A novel glycoprotein of feline infectious peritonitis coronavirus contains a KDEL-like endoplasmic reticulum retention signal.

(1)

0022-538X/92/084951-06$02.00/0

A Novel Glycoprotein of Feline

Infectious Peritonitis Coronavirus

Contains

a

KDEL-Like

Endoplasmic

Reticulum Retention

Signal

H. VENNEMA,* L. HEIJNEN,tP. J.M. ROTTIER, M. C. HORZINEK, ANDW.J. M. SPAANt Department of Virology, Faculty of Veterinary Medicine, State University of Utrecht,

Yalelaan

1,

P.O.

Boax

80.165,

3508 TD

Utrecht,

The Netherlands

Received 2April1992/Accepted 19 May 1992

Anewprotein of feline infectious peritonitis coronavirus (FIPV)wasdiscoveredinlysatesof [35Sjcysteine-labeled infected cells. Expression of open reading frame (ORF) 6b of FIPV in recombinant vaccinia

virus-infected cellswasused toidentifyitasthe 6bprotein. Further characterizationrevealed that it isanovel typeofviral glycoprotein whose function isnotclear. It isasoluble protein containedinmicrosomes;its slow export from thecell is caused by thepresence ofan endoplasmic reticulum (ER) retention signal at the C terminus.This amino acidsequence, KTEL,closelyresembles theconsensusKDELsignalofsolubleresident ER proteins. A mutant 6b protein with the C-terminal sequence KTEVbecame resistant to digestion by

endo-13-N-acetyWglucosaminidase

Hwithahalf-time thatwasreducedthreefold. In contrast,amutantwith the

sequenceKDELwascompletelyretained in the ER. The FIPV6bproteinis the firstexampleofaviralprotein

withafunctional KDEL-like ER retention signal.

The coronaviruses feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine aregenetically closelyrelated. However, FIPV

con-tainsacompleteopenreadingframe(ORF),ORF2,inthe 3' region of thegenomewhich isabsentfromTGEV(3). ORF

2is located 3' ofORF 1 of mRNA6, potentiallyencodes a

polypeptideof 24kDa,andwesuggested that it isproduced from a bicistronic mRNA (3). According to the recently proposednomenclature (2), ORF1 and ORF 2 of mRNA 6

are now called ORF 6a and ORF 6b, respectively. TGEV and FIPV have three structuralproteins: the 200-kDaspike protein (S),the46-kDanucleocapsid protein (N),and the 26-to29-kDa membraneprotein (M).TheproductoftheORF6a counterpartof TGEVwasrecentlyidentified in infectedcells (8). FIPV ORF 6b product contains a short N-terminal hydrophobic region (3), which may function as a signal

sequence, andone consensusNglycosylation site. ORF6b contains a single methionine codon, which might explain why itwentunnoticedinpreviousmetaboliclabeling

exper-iments performed with [ 5S]methionine (4, 20). In this

re-port, we show that the protein encoded by ORF 6b is

producedinFIPV-infectedcells and that it isanoveltypeof viralglycoprotein.

MATERIALS ANDMETHODS

Cells andviruses. FIPVstrain 79-1146 (12)wasgrown in Crandell feline kidney(CrFK) cellsorinFelis catuswhole fetus cells (fcwf-D, obtained from N. C. Pedersen). For vaccinia virus (strain WR, obtained from G. Wertz) infec-tions, HeLa, human 143 thymidine kinase-negative (TK-), and rabbit kidney (RK-13) cells were used. Cells were

maintained in Dulbecco modified Eagle medium (GIBCO Laboratories) containing 5% heat-inactivated fetal bovine

serum.

RIPA. Lysates ofFIPV- orvaccinia virus-infected cells

were prepared after metabolic labeling with

L-[35S]methio-*Correspondingauthor.

tPresent address:DepartmentofVirology, FacultyofMedicine,

UniversityofLeiden, Leiden,The Netherlands.

nineor

L-[35S]cysteine

(>1,000 Ci/mmol; Amersham Corp.).

