tothefelinespecies,wecharacterisedthebasicpropertiesofthe with Complete Protease Inhibitor Cocktail Tablet (Roche). Sam-
dif fere ntform sofgp3 inb othfelineand can inece lls.Theabse nc eof ples containing 100(cid:2)g o f total pr oteins w ere the n mixed with
culture-c ellada pt eds tra inso fCCoV -Ia ndofth enew lyd iscovere d an e qual volum e o f 5 × Laem mli buffe r and heat ed at 95◦C
atypicalFCo Vstrains impair ed studies ofth e diffe rentg p3proteins for 5min . Where m enti oned, 10% of (cid:4)- merc aptoeth ano l was

in cells culture infected with these specific CoVs. Through tran- addedtocelllysates.Proteinswereseparatedonsodiumdodecyl

sienttransfectionofplasmidsexpressingthedifferentproteins,we sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and

demonstratedthatthegp3deletionsinfluencetheirexpression.The transferredontoanitrocellulosemembrane(GEHealthcare).After
completegp3 prot ein isex pressedo nlyincan inec ells,gp3-(cid:2) 1is saturation with 5 % milk in PB S–Tween ( 0.01 %) buffer (P BST),
expressed ata highlev el inbothcan inea nd felinec ellsan dgp3-(cid:2) 2 the memb ranes wer e incu ba ted with ant i-Flag m onoclon al anti-

isfaintlyobservedinfelinecelllines.Incontrast,allproteinsassem- body(Sigma–Aldrich)dilutedat1/5000oranti-actinmonoclonal

bleintocovalentoligomersandaremaintainedintheendoplasmic antibody (Sigma–Aldrich) diluted at 1/2000 in PBST–milk buffer
reti culu m(ER)de spitecleav age oft heirpeptide si gnal andabsence overnigh t at 4◦C. The me mbrane s w ere wa sh ed three tim es in

ofaknownspecificretentionsignal.Incaninetransfectedcellsand PBST buffer and incubated for 1h at room temperature with

infected by CCoV-II, which does not harbour the ORF3 gene, the horseradish peroxidise-conjugated anti-mouse antibody (Roche)

gp3 proteins are also localised in the ER but none influence the diluted at 1/5000 in PBST-milk buffer. After three washes, blots

viralproductionofCCoV-IIstrains. wererevealedusingenhancedchemiluminescence(ECL)reagents

(Pierce) and analysed with a Fusion imaging system (Bio-Rad).

QuantificationwasperformedbyusingtheBio-1Dsoftware.
2. Materials and methods Forglycosid asea ssays,20(cid:2) lof cellly sate swere mixedwith1(cid:2)l

ofeitherpeptide-N-glycosidaseF(PNGaseF)orendoglycosidaseH
2.1. Cell culture and transient transfection (E ndoH) (Biolabs),and3(cid:2)Lof1 0 ×glycopr ot ein denaturingbuff er
(Biolab s) priortoin cub at ion at 37◦ Cfor3h.
Canine A72 and feline CrFK cells were maintained in Dul-

becco’s modified Eagle’s medium (DMEM) (Invitrogen, Life
2.4. Indirectimmunofluorescencemicroscopy
Technologies) supplemented with 10% foetal bovine serum, 1%

penicillin–streptomycinsolution,1%sodium-pyruvateand1%non-
A72 and CrFK were transfected with expression plasmids of
essentialaminoacids.
the different gp3 proteins. At 24h post-transfection, cells were
Subconfluent A72 and CrFK cells were transfected in 6-well
plates with 2.5(cid:2) g D NA p lasm id us ing Tr ansIT-LTI tr ans fection washed with PBS and fixed with 4% paraformaldehyde in PBS-4%
sucrose (Life Technologies). After two washes in PBS, a solution
reagent(Mirus),followingthemanufacturer’sinstructions.
of50mMNH4Clwasaddedfor10minatroomtemperature,then

fixed cells were incubated with PBS-0.5% bovine serum albumin

2.2. Plasmids (BSA)for30min.AfteronewashwithPBS-1%BSAandtwowashes

withPBS-1%BSA-0.5%saponine,amixofprimaryantibodyraised

OptimisedcodonsequencesofthecompleteORF3gene(Gen- againsttheFlagtag(Sigma–Aldrich)andeitherantibodyspecific

