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Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

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0022-538X/94/$04.00+0

Copyright © 1994,American SocietyforMicrobiology

Identification and Characterization

of the Cell

Surface

70-Kilodalton

Sialoglycoprotein(s)

as a

Candidate

Receptor for

Encephalomyocarditis

Virus

on

Human Nucleated Cells

YI-MING JIN, INGRID U.PARDOE,ALFREDT. H. BURNESS,t ANDTOMASZ I. MICHALAK*

Division of Basic Medical Sciences, Faculty of Medicine, Health SciencesComplex,Memorial University of Newfoundland, St. John's, NewfoundlandAlB 3V6, Canada

Received 23March 1994/Accepted 15 August1994

Theattachment ofencephalomyocarditis(EMC)virustohuman nucleated cellssusceptibletovirusinfection

was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively

blocked by unlabeled virions. The number ofbinding sites for EMC virus on HeLa and K562 cells were

approximately1.6 x 105and 3.5 x 105percell, respectively,and dissociationbindingconstantswere1.1and 2.7nM, respectively. Treatment ofcellswithcycloheximide afterpretreatmentwithtrypsineliminated EMC virusattachment, suggestingthat thevirus-binding moietyisproteinaceous innature.Digestionofcells,cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or

treatmentof cells with lectins demonstrated that theEMCvirus-cell interaction is mediatedbya

sialoglyco-protein.Proteins withamolecularmassof70kDawereisolatedfromdetergent-solubilizedcell membranes of

both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their

70-kDa-molecular-mass equivalentsdetected in intact surface membranes of HeLa and K562cells, specifically bound EMCvirusinavirusoverlay proteinblotassay,whereas membranes fromnonpermissiveK562D clone cellsdidnot.WesternimmunoblotanalysiswithglycophorinA-specific antibodyconfirmed thatthe identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on

virus-nonpermissive human erythrocytes (HeLa cellsdo notexpressglycophorinA). Theseresultsindicatethat

EMC virus attachment topermissive human cells is mediated by a cell surface sialoglycoprotein(s) witha

molecularmassof 70kDa.

Encephalomyocarditis (EMC) virus belongs to the genus

Cardiovirus of the picornavirus family. This small, nonenvel-oped,RNAvirusisanaturalpathogeninmice,although italso infectsmanyother species,including humans (21, 50, 58). The

picornavirusesareimportant in studiesonthepathogenesis of

human diseases, since in experimental animals they are

etio-logically associated with pathological processes that mimic

insulin-dependent diabetes mellitus (36), myocarditis (22), viral myositis (50), and demyelinating centralnervoussystem

diseases (31).

Toinfectacell, the virusmustattachtothe cellsurface. This usually highly specific virus-cell interaction involves the

recog-nition by a viral component of a cell membrane element, representing the cellular receptor for the virus. The

attach-mentisconsideredamajordeterminant of viral hostrangeand

tissue tropism. Diversecell surface moleculesor evenhostor

foreign moleculesinteractingwith cellmembranes have been identified as virus receptors. For a fewvirus receptors, their physiological function has also been defined. For example, intracellular adhesion molecules (ICAM-1), CD4 molecules, andclass Imajorhistocompatibility complex molecules,which all belong to the immunoglobulin superfamily, are receptors

for the major serotype of human rhinoviruses (59), human immunodeficiencyvirustype 1 (60), and simian virus 40 (12), respectively. The acetylcholine receptor for rabies virus (28) and theC3dreceptorCR2 for Epstein-Barr virus (9)areother

*Corresponding author. Phone: (709) 7301. Fax: (709)

737-7010. tDeceased.

examples of well-characterized cellular molecules acting as

viral attachmentsites.

The attachment molecule for EMC virus on human

eryth-rocytes is glycophorin A, the major sialoglycoprotein of the erythrocyte surface membrane, and sialic acid is the residue involved in thevirusbinding (4, 56). However, human eryth-rocytes do not support EMC virus growth, in contrast to a

number of human nucleated cells in which the virusreadily replicates (35, 47, 51). The nature of the cellular receptor determining EMC virus attachment to these cells remains unknown. Recently, vascular cell adhesion molecule 1 (VCAM-1)has been identifiedas areceptorfor theDstrainof EMC virus onmurine vascularendothelial cells derivedfrom

the heart(30).Since this molecule is restrictedtothe vascular endothelium(52),receptorsother thanVCAM-1mustexiston avariety ofother cells which are susceptible to EMC virus infection. In the course ofthe present study, we found that

EMC virusbindingto twotypesof viruspermissivehuman cell lines, HeLaandK562,whichdonotexpressVCAM-1(37, 55),

is mediated by 70-kDa cell membrane sialoglycoproteins but notby glycophorinA.

MATERMILS AND METHODS

Viruses. The K2 strain ofEMC viruswasgrown in Krebs ascites tumorcellsunder conditionsdescribedpreviously(14). Viruswaslabeledbyadding [3H]leucine (1.0 ,uCi/ml; DuPont, NEN Research Products, Boston, Mass.) to the culture

me-diumduringvirusgrowth (14).The viruswaspurifiedandthe

concentration was determined by UV absorbance

measure-ments.The number of virusparticleswas calculated by using

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the extinction coefficient and particle weight as previously published (13, 14). Specific radioactivity ranged from 800 to 1,000 cpm/,ug of virus.

Poliovirus serotype 1 Mahoney was kindly provided by Y. Svitkin and N. Sonenberg, Department of Biochemistry, McGill University, Montreal, Canada. This virus was grown in HeLa cells and purified essentially as described previously

(34). The number of virus particles was calculated from UV

A260 and was found to be in the same range as that of EMC virus, namely 9 x 10'sparticles per ml.

Celllines. Humanerythroleukemic K562 cells (44) and their mutantK562D clone cells resistant to EMC virus superinfec-tion (51) were grown in suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin per ml, and 100 ,ug of streptomycin per mlat37°C in ahumidified5% CO2 atmosphere. The cells were harvested by

centrifugationwhentheirdensitiesreached about106cells per

ml. HeLacells, an adenocarcinoma cell line (33),were prop-agated in monolayer cultures in Dulbecco's modified Eagle medium(DMEM) supplemented with 5% fetal calf serum and with the same antibiotics described above. HeLa cells were collected, when the monolayerswereabout90% confluent,by scraping them into DMEM with a rubber policeman. The number ofcells and theirviability were determined by using

0.1% trypan blue and countingunstained and total cells in a hemocytometer. Priortouse,the cellswerewashed three times with cold phosphate-buffered saline (PBS) (pH 7.4). The susceptibility of both K562 and HeLa cells to EMC virus infection has been establishedpreviously (35, 51).