Radioimmunoprecipitation assays (RIPA) and

endo-P-N-acetylglucosaminidase H (endo H; Boehringer Mannheim Biochemicals) treatmentwerecarriedout asdescribed

pre-viously (20). Neuraminidase (from Arthrobacter

urea-faciens; BoehringerMannheimBiochemicals)treatmentwas

performedin50mMsodiumacetatebuffer(pH 5) for 16hat 37°C. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done as described

elsewhere (9).

Membrane fractionation. The membrane fractionation methodwasadapted from procedures described previously

(7).Cellsweresuspendedin0.1x Tris-buffered saline(TBS; 25 mM Tris hydrochloride [pH 7.5], 137 mM NaCl, 5 mM KCl,0.7 mMCaCl2, 0.5 mMMgCl2,0.6 mMNa2HPO4)and disrupted byDouncehomogenization. Nuclei and cell debris

wereremovedby low-speed centrifugation. One half of the

lysatewas diluted withanequal volume of0.1x TBS; the

other half was mixed with an equal volume of 200 mM

Na2CO3 (pH 11) and incubated for 10min onice.

Microso-mal membraneswerepelleted by centrifugation in a

Beck-man TLA 100.2rotor at65,000 rpmfor 30 minat4°C. The supernatant fraction of the carbonate-treated material was

neutralized with HCl. The pellet and supernatant were

analyzed byRIPA.

Cloning and expression of ORF 6b. Recombinant DNA techniques were performed essentially as described

previ-ously (11). A cDNA fragment extending from a SpeI site

located84bpupstreamof the initiation codon ofORF 6bto

a Sall site in the polylinker of cDNA clone E7 (3) was

recloned inpTUG31 (21), aderivative ofpET-3 (15)which

contains bacteriophage T7 promoter and terminator tran-scription signalsand whichwasused in combination with the

T7 RNApolymerase-producing recombinant vaccinia virus vTF7-3 (6).Theexpressionvectorcontainsflankingvaccinia virus TKsequences whichwere used toprepare

recombi-nant vaccinia virus vTF6b by procedures described else-where (10).

Site-directedmutagenesis. TheC-terminalsequenceof the 6b protein was changed bysite-directed mutagenesis. The synthetic oligonucleotides 5'-CGGGTTGCCTTATACCTC

4951

on November 9, 2019 by guest

http://jvi.asm.org/

(2)

4952 VENNEMA ET AL.

AG-3' and5'-GCCTTATAACTCAT`ATG-3'

(Pharma-cia LKB)arecomplementary to theC-terminalend of ORF 6b and contain one and two mismatched nucleotides,

respec-tively (underlined).Theresulting codingsequencescontain a

GTA codon for valine insteadof TTA for leucineor a GAT

codonforaspartic acid insteadofACTfor threonine. These

oligonucleotideswereusedin thepolymerasechain reaction (PCR) together with the universal M13 sequencing primer

(Promega), with 10 ng of M13 mpl8 single-stranded DNA containing a 360-nucleotide XbaI-PstI fragment of cDNA

clone B12 (3) as a template and Taq DNA polymerase

(Promega) as specified by the manufacturer. The PCR prod-ucts of approximately 160 bp were treated with Klenow DNA polymerase in the presence of deoxynucleoside triphosphates, digested with

KprnI,

purified fromanagarose gel, and ligated with EcoRV- and

7pnI-digested

pBluescript SK- (Stratagene). The presence of the mutation and the

integrity of the rest of the fragment were confirmed by

sequence analysis. The inserts were cut with XbaI and BamHI and ligated in a three-fragment ligation with an

XhoI-XbaIfragment of theremaining part of the 6b gene and

XhoI- and BamHI-digested vector pTUG31 (21). The mu-tated constructs weredesignated pTF6bVandpTF6bD.