Bank accession numbers JN714200), and the two deleted gene ofthecalnexin(Sigma–Aldrich)orthegiantin(Abcam)atdilution

forms(GenBankaccessionnumbersJN714196andJN714199)were of1/500inPBS–BSA–saponinesolutionwasaddedfor1hatroom

synthesised in frame with the 3xFlag tag into pCDNA3.1 plas- temperature.Fortriplelabellingininfectedcells,aserumofCoV

mids (Invitrogen) between restriction site BamHI and NotI, by infectedcatatadilutionof1/250wasaddedtothemixofanti-Flag

Proteogenix(ProteoGenixSAS,France). andanti-calnexinantibodies.Afterthreewashes,cellswereincu-

MutantplasmidswithdeletionofthesignalpeptideoftheORF3 batedwithamixofanti-mouseAlexa488andanti-rabbitAlexa555

genesweresubsequentlygeneratedfromeachparentalplasmid. secondaryantibodies(Invitrogen)atworkingdilutionsof1/600in

Briefly,plasmidsconsistingofresidues15tothestopcodon,were PBS–BSA–saponinesolutionfor45minatroomtemperature.For
obtaine dbyPCR assayswit ht heforwa rd pr imer s((cid:2) SP-ORF 3F1: triplestaining,seco ndaryan tibo die suse d werea nti-catAlexa 488
5(cid:3)-GGACT AG TGG ATCCA TGCA CCC CTTTCAC G-3(cid:3),(cid:2)S P-ORF3-(cid:2)1/ (cid:2)2 (Jacks on),anti- mouseAle xa555and anti -rabb itAlexa 647(I nvi-
F1: 5(cid:3)-TCCGCCGCCACCATGCTCCACCCCTTTCAC -3(cid:3)) and reverse trogen),al latworking diluti onso f1/5 00.Afterth reew ashe swith
prim ers((cid:2)SP-ORF3R1:5(cid:3)-CGTGAAAGGGGTGCATG GATC CACTAG- PBS,cell sw er ecovered withaso lut ionofM owi olmix edwith DAPI
TCC-3(cid:3),(cid:2) SP-ORF-(cid:2)1 /(cid:2)2 R1:5(cid:3)-GTGAAAGGGGTGGAGCATGGTGG- fors tainin gof thenucle us.S li deswere e xamined at63× mag nifi-
CGGCG GA-3(cid:3)). To create gp 3 proteins with uncleaved signal cat ionbyap ot ome microsc ope(Z eiss) andimage sw ere analysed

sequence,theaminoacidscriticalforcleavagewerechangedinto usingtheZensoftware.

five Leucine residues by PCR assays using the following primers
(5L- ORF3 F2 : 5(cid:3)-ATC AG TGTC GTCCT TTTGT TAT TACTCCAC CCCTTT- 2.5. Infections
3(cid:3),5L-ORF 3R2 :5(cid:3)-AAAGGGGTGGAGTAATAACAAAAGAAGGACACT-
GA T-3(cid:3),5L- ORF 3-(cid:2)1/(cid:2)2F2:5(cid:3)-ATGATTAGTGTGGTCCTTTTGTTAT- For infection assays, subconfluent A72 cells were inoculated
TACACC CCTTTCAC-3(cid:3), 5L -OR F3-(cid:2)1/(cid:2)2 R2: 5(cid:3)-CGTGAAAGGGG- with t ype II stra in CCoV -1-71, from t he A meric an Ty pe Culture
TGTAATAACAAAAGGA CCACACTAAT CAT -3(cid:3)). PCR products were Colle ction (A TCC®V R-809TM), a t a m ultip licity of in fectio n (MOI)
submitted to digestion by DpnI en zyme (N ew E ngland Bi olabs) of10.Cell swerethenincubat ed fo r120minat 37 ◦C,with gentle
162 A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167