Enzyme and lectin treatment of cells. Samples containing about 2 x

106

K562 orK562 D clone cells in suspension or HeLa cells inmonolayerwere treated with different enzymes orlectins. Eachtreatment wasperformedin0.5 ml of PBS for 1 h at 37°C. After treatment, cells were washed three times with cold PBSorHanks' balanced salt solution without

Ca2+

and

Mg2+

(HBSS)to removeenzymesand reducepossiblecell

aggregation. The following enzymes and lectins were used:

trypsinfrom bovine pancreas(20,ug perreaction; activity, 110

U/mg; Worthington Biochemical Co.,

Freehold, N.J.),

a-chy-motrypsinfrom bovine pancreas (20,ug per reaction; activity,

90 U/mg; CalBiochem, La Jolla,

Calif.),

papain fromPapaya

latex (1 U perreaction; Sigma ChemicalCo., St. Louis,

Mo.)

preactivatedwith 0.38 ,ug ofL-cysteine hydrochloride

(Gibco

BRL, Grand Island,

N.Y.)

in 5

RI

of 0.1 MEDTA,

phospho-lipase C from Clostridiumwelchii

(40

U perreaction;

activity,

50 U/mg; CalBiochem), neuraminidase from Vibrio cholerae

(40mU perreaction;

activity,

20U/mg;

Boehringer

Mannheim

Canada, Laval,

Quebec)

andfrom Arthrobacter

ureafaciens (40

mU per reaction; activity, 25 U/mg; Boehringer

Mannheim),

both used in PBS adjusted to pH5.6 with 0.1 N HCl,wheat

germ agglutininfrom Triticum

vulgaris (0.5

mg per

reaction;

Sigma Chemical

Co.),

and

Limuluspolyphemus

agglutinin

from

thehorseshoe crab

(0.5

mg perreaction;

Sigma

Chemical

Co.).

Ascontrols, cellsampleswereincubated with PBS withouttest enzyme or lectin. In the case of protease

digestion,

which detaches theHeLacellmonolayerfromthe culture

dish,

cells

predestinedforcontrolexperimentswereremovedby

scraping

and then treated by the same method as that used for cells grown insuspension.

Cycloheximide treatmentoftrypsin-digested cells.

Samples

of 2 x

106

HeLa and K562 cells

pretreated

with 20

jig

of trypsin for1hat37°Cwerewashed in severalchangesof HBSS and incubated in 2 ml of fresh culture medium

containing

2to

12 ,ugofcycloheximide

(Sigma

Chemical

Co.)

for 8h ina5% CO2

atmosphere

at

37°C.

Control cellsweretreated

similarly

but without cycloheximide. Attachment of

3H-labeled

EMC virusto the cellswasdetermined aspresented below.

Virus-bindingassay.In mostcircumstances, approximately5

,ug

(5,000

cpm)of

3H-labeled

EMC viruswasaddedto2 x 106 K562orK562Dclone cells untreatedortreated with enzyme,

lectin, or cycloheximide, and the cellswerewashed and

sus-pended

in 100 ,ul of PBS. The same amount ofradiolabeled viruswasaddedtountreatedortreated HeLa cellmonolayers,

eachcontainingabout 2 x 106cells ina35-mm-diameter dish.

Priortovirusaddition,HeLacellswererinsed with cold PBS and then 0.5 ml of PBSwasaddedtoprovideavehicle for virus

dispersion.

After 30 min of incubationon

ice,

unless otherwise

indicated,allsamplesweredilutedto1ml withPBS. The K562

and K562D clone cellswerepelleted by centrifugation at300 xgfor 15 minat4°C, and the resulting supernatantwassaved, whereas the supernatants from HeLa cell monolayers were decanted and saved. Each cell

sample

waswashed with1mlof cold

PBS,

andthe washwassaved.

Finally,

the cellswere

lysed

in 1 ml of 1% Triton X-100 at ambient temperature. The

radioactivity

in

supernatants, washes,

and the

lysed

cell

sam-ples, constituting the total recovered

radioactivity,

was

mea-sured in a scintillation counter, after 10 ml of

Aquasol-2

(Dupont,

NENResearch

Products)

had been added. Attach-ment of virus to cells was

expressed

as a percentage of virus-bound

radioactivity

relativeto the totalrecovered

radio-activity.

To determine the time course of EMC virus

binding,

ap-proximately

5 ,ugof labeled viruswas incubated with 2 x 106

HeLa or K562 cells for 2.5 to 60 min on ice.

Saturability

of

binding

was

analyzed by

incubation of

increasing

amounts of

3H-labeled

virus

(0.4

to 50

jig)

with a constantnumber

(2

x

106)

of cells for 30 min on

ice,

followed

by

measurement of both bound and unbound virus.

Specificity

of

binding

was

demonstrated in

competition experiments,

in which 2 x

106

cells were

preincubated

on ice for 30 min with different amountsofunlabeled virus

(approximately

50 to500

,ug)

ina

final volume of 500

[lI,

then

supplemented

withlabeled virus

(approximately

5

pug),

and incubated for 2.5 to30 minonice.

Preparation and solubilization of cell membranes.

Mem-braneswere

prepared

from cells

disrupted by

hypotonic

shock and

subsequent

homogenization.

Nuclei and cellular debris wereremoved

by

centrifugation

at

1,000

xgfor 30 s, and cell membraneswere

pelleted

at

45,000

xgfor 60 min inaTiS0.1 rotor

(Beckman Instruments, Inc.,

Palo

Alto,

Calif.)

as de-scribed

by

Atkinson and Summers

(7).