RESULTS

Identification of the FIPV 6b protein. ORF 6b of FIPV contains onemethionine codon andsevencysteine codons. The electrophoretic patterns ofimmunoprecipitatedlysates from [35S]methionine- and [35S]cysteine-labeled FIPV-in-fected cells were compared to identify ORF 6b-derived polypeptides. After [35S]methionine labeling, the FIPV M protein appeared as a strong band of 29 kDa and a minor band of 26 kDa(Fig. 1A); thesebands correspondedto the

glycosylated and unglycosylated forms of the M protein, respectively (20). In [35S]cysteine-labeled

immunoprecipi-tates,adouble band of 26to26.5kDawasobservedat the

position of the unglycosylated M protein band. When the

sampleswere digested with endo H, which cleaves

aspar-agine-linked high-mannose oligosaccharide side chains, a 24-kDaproteinwas detected exclusively after[35S]cysteine

labeling(Fig. 1A).Theseexperimentsdemonstrate the pres-enceofahithertounidentifiedglycoproteininFIPV-infected cells whichwas readily immunoprecipitated byascitic fluid fromafieldcase of FIP. Thedifferential labeling suggested

thatthis novelprotein is encoded byORF6b; its apparent molecular weight is in close agreement with the weight

predicted from the amino acid sequence, and the shift after endo H treatment is consistent with the removal of one

oligosaccharideside chain (13).

Totestthehypothesisthat ORF 6b encodes this glycopro-tein,thecloned 6bgenewasexpressedineukaryotic cells;it wasreclonedfromcDNAclone E7(3)into abacteriophage T7 expression vector (Fig. 1B). The resulting construct,

designated pTF6b,wasused in the transient T7expression system with recombinant vaccinia virus vTF7-3, which producesT7RNApolymerase (6). The protein detected after immunoprecipitation comigrated with the 26.5-kDa protein from FIPV-infected cells (Fig. 1A). Digestion with endo H yielded a protein which comigrated with the 24-kDa protein mentioned above.

Inordertodetermine whether the recombinant 6bprotein and the 26-to26.5-kDaproteinin FIPV-infectedcellsarethe same, a competition RIPA was set up. Recombinant 6b

antigen was produced with recombinant vaccinia viruses vTF7-3 and vTF6b by the double-infection protocol (5).

A ["Simethionine [PSlcysteine

mock FIPV mock FIPV vTF vTF +

pTF6b

-.S + --+ - + - _ + - +

a

S

ma

a

am w

*-, __

O_

..

_61

JA keM

am~

d-b

B E7

N

M .6b

6a

6b

TK

L

T7

TKR

pTF6b

TK

L

6b

TKR

FIG. 1. Identificationofthe6bprotein.(A) Radioimmunoprecip-itationandSDS-PAGEanalysisoflysates from infected cells.The RIPAwascarriedoutwith asciticfluid containing FIPV anti-bodies. Virus-specific proteins S, N, M, and 6b areindicated. M protein bandsareindicatedwitharrowheads,and 6bprotein bands areindicatedwitharrows. Immunoprecipitatesweresplit; onehalf (+)wastreated with endoH,andtheother(-) servedas acontrol. FIPV-andmock-infectedcellswerelabeledat5 hpostinfection with [35S]methionineor[35S]cysteine.Recombinantvaccinia virus vTF7-3-infected cells andvTF7-3-infected,pTF6b-transfected cells (lanes vTFandvTF+pTF6b,respectively)werelabeled for 30minat16 h postinfection with [35S]cysteine. (B) Cloning of ORF 6b in a bacteriophage T7 expression vector. A SpeI-SalI fragment from cDNAcloneE7wasrecloned inpTUG31.Theresulting expression construct, pTF6b, contains FIPV ORF 6b flankedby a bacterio-phageT7 promoter(p) and terminator (T). Flankingvaccinia virus TKsequenceswereusedfor construction of recombinantvaccinia virus vTF6b.

Increasing amounts of lysate from unlabeled vTF7-3- and vTF6b-infected cells were added to constant amounts of

lysate from

[35S]cysteine-labeled

FIPV-infected cells. The samples were then immunoprecipitated. Addition of unla-beledrecombinant 6b proteinresulted inadecreased

inten-sity of the 26- to26.5-kDa doublet butnot of the FIPV M protein band(Fig.2A)orofthe bands of other FIPVproteins (19). In a control experiment, a similar competition RIPA wasdone withlysate from vTF7-3-infected cells transfected withpTFM, which contains the FIPV Mprotein gene under the control ofaT7 promoter(21).This resulted inadecrease in M protein band intensity only, demonstrating that both J. VIROL.

mm Mr

6t

so

on November 9, 2019 by guest

http://jvi.asm.org/

[image:2.612.317.552.76.416.2]

(3)