Fig.1. Schematicrepresentationofthedifferentgp3proteins.Gp3isthefulllengthproteinencodedbyCCoV-I,gp3-(cid:2)1andgp3-(cid:2)2arisefromatypicalFCoVandaredeleted
ofth e C-terminus partincompar iso nw ithgp3. Ade letionof nine a min oac idsdiff ersbetw eengp3 -(cid:2) 1andg p3-(cid:2)2.T hep otential signa lpep tideand N-gl ycos ylat ionsite

areindicatedbyalightgreyboxandastar,respectively.LengthofthedifferentproteinsisindicatedbythenumberofaminoacidsattheC-terminus.

rockingforoptimalviraladsorption.Inoculumwasthenreplaced detected.Aftertreatmentwith(cid:4)-mercaptoethanol,themonomeric

bystandardfreshmedium,supplementedwith10%FBS.Cellswere formsweremainlyobserved,suggestingtheassemblyofgp3,gp3-
lef ttoincub atefo rspecifie damountofti me.In pa rallel ,infe ction (cid:2)1an dgp3 -(cid:2)2in todimeric complexes thr oughdisu lfi deb onds
ofn on -transfect ed cellsusing thesam e protoc ol wascon ducted. (Fig . 2C ). For gp 3-(cid:2) 1, highe r order ass emblies were obs erved,

whichcouldcorrespondtotetramers.

2.6. QuantitativeRT-PCR

3.2. Post-translationalmaturationoftheproteins

ViralRNAwasextractedfromsupernatantsusingtheQIAamp

Viral RNA Mini kit (QIAGEN). RNA was reverse transcribed Thegp3proteinsbearaputativeglycosylationsiteattheAsn116

and amplicons were generated using the Quantitect SYBR position. This amino acid is conserved in all three proteins. In

Green RT-PCR kit (QIAGEN) and the following primers set: ordertocharacterisetheglycan,assayswithglycosidaseswereper-
CCoV- 7F: 5(cid:3)-GG CAA CCCGATGT TTAA AAC TGG-3(cid:3) and CCoV-7R : 5(cid:3)- forme d. At24hpost- tran sfection ,cellly sates wereincuba tedei ther
CACTAGA TCCAGACGTTAGCTC-3(cid:3), following the m anufactur er’s withPN Ga se F orEndoHormoc ktr eated. PNGa seFremov esall
instructionsatanannealingstepo f52◦C.The sepr imerstargetthe N-lin kedglyc an sw herea s End oHcl eaveson lypolym an nosecarb o-

accessorygene7boftypeIICCoV.Quantificationofviralproduc- hydratesthathavenotbeenfurtherprocessedintocomplexsugar.
tion in ce lls su per na tant w a s norm alised by the tot al am ount of Gp3,gp3 -(cid:2)1 andg p3- (cid:2)2a reallse nsitiveto PNG aseF(da tanot

RNA.Incellularextracts,quantitativeRT-PCRoftheactinhouse- shown)andtoEndoH(Fig.3A).Theseresultsshowthatthediffer-

keepinggenewasperformedinparallelusingthefollowingprimers entgp3proteinsareglycosylatedwithahighmannoseglycan.The
set:ACT IN-F: 5(cid:3)-C AGCACAAT GA AGATC AAGA TCA TC-3(cid:3)and ACTIN- sam ere sultswe reo btainedinbo thA 72 and CrFKcell lines(d ata
R:5 (cid:3)-CGGACT CATCGTACTCCTGCTT-3(cid:3).TheviralRNAcopyn umber notsh own).

incellextractswasnormalisedtotheexpressionoftheactingene InsilicoanalysesindicatedanN-terminalhydrophobicdomain
us ingt he2(cid:2)Ct met hod(Livaka nd Sch mittgen,20 01 ). funct ioning assigna lsequenc ec leavableaft erthealanin eamino

acidinposition15.Totestwhetherthispeptidesignaliscleaved,

mutants without the predicted signal peptide for each protein
3. Results ((cid:2)SP-gp 3,(cid:2)SP-gp 3-(cid:2) 1,(cid:2)SP-gp 3-(cid:2)2) werecon stru cted. Mutants