Isolationwas done in the presence of2 mM

phenylmethylsulfonyl

fluoride

(PMSF)

and 2mM

N-ethylmaleimide

asprotease inhibitors. Membrane

pellets

were

resuspended

in PBS

supplemented

withprotease

inhibitors,

and the

protein

contentwasmeasured

by

the

Lowry

method with bovine serumalbumin

(BSA)

as astandard

(43).

Cell membranes

(1.5

mgof

protein

per

ml)

were solubilized inan

equal

volumeof 12mMsodium

deoxycholate

(DOC)

in 20 mM sodium

phosphate

buffer

(pH 8.0)

on ice for 30 min. Insoluble membrane residueswereremoved

by

centrifugation

at

16,000

xgfor 5 min. Soluble membrane

proteins

contained in the

supernatant

were diluted with an

equal

volume of sodium

phosphate

buffer

containing

protease inhibitors and storedat

-20°C.

Cell membranes from human type 0

erythrocytes

were

prepared

from

recently

outdated

packed erythrocytes

provided

by

the Canadian Red Cross Blood Transfusion Service

(St.

John's,

Newfoundland,

Canada), by

hypotonic

lysis

(23).

Gly-cophorins

were

purified

from

erythrocyte

membranes

by

the lithium

3,5-diiodosalicylate-phenol

procedure

of Marchesi and Andrews

(45).

Radioiodination. EMC

virus,

isolated cell

membranes,

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cophorins, affinity-purified preparations of EMCvirus-binding

proteins (see below), and intact cells were labeled with

carrier-free Na1251 (200,uCi per labeling; activity, 10

,uCi/,ul;

Amer-sham Canada Ltd., Lachine, Quebec, Canada) by the IODO-GEN method (Pierce Inc., Rockford, Ill.) (46). Radioiodinated EMC virions, cell membranes, glycophorins, and

affinity-purifiedproteins were separated from free

125j

by gelfiltration

onSephadexG-25columns. In the case of surface radiolabeled

cells,morethan95% of the cells remained viable afterlabeling

asdetermined by trypan blue exclusion. The cells were washed three times with PBS to remove unbound 1251 and used

immediately to prepare cell membranes following the

proce-dure described above. Under our standard conditions, more than80% of theradioactivitywas recovered in cell membrane

preparations.

Preparation of EMC virusaffinity columns. Purified EMC

virus(1mg) was coupled with CNBr-activated Sepharose 4B (1

g)

(Pharmacia LKB, Uppsala, Sweden), by using conditions

recommended by the supplier. After conjugation, unoccupied sitesontheSepharose beads were blocked with 0.2 M glycine.

Non-covalently-bound virus was removed by three alternate

washes in 0.1 M sodium acetate buffer (pH 4.0) and 0.1 M sodium bicarbonate buffer (pH 8.3), both containing 0.1 M NaCl. Thevirus-Sepharose conjugate was packed into a Phar-macia column (220 by 9 mm) and stored at

4°C

in the presence of 20mM sodiumphosphate buffer (pH 8.0) until use.

Affinity chromatography. Chromatography was carried out as described previously (3). Briefly, DOC-solubilized unla-beled cell membranes or solubilized cell membranes derived from

1251I-labeled

cells were applied onto the EMC

virus-Sepharose column in the presence of 6 mM DOC and

incu-bated for 30 min at room temperature. Unbound material was removed byextensive washing with 20 mM sodium phosphate buffer (pH 8.0) until no further protein was detected when monitored at A280 in a UV monitor. Bound components were eluted in the same buffer containing 0.2 M NaCl. The column wasreused afterwashing with 0.1% Triton X-100 in phosphate buffer andreequilibrationwith phosphate buffer alone.

Chro-matography was performed at a flow rate of 0.4 ml/min.

Fractions(1 ml) were collected, and their protein content was monitored at A280. Forradiolabeledpreparations, radioactivity of thefractions was measured in a gamma counter. Protein or radioactivity peak fractions were combined, dialyzed against several changes ofdouble-distilleddeionized water at

4°C,

and

lyophilizedforfurther analysis.

Enzyme treatment ofcell membranes and affinity-purified EMC virus-binding proteins. Membrane protein

(about

280

jig)

orDOC-solubilized membranes derived from1'I-labeled

cells (3.5 x 105 cpm; approximately 30 jig of protein) were treated with 200jigoftrypsin in PBS (1 mg/ml) for 1 h at

37°C.

Subsequently, the enzyme was inactivated by the addition of

lima bean trypsininhibitor (200 jig per reaction; Worthington Biochemical Co.). Controls were treated similarly but without

trypsin. Following treatment, unlabeled membranes were

testedfor virusreceptor activity by a dot blot protein-binding assay,whereasradiolabeledmembranes were analyzed by virus

affinity chromatography. For the affinity-purified EMC

virus-binding material, about 1 ,ug (2 x 10 cpm) of

I251-labeled

proteinderived fromDOC-solubilizedHeLa or K562 cells was

treatedwith 20 jigoftrypsin or a-chymotrypsin as described

for cell membranes and then analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

autoradiography.

For neuraminidase treatment of cell membranes, approxi-mately 280 jig of membrane protein was incubated with 80 mU of Vcholerae neuraminidasein200jil of PBS, adjusted to pH

5.6, at

37°C

for1h. The enzymewasinactivated

by

theaddition of 100 jil of 0.1 M EDTA. The membranes were pelleted

by

centrifugation, and receptor activity was determined in both

pelletsand supernatantby adot blotprotein-binding assay. In some experiments, the membranepelletswere washed exten-sively in PBS containing 2 mM EDTA and 1 mM PMSF and digested again with neuraminidase, and the

virus-binding

activity was reexamined. Also, DOC-solubilized membranes from

125I-surface-labeled

cells (about 3.5x

105

cpm) were treated with V cholerae neuraminidase under the conditions described above. The enzyme was inactivatedbyboiling for 5

min,

and the sample was analyzed by chromatography on an EMC virus-Sepharose column. Neuraminidase treatment of the affinity-purified moiety was done by using

105

cpm of

125I-labeled

protein and 80 mU of enzyme under the condi-tions presented above. The enzyme was inactivatedbyboiling

and analyzedby chromatofocusing.