NOVEL FIPV GLYCOPROTEIN CONTAINS ER RETENTION SIGNAL

A rec. 6b protein

o '1/ 1 2 5 10

- M

~~

6b B rec. M protein

0 t/2 1 2 5 10

Omwvft~mw~ M

a*_

__

_m

6b

FIG. 2. Competition of immunoprecipitation with recombinant (rec.) 6b and M proteins. Lysate from recombinant vaccinia virus vTF7-3-infectedcells, superinfected with vTF6b (A) or transfected withpTFM(B),wasadded tosamples of[35S]cysteine-labeledlysate of FIPV-infected cells and processed forRIPA. Therelativeamount of unlabeled celllysateisindicated aboveeachlane. Virus-specific proteins(M and 6b)areindicated. Only the relevant parts of the gels areshown.

bands of the doubletareunrelatedtothe Mprotein (Fig.2B).

A second control experiment was performed with lysate

from vTF7-3-infected cells; at concentrations similar to those used forlysatefromvTF6b-and vTF7-3-infectedcells,

band intensities were not diminished for any FIPV protein (19). Theseexperiments demonstrate that ORF 6b encodes the26.5-kDa FIPVglycoprotein,which will be referredto as the 6bprotein fromnow on.

The 6bprotein isnot anintegralmembraneprotein. Apart

from the N-terminalsignalsequence,nohydrophobicregion which might serve as a membrane anchor was predicted from the 6b amino acid sequence. Unless thesignalsequence itself anchors the 6b protein, it is not expected to be an

integral membrane protein. Membrane fractionation was carriedout toinvestigatethispossibility.Crude fractionation of membranesby centrifugation resulted incomplete recov-ery of the recombinant 6b protein in the pellet (Fig. 3). Closed microsomal membrane vesicles were converted to open membrane sheets by carbonate treatment, leaving integral membrane proteins in the membrane and

releas-ing solubleproteins and peripheral membraneproteins (7).

After adjustmentof thelysate to100 mMNa2CO3(pH 11),

most of the 6b protein was detected in the supernatant

after centrifugation. The same analysis carried out for FIPV-infected cells resulted in the same fractionation pat-tern. The S and M proteins were convenient controls for

integralmembraneproteins,and bothwererecovered in the

pelletfractionatbothpH11 and7.5. Alargepartof the N

protein from FIPV-infected cellswas found in thepellet at

pH7.5;this resultprobablyreflects the association of theN

protein with viral membrane proteins. Similar resultswere obtained recentlyfor the N

protein

ofinfectiousbronchitis virus(16). Ourexperiments show that the6bprotein isnot an integral part ofmicrosomal membranes, but we cannot exclude thepossibilitythat it isperipherallyassociated with membranes.

The 6bprotein isasecretoryprotein.Preliminary

observa-tions indicated that the 6b

protein

is released into the medium of FIPV-infected cells. It remainedtobedetermined

a

_-~~~~i

_

A

M _0

FIG. 3. Membrane fractionation of 6b protein-producing cells. Cells transfected with pTF6b and infected with vTF7-3 [lanes pTF6b(+)]or infectedwith FIPV were labeled with[35S]cysteine. Membranes were pelleted after treatment at pH 7.5 or 11 as indicated. Membrane pellets and supernatants (lanes p and s, respectively)wereprocessedfor RIPA. FIPV S, N, andM struc-turalproteinsareindicated. The gels contained different concentra-tions of acrylamide, which explains why the 6b protein bands (indicated with arrowheads) did not comigrate.

whether the 6b protein is a structural protein. It might be attachedtovirusparticlesas aperipheralmembraneprotein

or by protein-protein interactions, which may have been disrupted in the carbonate extraction assay.

Therefore,

[35S]cysteine-labeled

viruswaspelleted from the medium of infected cells bycentrifugation; the pellet and supernatant

fractions were processed for RIPA. The 6b protein was found in the supernatant fraction but not in the pellet fraction,wherethe structuralproteinsaccumulated(Fig. 4).

Themigrationof 6bwasconfirmedbycoelectrophoresis of secretedrecombinant6bprotein. Itwasalsonot detectable insucrosegradient-purifiedviruspreparationsbyaWestern blot (immunoblot) assay(19). Consequently, the6bprotein

is not stably associated with extracellular virus particles producedintissue culture.