3.1. Expression of gp3, gp3-(cid:2)1 and gp3-(cid:2)2 in A72 and CrFK cells 5wLi-tghp t3h-e(cid:2) c1le,a5vLa-ggpe 3s-i(cid:2)te2 r)ewplearceeda lsboy cfiovnes tLreuucctiende inreosirddueerst o(5iLm-gppa3ir,

thepotentialcleavageofthesignalpeptide.At24haftertransfec-
TheORF3geneswereclonedintoexpressionplasmidsunderthe
tion,cellslysateswerecollected,treatedornotwithEndoHand
tchoent3r(cid:3)oel nodf tohfeO CRMF3V( pFirgo.m1)o.tEear cwhipthla as m3xid-Fwlaags ttarga nfusfseecdt eind efriathmeer iant analy sed b y immu noblo tting. All (cid:2) SP prot ein s mi grate d faste r t han
thewildtypeproteins(Fig.3B).Theestimateapparentmolecular
A72 canine cells or in CrFK feline cells. Both cell lines were chosen wei ghto fthe (cid:2)SPpro teins was abo ut3kDa lessthan thenative
for their susceptibility to CCoV-II and FCoV-II infections. At 24h
proteins.Withoutasignalpeptide,proteinscannotentertheERand
post-transfection,cellswerelysedandproteinsweredetectedby
thereforecannotbecomeglycosylated,asconfirmedbythesame
immInu Ano7b2l octetlilns,g awlli tthh raene apnrtoi-tFeliangs ,a gnpti3b,o gdpy3.-(cid:2)1 and gp3-(cid:2)2 are eglpe3c-t(cid:2)ro1pha onrdetgicp 3p- r(cid:2)ofi2lep orof (cid:2)t eiSnPs pwrohtiecihnsh ta rreba oteudr oanr nuontc b leya E vnadbol e Hs. i5gL-
detected. They migrated according to their predicted molecular
nalpeptidedisplayaslowermigrationandanapparentmolecular
weightat28,22and21kDa,respectively(Fig.2A).InCrFKcells,
express ion of gp3 and gp 3-(cid:2)2 waslow,wh erea sgp3 -(cid:2) 1wa sstill weight of approximately 3 kDa higher than the wild type proteins.
Someunglycosylatedformsaredetectedasshownbycomparison
detected(Fig.2A).Quantificationdemonstratesthatexpressionof
the gp3- (cid:2)1 i s abo ut fivefold hig her than gp3 and tenfold mo re with lysates treated with Endo H. 5L-gp3 migrated faster than the
than gp3-(cid:2)2 ( Fig.2B ).InA72 cells,d iffere nces ofe xpressio nlev- wild-type protein.

els between the three proteins are less pronounced. Quantity of
gp3 -(cid:2)1ison lytw ofold moreele vat edth angp3andg p3-(cid:2)2. 3.3. Subcellularlocalisationoftheproteins
Asgp 3 poss essessix cyste ineresidu esw hile gp3 -(cid:2)1andgp3-
(cid:2)2 co ntai n only fou r, w e invest igated th e poss ible form atio n of Next,thesubcellularlocalisationoftheproteinswasexamined

covalent homo-oligomers. At 24h post-transfection, cell lysates by colocalisation assays of gp3 proteins with different markers

were treated and migrated onto SDS-PAGE gels with or without of the cellular compartments in both A72 and CrFK. 24h post-
(cid:4)-me rcaptoe than ol.Under non-r educingco nditi ons,s up raforms tra nsfe ction,du allabellingwer eca rried outw itha nti-Fla ga nt ibody

ofapproximatelytwicethemolecularweightofeachproteinwere to visualise the gp3 proteins and specific antibodies directed
A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167 163