Dot blot protein-binding assay. Twofold serial dilutions of proteins of intact and detergent-solubilized cell membranes, enzyme-treated and control membranes, and preparations of cellular proteins purified on EMC virus affinity columnswere applied onto a nitrocellulose membrane (0.45-jim pore size; Bio-Rad Laboratories, Richmond, Calif.) by using a dotblot microfiltration apparatus (Bio-Rad Laboratories) (49). After immobilization of samples by gravity filtration for 30

min

and removal of excess liquid under vacuum, the nitrocellulose was removed from the apparatus, incubated in PBS containing 3% BSA and 0.05% Tween 20 for 2 h to prevent nonspecific binding, washed, and probed with 2x

106

cpm of

"25I-labeled

EMC virus suspended in PBS with 1% BSA and 0.05% Tween 20. After extensive washing, the blots were air dried and exposed to Kodak XAR-5 film.

SDS-PAGE. Membranes, preparations eluted from EMC virus affinity columns, and purified glycophorins were sepa-rated under reducing conditions on 10% separating gels, essentially as described by Laemmli (40). Most experiments were performed by using a Protean II apparatus, but some employed a Mini-Protean II apparatus (Bio-Rad Laborato-ries). Prestained high-molecular-weight protein standards (BRL, Gaithersburg, Md.) or

14C-methylated

molecular weight protein standards (Amersham Canada) were run in parallel on all gels. Protein bands were visualized by Coomassie blue staining or by autoradiography.

Virus overlay protein-binding assay (VOPBA) and Western immunoblotting. Following SDS-PAGE, proteins were elec-trophoretically transferred from the gel onto 0.45-jim-pore-size nitrocellulose filters using either a Trans-blot cell appara-tus (Bio-Rad Laboratories) at 60V and 200 mA for 6 h or a semidry Bio-Trans Maxi unit (Gelman Sciences Co., Ann Arbor, Mich.) at a constant current of 180 mA for 1 h. The nitrocellulose was incubated with PBS containing 3% BSA and 0.05% Tween 20 for 2 h to block nonspecific binding sites and rinsed in a few changes of PBS. To determine molecular species of proteins specifically recognized by EMC virus, the blot was incubated with 2 x 106

cpm

of

1251I-labeled

EMC virus (approximately 1 jig) in 10 ml of PBS with 1% BSA and 0.05% Tween 20 for 16 to 18 h at

4°C

or for 2 h at ambient temperature on a rocking platform. In competition experi-ments designed to determine the specificity of

1251I-labeled

EMC virus binding to the identified protein bands, the nitro-cellulose blot was first incubated with an excess (30 jig) of unlabeled EMC virus or poliovirus suspended in 10 ml of PBS containing 1% BSA and 0.05% Tween 20 for2 h at ambient temperature and then probed with

1251I-labeled

virus by using the conditions described above. The blot was washed three

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times with 100mlof 0.05% Tween 20 in PBS foratotal of 45

min,air

dried,

andexposed tofilm.

ForWestern blotidentificationofglycophorin A expression,

the nitrocellulose blots were incubated for 2 h at ambient

temperaturewithmousemonoclonal glycophorin A

anti-body (generously

provided by D. J. Anstee, Blood Group Reference

Laboratory,

Radcliffe Infirmary, Oxford, United

Kingdom)

diluted 1:10 with PBS containing 1% BSA and 0.05% Tween 20. The

antibody

recognizes amino acids in

positions

34to39on

glycophorin

A

(6)

andblocks EMC virus attachment to human

erythrocytes

(16).

After being washed,

the membranes were incubated with

12I1-labeled

sheep

anti-mouse

immunoglobulins (Amersham

Canada) diluted 1:1,000 in PBS

containing

1% BSA and 0.05% Tween 20 for 2 h at ambient temperature or

overnight

at 4°C. Subsequently, the

blots werewashedas described for theVOPBA, andbinding

was visualized

by autoradiography

after 3 days of exposure. Human

erythrocyte

membranes and purified glycophorins were usedas

positive

controls. Substitution of the

anti-glyco-phorin

A

antibody

withPBS

supplemented

with1% BSA and

0.05% Tween 20wasusedtoinsure that anti-mouse antibody

didnot reactwith cellmembraneproteins.

Chromatofocusing. Chromatofocusing

of

affinity-purified

vi-rus-binding proteins

derived from both HeLa and K562 cells was

performed

on columns

(220 by

9

mm)

of

Polybuffer

exchanger

PBE 94

(Pharmacia).

About 0.5 ,ug

(105 cpm)

of

125I-labeled proteins purified by affinity chromatography

on EMC virus columns and radiolabeledpreparationsof thesame

purified

proteins desialylated by

treatment with V cholerae

neuraminidase were

separately applied

onto

exchanger

col-umns and allowed to adsorb for 5 min. The columns were washed with

Polybuffer

74

(Pharmacia),

diluted 1:8 with deion-ized water, and

adjusted

to

pH

4.0 with 0.1 N

HCl,

at aflow rateof 0.4

ml/min.

Fractions of1mlwere

collected,

and their

pHs

and radioactivitieswere measured. RESULTS

Kineticsof EMCvirus

binding.

The

dynamics

of the EMC virus interaction with HeLa and K562cellswas

analyzed by

a

virus-binding

assay in which 3H-labeled

purified

virionswere incubated on ice with intact cells. The results showed that EMC virus

binding

to HeLacells increased almost

linearly

in theinitial 10to 12minandachievedamaximumwithin 20to 30min,with about 50% of the virions bound

(Fig. 1). Longer

incubation times didnot increase theamountof virus

bound,

indicating

that the

binding

was

complete

in

approximately

30 min.Thetimecourseof EMC virus

binding

toK562 cells

(data

not

shown)

was

closely comparable

tothat

presented

for HeLa cells.

Nonpermissive

K562Dclone cellswereresistanttoEMC virus

binding

under all testedconditions.

Competition

assayswereusedtodemonstrate the

specificity

ofthe EMC virus interaction with the cell surface. Preincuba-tion of HeLa cells withanexcessof unlabeled

virus,

followed

by

measurement of the 3H-labeled virus

binding

at different

time

points,

showed that a 10-fold excess ofunlabeled virus

(approximately

50

,ug)

inhibited

binding

by approximately

70% within 20 min, whereas a 30-fold excess of unlabeled virus

(approximately

150

,ug)

blocked about95% of the

binding

in thesametime

period

(Fig.