Posttranslational modificationsofthe 6b protein.As dem-onstratedabove, pulse-labeled6b proteinwas endo H sen-sitive (Fig. 1A); this sensitivity isindicative of N

glycosyl-ation. Inhibition ofglycosylationwith tunicamycinresulted in a 6b protein band of 24 kDa (19). After a 1-h labeling period, the 6bprotein appeared as adouble bandin FIPV-infected cells

(Fig.

1A). By using shorter pulse-labeling times, we found that the upper band of the doublet is the precursorand that the lower band isa processing interme-diatewhich appearedduringthesubsequent chase

(Fig.

5A and B) or during longlabeling periods. Afterlonger chase

times, the intermediate form was converted to the mature form. The precursor was fully

susceptible

to

digestion

by

endo H, whereas the intermediate and mature forms were resistant. Theintermediate andmatureformswereresolved best in the endo H-treated

samples.

Their

subsequent

ap-pearance in these

samples

showed the

precursor-product

pTF6b(+) 7.5 11 p s p s

FIPV

7.5 11

p s p s

dab

--*A0a

N

VOL. 66,1992 ₄₉₅₃

on November 9, 2019 by guest

http://jvi.asm.org/

[image:3.612.370.502.76.305.2] [image:3.612.137.238.77.247.2]

(4)

4954 VENNEMA ET AL. mock FIPV

14C

p s

p)

_{_~~le}6b

_w-

u r

A 0 15 30

_ + _ + - +

mm am

_U

_

_046A

_

60 90 120

_ +- + A+

ia'§

IA&-m

_ml

_

B 0 30 60

- + - + - 4.

FIG. 4. Viruspurification. Radioactivelylabeled viruswas puri-fied by centrifugation. Thepellet and supernatant (lanesp and s,

respectively) of mock-andFIPV-infected cellswereprocessedfor

RIPA. Secreted recombinant 6b protein and 14C-labeled marker

proteinswere run in parallel for comparison (lanes 6b and 14C, respectively).

relationshipdescribed above. Theinterpretationofthe anal-ysisof FIPV-infected cellswashampered bythepresenceof the Mproteinin thesame regionof thegel. To reducethis problem,we madeuse ofthe hydrophobicnature oftheM protein, which allowed extraction with Triton X-114 (17). Nevertheless, asmallamountof Mproteinremained inthe samples (Fig. 5A).Thematureform and thedeglycosylated form of the 6bproteincould beunequivocallyidentified.The mature secreted 6bproteinhadahigherapparentmolecular weight than the intermediate form (Fig. SC), which we

assumedtobe the result of terminalsialylation.Inagreement with this assumption, digestion with neuraminidase in-creased itselectrophoretic mobility (Fig. SC). Theanalyses whose resultsare showninFig. 5A and Bwere carriedout with lysates of cells combined with medium which were

prepared by addingconcentrated lysisbuffertothe culture medium (20); this was done to avoid underestimation of productformation due tosecretion into the chase medium. Acquisitionof resistancetoendo Hdigestionwasestimated

to be 80 to 90% complete after a 2-h chase period in FIPV-infected cells. Accurate measurements could not be made because of theinterferingM protein.In recombinant 6bprotein-producing cells, theprocesswas slower; aftera

3-h chaseperiod,morethan50%of the 6bproteinwasstill

endo H sensitive (see below).

ExpressionofC-terminally mutated 6bprotein genes.The 6bproteinC-terminalsequence,KTEL(3),isverysimilarto the KDELsignalofcellular residentendoplasmicreticulum (ER) proteins (14). We hypothesized that the KDEL-like

sequenceof the 6bproteincaused itspartialretention inthe ER.Therefore,we constructedamutated version of the6b proteingene encodingtheC-terminalsequenceKTEV. The alteration from leucine tovaline merelyremoves a

methyl-enegroupandpreserves thehydrophobicnature. Thesame

change has been demonstrated to abolish ER retention (1,

90 1 20 180

- + _ + -4.