Fig.2. Proteinexpressionincanineandfelinecelltypes.(A)DetectionoftheproteinsintransfectedcanineA72andfelineCrFKcellsbyimmunoblottingassays.Cellswere

transfectedwiththedifferentgp3expressionplasmidsorwithanemptypCDNAvector(Control).At24hpost-transfection,cellswerelysedandprocessedontoSDS-PAGE
gelsforimm unob lot ting.Gp3 prot einswered etectedu sin gana nt i-Flaga ntibody .Cellul ar(cid:4)-actin wa sr ev ealedinparallelb yasp ecific mono clon alanti-act inan tibody.(B)

Levelsofexpressionofthedifferentgp3proteins.Immunoblotsofthedifferentgp3proteinswerequantifiedusingtheBio-1Dsoftware.Quantityofgp3wasarbitrarysetto
1andt he relativequ an tity ofgp3-(cid:2) 1an dgp3-(cid:2)2 areexpressed in com parison with gp3.All dataa rereprese ntativ eof threein dependen texperim e nts.E rror barsrepr ese nt

standarddeviations.(C)Multimerisationofthegp3proteins.A72andCrFKcellsweretransfectedwiththeemptyvectorpCDNA(control)orplasmidsencodingthedifferent
gp3prote insandlyse da t24hpost-transf ec tion .Cel llysatesw ere trea tedw ith(+ )orw ithout(−)(cid:4) -Mer cap toethan ol((cid:4)-M E),prio rtomigr ati onontoS DS-PAGEg els .Proteins

weredetectedbyimmunoblottingwithanti-Flagantibody.Positionsandmasses(inkDa)ofthemolecularmassproteinmarkersareindicatedontheleft.

againstcalnexin,aresidentproteinoftheER,orgiantin,amarker gp3proteinscolocalisedwiththecalnexinsignalininfectedcells

oftheGolgiapparatus.Byapotomemicroscopyanalyses,thefluo- (Fig.4C).

rescencesignalsofthegp3proteinsandthegiantinweredistinctin

bothA72andCrFK,indicatingthattheproteinshadnotreachedthe 3.4. CCoV-IIviralproductionincellsexpressingthedifferentgp3

Golgiapparatus(datanotshown).Incontrast,thegp3proteinscolo- proteins

calisedwithcalnexinsignalsincanineandfelinecelllines(Fig.4A

andB). Toevaluatetheimpactofthedifferentgp3proteinsduringthe

Subcellular localisation of the proteins in a viral context was viral life cycle, 12h after transfection with the empty pCDNA3.1
investigated. A 72 cells we re infe cted with th e CCoV -II strai n at plasm id orplas mi ds enco dinggp3,gp 3-(cid:2)1 ,or gp3-(cid:2)2 ,A72cells

MOI of 10 and transfected 20h post-infection, with either the were infected with CCoV-II strain at MOI of 10. A72 cells were

empty vector pCDNA3.1 or with plasmids encoding the differ- alsoinfectedatthesameMOIwithoutpriortransfection.At24h,

ent gp3 proteins. Cells were then fixed 72h post-infection and 48h and 72h post-infection, supernatants and cells were col-

processedforsubcellularstudies.Thefluorescencesignalsofthe lectedandanalysedbyquantitativeRT-PCR.Quantityofviralcopy
164 A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167

Fig.3. Processingofthegp3proteins.(A)MaturationoftheN-linkedoligosaccha-

rides.A72cellsweretransfectedwithplasmidscontainingtheindicatedsequences
andly seda t24 hpost -transfectio n.20 (cid:2)Lofwho lecelllysa tesw erethen treated(+)
with Endo H or m ocktreated(−).A fte rm ig ration on SDS-PA GEge ls,gp 3prote ins

wererevealedbyimmunoblottingwiththeanti-Flagantibody.(B)Cleavageofthe

signalpeptideofthegp3proteins.A72cellsweretransfectedwithexpressionplas-
midsc ontainin g diff eren tconstru ctso fthe gp3 proteins.(cid:2) SP-gp 3,(cid:2)SP-gp 3-(cid:2)1
and(cid:2) SP-gp3-(cid:2)2 aredele tedofthefi rst 14N -ter minalam inoacids. In5L-gp3,5L-
gp3- (cid:2)1and5L-gp 3-(cid:2) 2const ru cts, thep uta tivecleava gesite wasre pla cedby five