1).

Nofurther inhibitionof

3H-virus

binding

wasobserved

by

preincubation

of cells witha100-fold

excess of unlabeled virus

(approximately

500

,ug) (data

not

shown).

Increasing

the incubation time with labeled virus did notaffect theinhibition rates,

suggesting

that the

competitive

displacement

of unlabeled virions

by

labeled virus from the cell surfacewasveryslow

and,

thus the virus

binding

was

relatively

60-

50-0

-2

30-

20-

10-0 10 20 30 40 50 60

Time (minutes)

FIG. 1. Time course and competition of EMC virus binding to HeLa cells. Aliquots of2 X 106 cells were incubated on ice with purified3H-labeledvirus (approximately5

Rg)

(0) orpreincubated witha10-fold(A)or30-fold(A)excessofcoldvirus(approximately50 and150 ,ug,respectively)onice for 30min,washed, and incubated with 3H-EMC virus (approximately 5 ,ug). Virus binding to cells was determinedatthe timesindicated andexpressedas apercentageof the virus bound relativeto the total recoveredradioactivity. Eachpoint representsthemean± standarddeviationfor four determinations.

tight. Virtually identical resultswere obtainedby using K562 cells(datanotshown).

Insaturation assays, increasingamountsof3H-labeledEMC virus were incubated with a constant number (2 X 106) of HeLacells orK562 cells, and bound and unbound viruswas measured.Plottingthe number of bound virusparticlesagainst the number of unbound virus particlespercell, asshown for HeLa cells in Fig. 2, gave a monophasic saturation binding

curve, suggesting thatvirusbindingwas mediatedbya single class of saturable receptor. When the data in Fig. 2 were

replotted in the form of a Scatchard plot (Fig. 2, inset), a

straight line was obtained, supporting the conclusion that a

single class of EMC virus-binding sites is present on HeLa

cells.The total number ofreceptorsites thatcanbeoccupied on aHeLacellwasestimated from the maximalbindingvalue

(Bm.)

and was found to be 1.6 X 105. The equilibrium

dissociationconstant(Kd)wascalculated from theslopeofthe Scatchardplottobeapproximately1.1 nM. These resultswere

comparable to those obtained for K562

cells,

which gave an

estimated 3.6 X 105bindingsites per cell withaKdof 2.7 nM.

Enzymaticsensitivityand lectin interference of EMC virus

bindingtoHeLa and K562 cells. The biochemicalnatureof the

virus-bindingsubstanceon the cellsurfacewas

partially

char-acterized byincubation ofHeLa andK562 cellswith trypsin,

aL-chymotrypsin, papain, phospholipase C,or neuraminidases.

Cell viability after enzyme

digestion

was not impaired, as

determined by trypan blue

staining.

As shown in Table 1,

treatmentwiththree different proteases resultedinan

approx-imately80% reduction of EMC virusbindingtoHeLa cells and

in a 60 to 80% reduction for K562

cells,

compared

with untreatedcontrol cells. These data indicate thatacellsurface

protein is an essential component of the EMC

virus-binding

moietyoneither cell type. Incontrast,

phospholipase C,

which

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0

a.

~0

0

1.4 1.2

1.0

0.8 0.6 0.4 0.2

0

FreeParticles, 105/Cell

FIG. 2. Saturability ofbinding of EMC virus to HeLa cells. In-creasedamountsof3H-EMC virus(specific radioactivity,1.1 X10-8to 1.4 x 10-8 cpm per virus particle) were incubated with a constant number of HeLa cells(2x106)onice for 30 min. The kineticsofEMC virusbinding wasdeterminedby plotting the number of bound virus particlesagainstthe number of unbound virusparticlespercell. Each point is the mean ± standard deviation for four determinations. (Insert)Scatchardplot ofbindingdata. The line of bestfittothedata pointswasdeterminedbyacomputerizedlinearregression.

affectslipid components of cell membranes, had noeffecton virus binding. Treatment of cells with V cholerae neuramini-dase, which hydrolyzes bothox(2-3)and

c(x2-6)

linkagesof sialic acid, and with neuraminidase from A. ureafaciens, which preferentially cleaves ot(2-6)-linked sialic acid, resulted in a comparable reductioninvirusbinding ranging from 70to80%

in both cell lines, demonstrating that sialic acid residues are required for the attachment of EMC virions. In agreement with thisresult, the pretreatment of HeLa and K562 cells with wheat germ agglutinin and L.polyphemus agglutinin, both of whichrecognize and bindtosialicacids, produced,onaverage,

90% reduction of the cell-binding capacity for EMC virus compared with untreated cells (Table 1). The sensitivity of EMC virusbinding to both proteases and neuraminidases and

TABLE 1. Effectof enzymes and lectinsonEMC virus bindingtoK562 and HeLacellsa

%Virusbindingto:

Treatment

HeLacells K562cells

None 100.0 100.0

Enzyme

Trypsin 13.1 ±4.1 25.4±4.9

Chymotrypsin 14.6 ±1.4 38.4±6.6

Papain 15.4±2.5 17.1±2.2

PhospholipaseC 98.6 ± 2.0 98.4±4.7

Vcholerae neuraminidase 29.2 + 4.8 29.2 ± 4.8 A. ureafaciensneuraminidase 22.5 ± 4.0 25.0 ± 4.4 Lectin

WGAb 11.8 ±3.0 10.6± 3.0

LPAC 10.6±1.5 8.1 + 2.9

1K562orHeLa cells were treated with the indicated enzymes or lectins under conditions described in Materials and Methods.Bindingof3H-labeledvirus to the treated cellswas expressed as apercentage of the binding to untreated controls. Each value is the mean±standard deviation forfour determinations.

bWGA,wheat germagglutinin.

'LPA,L.polyphemusagglutinin.

inhibition of virus

binding by

lectins indicate the involvement ofa

sialoglycoprotein

in the

virus-binding

site.