JWLjML

C cells medium

[image:4.612.388.483.71.438.2]

- H N - H N

FIG. 5. Posttranslational modifications of the 6bprotein.EndoH resistance acquisition by the FIPV (A) and recombinant (B) 6b proteins isdepicted. Cells werepulse-labeled and chased for the indicated periods (in minutes). RIPAand endo H analyseswere

carriedout asdescribedinthelegendtoFig.1. Asmallamountof Mprotein remained in theFIPVsamples. (C) Oligosaccharideside chains of intracellularand secreted recombinant 6bprotein. Recom-binant vTF7-3- and vTF6b-infected cellswere pulse-labeled and chased for 3 h. Cells and mediumwere processedseparately for RIPA. Samplesweremocktreated,endoHtreated,or neuramini-dase treated,as indicatedby -, H, andN, respectively. Onlythe relevant partsof thegelsareshown.

22). The mutant construct, designated pTF6bV,was

com-paredwith theoriginalconstructinapulse-chaseexperiment (Fig. 6).Theproteinencodedby pTF6bV, the 6bVprotein,

wassecreted into the medium and became endo Hresistant

faster than thewild-typeexpression product.

Quantitation

of the resultswasdonebyliquidscintillationcountingof bands excised fromgels(Fig.

7).

The half-times of endo H resis-tance acquisitionwere 3 and 1 h for the

wild-type

and the mutated 6bprotein, respectively. After a3-h chaseperiod, 17%of thewild-type6bproteinwassecreted,compared with

50% of the 6bVprotein. Next, we changed theC-terminal

sequencetoKDELtodetermine whether this would confer

complete ER retention. The resulting

protein (the

6bD

protein)wasanalyzedinapulse-chaseexperiment

(Fig. 6C).

It was completely retained in the ER during a 3-h chase

J. VIROL.

VW _E

i,

40M &0464

owsm

on November 9, 2019 by guest

http://jvi.asm.org/

[image:4.612.134.220.75.300.2]

(5)

A KTEL KTEV

0 1 3 0 1 3

intra- _ d

cellular

secreted - _

B

₀ ₁ ₃

_ + _ + - +

KTEL w _ _

=

a S

_ _m _i

0 1 3

- + - + - +

KTEV ~=E

C

0 1 3

c m c m c m

0 1 3

_ +-_ + _-+

FIG. 6. Pulse-chase analysis ofwild-type and mutated 6b

pro-teins. Cellsinfected with vTF7-3were transfected withpTF6bor

pTF6bV (indicated by KTEL and KTEV, respectively),

pulse-labeledfor1h,andanalyzed immediatelyorafter chase times of 1

and 3h,asindicated.Analysiswasperformedas described in the

legendtoFig. 1. (A) Intracellular and secreted polypeptideswere

analyzed separately. (B) Lysates includingthe culture mediawere

analyzed byendo Hdigestion. (C)Similar experiments were

per-formed withpTF6bD. Cells (lanes c) and medium (lanes m)were

processed separately. Intracellularmaterialwassubsequently

ana-lyzed after treatmentwith endo H (lanes +) or mock treatment

(lanes -).

period;it couldnotbe detected in the medium and remained entirely endo Hsensitive. These experiments demonstrate that theoriginalC-terminalsequenceof the FIPV6bprotein conferspartialERretentionbythesamemechanismused for cellular ER-residentproteins.

100

100 -- E KTEL

90

KITEV

80 78

70

E ₆₀

-60~~~~~5

endo resistantsecred48

00

40 j30

0.

20 -16 1

12 15 1

105

0

pulse lh 3h lh 3 h

endo H resistant secreted FIG. 7. Quantitationof wild-type and mutated 6b protein trans-port.Immunoprecipitated6bproteins fromatleast twoexperiments werelocalizedbyfluorography;the bands were excised and quan-titated by liquid scintillation counting. The percentage of endo H-resistant materialwascalculatedasthe ratio of endoH-sensitive countstoundigestedcounts,multiplied by 100 andsubtractedfrom 100%.The secretedfraction,expressed as a percentage, was calcu-lated as 100times the ratio of secreted counts to total counts.