Leucineresidues,topreventsignalpeptidecleavage.At24hpost-transfection,cell
lysatesw erereco ver edandt reated with(+) orwithou t( −) En doHtreatment,p rior

tomigration.Thedifferentproteinsweredetectedbyimmunoblottingwithananti-

Flagantibody.Positionsandmasses(inkDa)oftheproteinmarkersareindicated

ontheleft.

genomeincellssupernatant(Fig.5A)andcellularextracts(Fig.5B)

was calculated and normalised to total RNA amount and the

actinhousekeepinggene,respectively.Nodifferencewasobserved

betweeninfectedcellsandinfectedcellsexpressingthedifferent

gp3proteins.

4. Discussion

Thepresenceofanadditionalaccessorygene,ORF3isspecificto

CCoV-I.InCCoV-IIandFCoV-II,onlyveryshortsequences,lessthan

71-ntandcorrespondingtoORF3,arepresentandwereinterpreted

byLorussoetal.(2008)asresidualsequencesfromaCoVancestor.

InFCoV-I,nonucleotidecorrespondingtoORF3hasbeendescribed

untilourrecentstudy,inwhichtheORF3genewasidentifiedin

atypicalfelineCoVstrainsharbouringanNgenerelatedtoCCoV-
Fig. 4. Subcellular localisation of gp3, gp3-(cid:2)1 and gp3-(cid:2)2. A72 cells (A) and
I and an S gene close to FCoV-I (Le Poder et al., 2013). In these
a typic al fe line st rains, th e ORF3 s ar e 595- nt or 5 68-nt i n l ength CmriFdKs ceenllcso d(Bin) gwtehree tdriaffnesrfeencttegdp w3itphr oatne inems.pAtty 2p4ChDNpAo spt-latrsamnisdfe (cctoionntr,ocle) lolsr wplearse-
and are preced ed by s peci fic tran scri ption-re gu lating s equ ences, dual labelledw itha ntibodies again sttheFla gta g((cid:3) - Flag)andagainstc alnex in((cid:3)-
sugg esti ngthepos sib letransl ationintofunctionalprot einsduring calne xin),am arke roftheER. Alexa-48 8a nti-m ou seandale xa- 555anti -rabbitw ere

usedassecondaryantibodies.(C)A72cellswereinfectedwiththeCCoV-II1-71
infections.
strainandtransfected20hpost-infectionwithplasmidscontainingtheindicated
AllORF3sequencesrecoveredinthesefelineCoVstrainswere seque nces .At72hpos t-in fe ction,cellswer efixe dandtrip lestainedw ith anti-Flag,
delete dthes ameway,l eadingeith er toap utative gp3 -(cid:2)1pr otein, anti-calnex in and ananti-CoVser umo rigin ating from anin fected cat.C ellnuclei
compris ing thefi rst15 8a.a.o fgp3o rt o gp3-(cid:2)2 protein, withan werecounter stain ed withDAP I.Allsli deswereob serve dw itha63× oil imm ersion
additional 9 a.a . dele tion com p ared to gp 3-(cid:2)1 (F ig. 1). A p revio us tohbejeacn titvi-eF llaegn,st hbey aannt ia-pcoaltno emxein ma nicdr otshceoDpeA P(ZIesiigs sn)a. lAits tshheo wringh.Bt ,a ar cmhea rrgt:e 1d0 im(cid:2)mag.e of
studyexploredthebiochemicalpropertiesofthecompletecanine
A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167 165

gp3proteinusinganinvitrotranslationsystemandconcludedthat statusofCoVaccessoryproteinshasbeeninvestigatedforsomeof

gp3isaprobablysecreted,solubleglycoproteinof28kDa(Lorusso them.Multimerizationhasalsobeendemonstratedfortheacces-
et a l., 2 008). Her e, the ex pression of the diffe re nt gp3, gp3-(cid:2)1 soryO RF3fromPEDV( Wan get al.,20 12),ORF4afro mH CoV -229E
an dgp 3-(cid:2)2 protein sw asanalysed in cell culture.P rote inswere (Zha ngeta l.,20 14)or ORF8ab fr om SARS- CoV(Oo strae tal.,2007).