Effect ofenzymatictreatmentof isolatedcellmembraneson

EMC virus recognition.To be moreconfident that the virus attachment ismediated

by

cell surface

sialoglycoproteins,

cell membranes isolated from HeLa and K562 cellswere treated

with trypsin or V cholerae

neuraminidase,

and the

virus-binding activity

was determined in adot blot

protein-binding

assay.

Trypsin

treatmentdecreased the

binding activity by

8-to 16-fold both in HeLa and K562 cell membranes

compared

with that in untreated

membranes,

whereas

digestion

with

neur-aminidase resulted in 8- and64-fold reduction of EMC virus

bindingtoHeLa and K562 cell

membranes,

respectively

(Fig.

3). Although

virus

binding

to cell membranes was

greatly

reduced aftera

single digestion

with

neuraminidase,

itwasnot

totally

abolished. Double neuraminidase

digestion

was per-formed to test whether the

remaining

activity

could be

de-stroyed.

The results showed that the second

digestion

of the same HeLa or K562 cell membranes further reduced virus

binding

by

two-to

eightfold

toalmost

background

levels

(Fig.

3).

Moreover, supernatants

resulting

from either

single

or

double neuraminidase

digestion

of the membranes did not

show EMC

virus-binding

ability

(Fig. 3).

As determined

by

high-performance

liquid chromatography

(data

not

shown),

these supernatantscontained released sialic acids. This obser-vation may suggest that free sialic acid alone doesnotserve as

the virus receptor

moiety.

Overall,

the data obtained from

digestion

of cell membranes with protease and sialidasewere

consistent with thoseobtained from the

enzymatic

treatment of intact HeLa and K562cells and confirmed that the EMC

virus-binding

site isacell surface-associated

sialoglycoprotein.

Blockage of EMC virus binding to the cell surface by

cycloheximide. It was found that both

trypsin-treated

HeLa

and K562 cells were able to

fully

restore their EMC

virus-binding capacity

within6to8 h of incubation in fresh medium after

trypsin

had been removed

(data

not

shown).

To deter-mine whether such recovery was due to

synthesis

of new

proteins

or to

replacement

of receptor

protein

from a

preex-isting

intracellular

pool

to thecell

surface,

various concentra-tions of

cycloheximide

wereaddedtothe culture medium and cellswereincubated for 8 h after

trypsin

treatment.Asshown in

Fig.

4,the

virus-binding capacity,

which

fully

recovered after

trypsin

treatment inthe absence of

cycloheximide,

was inhib-ited in a

dose-dependent

mannerinthe presence of

cyclohex-imide,

producing

almost

complete

inhibition of receptor

recov-ery at aconcentration of 8 to 10

,ug/ml.

Cell

viability

was not

impaired by trypsin

and

cycloheximide

under the conditions

used,

as determined

by

trypan blue exclusion. These data indicate that

regeneration

of the EMC virus

binding

depends

on active

protein synthesis

butnot on

recycling

ofthe

preex-isting protein.

Isolation and biochemical characterization of cellularEMC

virus-binding moiety by

affinity

chromatography. In

prelimi-nary

experiments,

it was established that the bond between EMC virus and its

binding moiety

onHeLa andK562cells isa

relatively

weak ionic interaction whichcanbe broken

by using

0.2 M

NaCl,

similar to the bond between EMC virus and

glycophorin

A, as determined

previously

(3). Thus,

it was

found that treatment of cell membranes derived from HeLa and

K562

cells with 0.2MNaCl in 20mM sodium

phosphate

buffer

(pH 8.0)

for 15 min at ambient temperature released morethan90% of the bound

3H-labeled

virus. Onthe basis of the

protocol

for

glycophorin

A

purification

from human

eryth-rocytemembranes

by

EMC virus

affinity chromatography

(3),

DOC-solubilized membranes

(approximately

6mg of

protein)

derived fromHeLa,

K562,

and controlK562Dclone cellswere

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(6)

HeLa K562

Untreated

, * * 0* , ,# Buffer alone

Trypsin

* * *. *

0 * 0. .0

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.0

* , tw.

.::0

k I* I.* .;.

1 2 4 8 16 32 64 128 256

*..

0:

* S * * o

.eV: N ;. * *, 0 .i

* 0 * ao *. * 0 a.

Untreated Bufferaloniex 1

Buffer alonex2 Neur.x1

supernatant pellet

Neur. x 2

supernatant

pellet

1 2 4 8 16 3264 128 266

FIG. 3. Effect oftrypsin andneuraminidase treatment on binding of EMC virus to HeLa and K562 cell membranes. Membrane proteins from HeLa and K562cells(280,ug) were incubated for 1 h at 37°C with 200 ,ug of trypsin or subjected to single (Neur. x 1)or double (Neur. x 2) treatmentwith 80 mUof neuraminidase (Neur.).Untreated membranes and membranes treated with appropriate buffer withoutenzymes(Buffer alone)wereusedascontrols. Virusbinding was analyzed by using serial twofold dilutions (presented in reciprocal values) of treated membranes (valueof 1, 140

jig

protein) andsupernatants(value of 1, 150

[il

from single ordouble neuraminidase treatment of 140 ,ug of membrane proteins) andadotblotassay.

chromatographed on EMC virus-Sepharose columns. As

shown inFig. 5, when HeLa and K562 cell membranes were analyzed, three protein peaks, which presumably represented unbound, specifically bound, and aggregated cell membrane components, were recovered from the column with 20 mM

L K562

100 - HeLa

80-CD

c

60-40 A

20-...