DISCUSSION

In this article, wereport theidentification and character-ization ofa newvirus-specific protein in FIPV-infected cells. Aglycoprotein of 26.5 kDa was detected in [35S]cysteine-labeledbutnotin[35S]methionine-labeled cell lysates, which explains whyit hadnot been observed beforesince previous labeling experiments were performed with [35S]methionine (4, 20). The protein was identified by recombinant gene expression of the second ORF of mRNA 6 (3), currently

designated ORF 6b. Further studies showed that the 6b protein isasolubleprotein contained in microsomes, that it is secreted from infected cells, and that it is not stably associated withvirusparticles in tissue culture medium.

Numerous ascitic fluid and serum specimens from natu-rally and experimentally infected cats immunoprecipitated the 6bprotein, whereas preinfectionseradidnot(19),which provesthat itis also produced in vivo and confirms its virus specificity. In infected cats, secreted 6b protein and antibod-iesagainst it may form immune complexes which play a role in the immunopathology of FIP. The function of the 6b protein in the life cycle of FIPV isnotclear. Itis apparently nonessential forTGEV, from which it iscompletely absent; the closely related feline and canine enteric coronaviruses bothproduce 6b-like proteins and have 6b-like ORFs (18). The canine coronavirusORF 6b is colinear withthat ofFIPV and has 58% identity at the amino acid level. The feline enteric coronavirus ORF 6b contains a deletion in the C-terminal half, reducing the colinear part to 60% of the FIPVORF 6b.

Detailedanalysisof its intracellular transportshowedthat the6bproteinwasreleasedslowlyfrom theER, particularly

in recombinantvaccinia virus-infected cells. The 6bprotein

C-terminal sequence, KTEL, is almost identical to the KDEL signal of resident ER proteins. This signal or a

closelyrelated sequenceat the C terminus ofaproteinwith

on November 9, 2019 by guest

http://jvi.asm.org/

[image:5.612.322.555.76.280.2] [image:5.612.108.253.77.531.2]

(6)

4956 VENNEMA ET AL.

anN-terminalsignal sequence is a strong indication that it is a resident luminal ERprotein (14). Proteins with a KDEL signal arerecognized by a receptor located in a compartment between the ER and the

Golgi

apparatus andrecycled back tothe ER.Site-directed mutagenesis of the signal to KTEV abolishedretention; the half-life of endo H resistance acqui-sition of the 6bVprotein was reduced threefold compared with that of thewild-type protein. The wild-type 6b protein wasnotcompletely retained in the ER, while mutagenesis of its C terminus to KDEL resulted in complete ER retention. Incomparison with transport of the recombinant expres-sion product, 6b protein transport was faster in FIPV-infected cells. Interference with the retrieval mechanism may be due to

assembly

of coronavirus particles in an intermediate compartment between the ER and the Golgi apparatus.Alternatively,the 6bproteinmay bindtransiently toimmature virions or viralproteins in the ER and thereby escape from retrieval.

TheFIPV6b protein is the firstexampleof aviralprotein

with afunctional KDEL-likeERretentionsignal.

ACKNOWLEDGMENTS

We thank RaouldeGroot for criticalreading of the manuscript. B. Moss is gratefully acknowledged for providing the recombinant vaccinia virusT7expression system.

H.V. and L.H. were supported byagrantfromSolvay-Duphar BV,Weesp, The Netherlands.

REFERENCES

1. Andres, D. A., J. D. Rhodes,R.L. Meisel, and J. E.Dixon. 1991. Characterization of the carboxyl-terminal sequences responsi-bleforproteinretention in theendoplasmic reticulum. J. Biol. Chem. 266:14277-14282.

2. Cavanagh, D., D. A. Brian, L. Enjuanes, K. V. Holmes, M. M.C.Lai,H.Laude, S. G.Siddell,W.Spaan,F.Taguchi, andP.J. Talbot. 1990. Recommendations of the Coronavirus Study Groupfor the nomenclature of the structural proteins, mRNAs, and genes of coronaviruses.Virology 176:306-307. 3. deGroot,R.J.,A. C.Andeweg,M.C.Horzinek,and W.J.M.

Spaan. 1988. Sequence analysis of the 3' end of the feline coronavirus FIPV 79-1146 genome: comparison with the ge-nome ofporcine coronavirus TGEVreveals large insertions. Virology167:370-376.