expressedeitherincanineorfelinecellsinaccordancetothehost Post-translationalmodificationsarealsocommontothethree

tropismfromwhichtheywerederived.Thelevelofexpressionis proteinsinthestudiedcelllines.Invitrotranslationindicatedthat

differentnotonlybetweenthetwocelllinesused,butalsobetween gp3containsanN-linkedglycosylationsite(Lorussoetal.,2008).

thedifferentproteins.IncanineA72cells,thethreeproteinsare In cell culture and through the use of different deglycosidases,

expressedatapproximatelythesamelevel,butinfelinecells,dis- we were able to identify the glycan residue as a high polyman-
crepancies be tweenthemar eve ryma rked. Exp res siono fgp3 -(cid:2)1 nos e (Fig . 3A) , w hich cor robo rates w ith thei r l oc alisat ion in the
is fivefold higher th an gp 3 an d gp 3-(cid:2)2 (Fi g. 2B). Whe th er these ER(F ig.4A and B).Ind eed,maturat ionin toco mplexsugar ne eds

differentexpressionslevelsareduetodifferentdegradationpro- processingbyenzymesfromtheGolgiapparatusandpresenceofa

cessesormodulationsatthetranslationstepremaintobeexplored. polymannoseischaracteristicofproteinsresidingintheER.Itisstill

Expressionlevelsmightbeinfluencedbycellularfactorsinvolvedin unclearhowthegp3proteinsaremaintainedintheER,asnopecu-

regulatingproteintranslation.Interestingly,thegp3isexpressedin liarsignalforERretentionhasbeenidentified(Gaoetal.,2014).

caninecellsbutfaintlydetectedinfelinecellswhichmaycorrelate Retentionofthegp3proteinsintheERcouldhavebeenexplained

withthecaninevirusoriginofgp3. byanon-cleavageofthesignalpeptide.Thishypothesiswastested

Beside these discrepancies, the proteins have common bio- bymolecularmasscomparisonbetweennativegp3proteinsand

chemicalproperties.Ininvitrotranslationassay,gp3wasexpressed proteinseitherdeletedoffthesignalpeptideorharbouringanon-

inamonomericform(Lorussoetal.,2008).Here,incellculture,we cleavablesignalpeptide(Fig.3B).Themigrationdifferencebetween
de m onstratedth atgp 3,butal so gp3 -(cid:2)1a ndgp 3-(cid:2) 2o ligomer ise 5Lmutan tsand nativep rotei nss tron glysugges tanefficie ntcleav-
in,atleast,co valen tho mo- dime rsandp roba blyinh ighermulti- age ofthesi gnal peptid einnativ egp3pr oteins.In de ed,5L-gp 3-(cid:2)1
me ric form s,asshow nforgp3-(cid:2)1 (Fig. 2C).Thea bs enceof detec- and 5L -gp 3-(cid:2)2h aveam ol ecular mas shighert hanthe nativegp3-
tiono fhigher m ultimer icf ormothe rtha ndi mer sforgp3 an dgp3- (cid:2)1a ndgp3-(cid:2)2 .The 5L -gp3muta ntmi grated atalo we rmole cular
(cid:2)2 cou ldbed uetotheir faint erexp ressi on.The oli gom erisa tion mas sth anthena tive gp3.Lor ussoeta l.(2008)h ad a lready observed