0 I I

0 2 4 6 8 10 12

Cycloheximide Concentration

(gg/ml)

FIG. 4. Inhibition of EMC virus receptor recoveryonHeLa and K562 cellsbycycloheximide.HeLaand K562 cellsweretreated with 20 ,ugoftrypsinfor 1 hat37°C,washed inHBSS,and thenincubatedwith fresh medium containingthe indicated concentrations of cyclohexi-mide for8 hat37°Cin5%CO2. Control cellsweretreatedsimilarly but without cycloheximide. Binding of 3H-labeled EMC virus was

determinedandexpressedas apercentageofbindingtocontrol cells recoveringfor 8 hat37°Cina5%CO2atmosphere following trypsin

treatment. Pointsarethemeans ± standard deviations forduplicate

determinations. Dashed and dottedlinesarethe levels ofvirusbinding remaining directly after trypsin treatment andwashingof K562 and HeLacells,respectively.

sodium phosphate buffer (pH 8.0) or buffer containing 0.2M NaCl or 0.1% Triton X-100, respectively. In contrast, when membranes of K562 Dclone cells were chromatographed, 0.2 MNaCl failedtoelute anyprotein(Fig.5,bottom), suggesting

that only membrane components derived from

virus-permis-sive cell lineswereabletobindto thecolumn. Increasing the

quantity of HeLaorK562cell membrane proteinsapplied to

the column did not increase theamountof the material eluted with 0.2 M NaCl, indicating that the column was saturated under the conditions used. Chromatographywas repeated by

usingDOC-solubilized membranes isolated from surface

12511

labeledHeLa, K562,orK562Dclone cells (approximately30 ,ugoftotalproteinpercolumn;specific radioactivity, 1.2x 104 cpm/,ug ofprotein).As shown for untreated HeLacell mem-branes inFig.6, elutionradioactivity profilesof the membrane components adsorbed to the columns were identical to the protein profiles given in Fig. 5, indicating that cell surface proteins were present in the material dissociated with 0.2 M NaCl.

It has been proposed that enzyme digestion of solubilized receptorpreparations givesamorereliable characterization of their biochemicalnaturethandigestionof whole cells

(18, 29).

Therefore, solubilized surface-iodinated membranes of K562

or HeLacellswere treated with trypsin or V cholerae neur-aminidase and then chromatographedon EMC

virus-Sepha-rose columns. Controls were treated similarly, but without enzymes.Asshown for HeLacellsinFig. 6,thepeak normally

elutedby0.2MNaClwaslost in bothtrypsin- and neuramin-idase-treatedsamples,indicatingthatbothenzymes eliminated cell membrane components capable of specific binding to EMC virus. These resultswere compatiblewith the effect of

proteolytic digestions of the

"251-labeled

proteins recovered

with 0.2 M NaCl from affinity columns, as shown by SDS-PAGE andautoradiographyforHeLacell membrane

proteins

inFig.7.Overall,these datawerein full agreement with results

from experiments with enzyme digestion of intact cells and

nonradiolabeled cell membranepreparations and

consistently

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a

E

c

0.3

0.2

-0.1

-

0-0.3

-0.2

-0.1

-0

-0.3

-02

-0.12

o

I I I I I I I

0 30 60 90 120 150 180 210 240 270

Tun (mbnubs)

FIG. 5. Virus affinity chromatography of cell membranes from permissiveHeLa andK562cells and from controlnonpermissiveK562 D clone cells.Aliquots of DOC-solubilized membrane proteins (ap-proximately 6 mg) from HeLa, K562, or K562 D clone cells were

appliedonEMCvirus-Sepharose columns in thepresence of 6 mM DOC and incubated for 30 minatambienttemperature.The columns

werewashed with 20mMphosphatebuffer(pH 8.0) untilnofurther

protein appeared in thewashes atA280, elutedwith 30 ml of buffer containing 0.2 M NaCl, and then washed with 0.1% Triton X-100 appliedatthepoints indicated byarrows.The flowratewas0.4ml/min. Protein absorbanceprofilesweremonitoredatA280andpresentedas

optical density (O.D.) values.

suggest that the moiety recognized by EMC virus on the surface of HeLa andK562 cells isasialoglycoprotein.

Molecular mass of EMC virus attachment protein. The

material eluted with0.2 M NaCl from EMCvirus-Sepharose columnswasanalyzed,eitherdirectlyorafterradioiodination, by SDS-10% PAGE with Coomassie blue staining or by

autoradiography, respectively. Figure 7 shows that a single

protein bandwithan approximate molecularmass of 70kDa wasdetected in theaffinity-purifiedmaterialfrom either HeLa or K;562cells but not from the virus-nonpermissive K562 D clone. Failure to detectthe 70-kDa protein in membranes of K562Dclone cells (Fig. 7,lanes 12and 13) stronglysuggests

that the detected protein species maydetermine EMC virus

bindingtopermissive human cells. However, the protein band inthe eluate ofK562 cell membranes appeared broader than that obtainedfromHeLa cells, perhapssuggestingadifferent

degree ofglycosylation. In addition, theanalysis showed that theidentified 70-kDaprotein band doesnotmatch the molec-ularmassofanyconstituentpresentinthepurified glycophorin preparation (Fig. 7, lanes 8 and 9). Identical molecular protein

80

40 -10:

5-0

80- 40-10:

E

0

0

0

ar

5

O

-80 -40 -10-7

5

-n

80

40]

10

5

0

Phosphate Buffer 0.1% Triton X -100 0.2 M NaCI

+

+

+ jTrypsin~

1

.

A

jNeurami~nidas~eI

rControlI

A

0 20 40 60 80 100 120

Fraction Number

FIG. 6. Effect of trypsin and neuraminidase treatment on the

bindingof HeLa cell membrane componentstoEMCvirus-Sepharose. DOC-solubilized cell membranes derived from surface 125I-labeled HeLa cellsweretreated for 1 hat37°Cwith 200,ugoftrypsinor80 mU

of neuraminidase andappliedonEMC virusaffinitycolumns

(approx-imately30 p,g ofprotein; specific radioactivity, 1.2 x 104cpm/uxg).

Control membranes from thesamecellswereincubatedunder

iden-tical conditions but without enzymes and similarly processed. The

columnsweretreatedasdescribed forFig.5. Fractionsof 1 mlwere

collectedat aflow rateof 0.4ml/min, and theirradioactivitieswere

determined andplotted againstthe fraction number.

profiles wereobtained by direct stainingof SDS-PAGE gels

withCoomassie blue(data notshown).

Specificity of EMC virus binding to affinity-purified cell membrane proteins. To verify whether the purified 70-kDa proteins express receptor activity for EMC virus, a VOPBA

wasperformed.In thisexperiment, approximately25 to 100,ug ofDOC-solubilized cell membraneproteinand 3,ugofprotein of the affinity-purified material from HeLa and K562 cell membranes were separated by SDS-PAGE, transferred onto nitrocellulose, andprobed with 125I-labeled EMC virus. Cell membranes and glycophorins derived from human erythro-cyteswereexamined inparallel.As shownby autoradiography (Fig. 8a),EMC virusrecognizedbands withidentical molecu-lar masses both in the isolated membranes from HeLa and K562 cells and in the receptor material purified from these

PhosphateButr 0.1%TrtonX-100 HSU 02M NaC

K562DCln

iCk

l

L.