4. deGroot, R.J.,R.J.terHaar,M.C.Horzinek,and B.A. M. van derZeUst. 1987.Intracellular RNAs of the feline infectious peritonitis coronavirus strain 79-1146. J. Gen. Virol. 68:995-1002.

5. Fuerst, T. R., P.L. Earl,and B. Moss. 1987. Use ofa hybrid vaccinia virus-T7 RNApolymerase system for expression of target genes.Mol. Cell. Biol. 7:2538-2544.

6. Fuerst, T.R.,E. G. Niles, F. W. Studier, and B. Moss. 1986. Eukaryotic transient-expression system basedonrecombinant

vacciniavirus thatsynthesizes bacteriophageT7 RNA polymer-ase. Proc.Natl. Acad. Sci.USA 83:8122-8126.

7. Fujiki,Y.,A.Hubbard, S.Fowler, and P. B. Lazarow. 1982. Isolation of intracellular membranes bymeansof sodium car-bonate treatment: applicationtotheendoplasmic reticulum.J. Cell Biol.93:97-102.

8. Garwes, D.J., F. Stewart, andP.Britton.1989. Thepolypeptide ofMr 1400 ofporcine transmissiblegastroenteritis virus: gene assignment and intracellular location. J. Gen. Virol. 70:2495-2499.

9. Laemmli, U.K.1970.Cleavage of structuralproteins during the assembly of the head ofbacteriophage T4. Nature (London) 227:680-685.

10. Mackett, M., G.L.Smith, andB.Moss.1984. General method forproduction and selection of infectious vaccinia virus recom-binantsexpressing foreign genes. J. Virol. 49:857-864. 11. Maniatis,T., E. F. Fritsch,andJ.SambrooL 1982.Molecular

cloning: alaboratory manual. Cold Spring Harbor Laboratory, ColdSpring Harbor,N.Y.

12. McKeirnan, A. J., J. F. Evermann, A. Hargis, L. M. Miller, and R L. Ott. 1981. Isolation of feline coronavirus fromtwo cats with diverse disease manifestations. Feline Pract.11:16-20. 13. Neuberger,A.,A.Gottschalk,R.0. Marshall,andR. G. Spiro.

1972.Carbohydrate-peptide linkages in glycoproteins and meth-odsfor theirelucidation, p. 450-490.In A.Gottschalk(ed.), The glycoproteins: their composition, structure and function. Elsevier, Amsterdam.

14. Pelham, H.R.B. 1990. The retention signalfor solubleproteins of theendoplasmic reticulum. Trends Biol. Sci. 15:483-486. 15. Rosenberg,A.H.,B. N.Lade, D.-S.Chui, S.-W. Lin, J. J. Dunn,

andF. W. Studier. 1987. Vectors for selective expression of clonedDNAsbyT7 RNApolymerase. Gene 56:125-135. 16. Smith, A. R., M. E. G. Boursnell, M. M. Binns, T. D. K. Brown,

and S. C. Inglis. 1990. Identification of a new membrane-associatedpolypeptide specified by the coronavirus infectious bronchitis virus. J. Gen.Virol. 71:3-11.

17. Sturman,L.S.,K.V.Holmes,andJ.Behnke.1980.Isolation of coronavirus envelope glycoproteins and interaction with the viralnucleocapsid. J. Virol. 33:449-462.

18. Vennema, H.1991. Ph.D. thesis. State University of Utrecht, Utrecht, The Netherlands.

19. Vennema,H., and L.Heinen.Unpublisheddata.

20. Vennema,H., L.Heinen,A. Zlderveld,M. C. Horzinek, and W.J. M. Spaan. 1990. Intracellular transport ofrecombinant coronavirus spike proteins: implications for virus assembly. J. Virol. 64:339-346.

21. Vennema, H., R.Rjnbrand,L.HeUnen,M. C. Horzinek, and W.J.M.Spaan.1991.Enhancement of the vaccinia virus/phage T7 RNA polymerase expression system with encephalomyo-carditisvirus 5'-untranslated region sequences. Gene 108:201-210.

22. Zagouras,P.,andJ.K. Rose.1989.Carboxy-terminal SEKDEL sequences retard but donotretaintwosecretoryproteins in the endoplasmic reticulum. J.Cell Biol. 109:2633-2640.

J.VIROL.