Fig.5. ViralproductionofCCoV-IIinA72cellsexpressinggp3proteins.A72cellsweretransfectedwithplasmidscontainingtheindicatedsequencesandinfected12h

post-transfectionwiththeCCoV-II1-71strain.Supernatant(A)andcellularextract(B)werecollectedat24h,48hand72hpost-infectionandprocessedforviralgenome

quantificationbyquantitativeRT-PCR.Insupernatants,quantityofviralRNAwasnormalisedbythetotalamountofRNA.MeanexpressionofintracellularviralRNAcopy
numbersnorm ali sedtoactina regiven ,us ingthe2(cid:2)Ct method.M e ansf romt ripli cateexperim en tsa regiv en.Error ba rsrep resen tstandardd ev iations.
166 A.-L.Pham-Hungd’Alexandryd’Orengianietal./VirusResearch202(2015)160–167

thatthepresenceofthesignalpeptideingp3inducedanaberrant ofgp3conservethesamebasicfeaturesandcouldhaveasimilar

fastermigrationsimilartoourobservationherein.Thus,retention functionthanintactgp3.

intheERofthegp3proteinsisnotduetoadefaultinthesignal In concordance with other studies, our data underline the

peptidecleavage.Toinvestigatewhetherviralproteinsmayinflu- importancetoinvestigatetheroleofCoVaccessoryproteinsdur-

encethesubcellularlocalisationofgp3proteins,A72wereinfected ingtheviraladaptationtonewhosts.Inthecontextofemergence

withaCCoV-IIstrain,whichdoesnotexpressgp3,thentransfected ofhighvirulentCoVssuchasSARS-CoVorMERS-CoVarisingfrom

withplasmidsencodingthedifferentgp3.Againinthisviralcon- animals, it remains important to understand the mechanisms of

text,gp3proteinsarelocalisedintheER(Fig.4C).Altogether,these CoVpathogenicityandcross-speciestransmissions.

resultsstronglysuggestthatthegp3proteinscouldbemaintained

intheERthroughinteractionswithcellularfactors,asproposedby

studiesontheORF8abproteinoftheSARS-CoV(Oostraetal.,2007). Acknowledgments

Fromthecomparativestudyofthecompletegp3andthedeleted
mutantg p3- (cid:2)1and(cid:2)2, weca n ded ucethat thefi rst 158 amino WethankfullyacknowledgeDrSophieRogéeforthepCDNAvec-

acidscontainthenecessarysignalsforthebasicfeaturesofthepro- torandextendourgratitudetoDrMyriamErmonvalandDrMarie

teins:thecysteineresiduesforoligomerisation,theN-glycosylation Flamandforthedifferentcellularcompartmentmarkers.Thiswork

siteandtheunknownretentiondeterminantintheER.Thesecon- wassupportedbyagrantfromtheFrenchAgenceNationaledela

servedpropertiesmightbeimportantfortheirfunction. Recherche(ANR)throughtheprojectentitled“Eco-epidemiology

IntheabsenceoflaboratorystrainsharbouringORF3gene,i.e. of Coronaviruses, from wildlife to Human: Emergence threat

CCoV-IstrainsortheatypicalFCoVstrains,theonlywaytostudy assessment” (Grant ANR-13-BSV3-0013-EPICOREM). Anne-Laure

thedifferentgp3proteinsinvirusinfectedcellswastoinfecttrans- Pham-Hung d’Alexandry d’Orengiani was supported by a PhD

fectedcellswithaCoVstrainthatdoesnotencodegp3.Asthegp3 fellowship from the Agence Nationale de Sécurité Sanitaire, de

proteinsarebetterexpressedincanineA72cellsthaninfelineCrFK l’Alimentation,del’Environnementetdutravail(ANSES)andthe

cells,theimpactofgp3onviralreplicationefficiencywasinvesti- Direction Générale de l’Enseignement et la Recherche from the

gatedintransfectedA72infectedbyaCCoV-IIvirus.Inthiscontext, FrenchministryofAgriculture(DGER).

theviralproductionissimilarincellsexpressinggp3incompari-

sonwiththeemptyplasmidtransfectedcellsoronlyinfectedcells

(Fig. 5). As gp3 is present only in CCoV-I strain and deleted gp3 References

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