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(8)

mol wt(kD)

9 10 11 12 13 14

-200

_ 97.4

!vX . 69

-- 46

_ --30

14.3

FIG. 7. Molecularprofiles and effect of trypsin anda-chymotrypsin

treatmentonaffinity-purified EMC virusreceptorproteins. Proteins eluted with 0.2 M NaCl from EMC virus-Sepharose columns after applicationof DOC-solubilized cellmembraneswere radioiodinated,

treated for1hat37°Cwith trypsinora-chymotrypsin, and separated

inSDS-10%polyacrylamide gel (2 x105 cpm,approximately 1,ug). The bandswerevisualizedbyautoradiography. Affinity-purified

pro-teins fromHeLa cells (lanes 1 and 2), thesame proteins treated with trypsin (lanes 3 and 4) ora-chymotrypsin (lanes 5 and 6), control

125I-labeledglycophorinpreparation (2 x105 cpm,approximately 2,ug

ofprotein [lanes8 and9]), affinity-purified proteins fromK562 cells (lanes 10 and 11), and corresponding column fractions from virus

nonpermissive K562 D clone cell membranes (lanes 12 and 13). 4C-labeled proteinmolecularmassstandardsareshown in lanes 7 and

14.Molecularmasses(in kilodaltons [kD])areindicatedonthe right.

membranes by chromatography on the virus-Sepharose

col-umn. Furthermore, the molecular masses of the bands were

the same as those detected in the affinity-purified material analyzed directly by SDS-PAGE and autoradiography, as

presentedin Fig.7.Also, inagreementwiththe resultsshown in Fig. 7, the 70-kDa proteins recognized by EMC virus ina

VOPBAdidnotmatchthe molecularmassofanyglycophorin

orerythrocytemembranecomponents. However, the virusdid interact with glycophorin by visualization of multiple protein bands (Fig. 8a, lanes 11 and 12) and with membranes of erythrocytes by bindingtobands with molecularmassesof86

and 43 kDa (Fig. 8a, lanes 11 to 14). The binding between EMC virus and the affinity-purified putative 70-kDareceptor

proteinorits molecularmassequivalentspresentin HeLa and K562 cell membraneswascompletely inhibited by

preincuba-tionof blotswitha30-foldexcessof cold EMC virus,asshown

for HeLa cells (Fig. 8b). The sameamountof cold poliovirus did not interfere with 125I-EMC virus recognition of the 70-kDa protein band in HeLa cell membranes (Fig. 8c), demonstrating that the bindingis EMC virus specific.

Isoelectric points of purified EMC virus attachment

pro-teins. Itwassuspected that the 70-kDaprotein on K562cells

was moreheterogeneously glycosylated,asthe band appeared

broader than that for HeLa cells on SDS-PAGE (Fig. 7).

Therefore, the isoelectric point (pl) of the K562cell protein specifically recognizedby EMC virus might be expected tobe lowerthanthat ofHeLa cells. Chromatofocusingon a

PBEion-exchangecolumn elutesproteins accordingtotheirpIswith the

useofaninternally generatedpHgradient. Analysis of

affinity-purified

1251I-labeled

EMC virus-binding protein derived from HeLa cells by chromatofocusing resulted in a sharp peak at pH 6.4. Desialylation of the protein by neuraminidase resulted in a shift of the peak from pH 6.4 to 6.7 (Fig. 9). On the other hand, chromatofocusing of the K562 cell protein interacting with EMC virus resulted in a broad peak eluted at pH 4.8, which may reflect a greater degree of protein glycosylation, and desialylation of the protein shifted the peak from pH 4.8 to 5.6 (Fig. 9). Therefore, it appears that the

K;562

cell protein for EMC virus may in fact be more sialylated than that on HeLa cells, as it had a lower initialpl and a greater shift of

pI

after desialylation.

Western blot analysis of EMC virus receptor with anti-glycophorin A antibody. Glycophorin A, which is expressed on K562 cells because of their erythroid origin (32), has been reported to be a receptor for EMC virus on human erythro-cytes (2, 4). To confirm that the 70-kDa EMC virus-binding protein onK562 cells is not glycophorin A, as the results of SDS-PAGE and VOPBA analyses suggested (i.e., Fig. 7 and 8),

Western

blot analysis of the affinity-purified EMC virus-binding material and DOC-solubilized

K562

and erythrocyte cell membranes was performed by using anti-glycophorin A antibody. This antibody is known to block EMC virus attach-ment to human erythrocytes (16). As shown in Fig. 10, the antibody recognized glycophorin A in the isolated

K562

and erythrocyte membranes but failed to react with the 70-kDa protein ofK562 cells. This result strongly suggests that the affinity-purified 70-kDa sialoglycoprotein from

K562

cells is not glycophorin A. This is consistent with the detection of the 70-kDa binding protein for EMC virus on HeLa cells, which do not express glycophorin A.

DISCUSSION

We investigated the attachment of EMC virus to two permissive human cell lines, HeLa and

K562

cells, and we report here findings on the isolation and biochemical nature of a novel class of cell surface proteins specifically recognized by EMC virus. These candidate cellular receptors for EMC virus are sialoglycoproteins with a relative molecular mass of 70 kDa, and their presence was not detected in membranes from cells nonpermissive for virus infection.

Our observation that the EMC virus binding to HeLa and K562 cells was saturated by excess virus and that unlabeled EMC virus competitively inhibited the attachment of iodinated virions satisfied two major criteria for receptor specificity: saturability and competition. Analysis of data from saturation experiments revealed that the number of cellular binding sites per cell for EMC virus were 1.6x 105on HeLa cells and 3.5x 105 onK1562 cells. This is consistent with previous reports on the binding of EMC virus to HeLa S3 (47) and most reports on other picornaviruses (20, 24, 42, 48), with ranges of

104

to

106

binding sites per cell. Scatchard analysis also revealed that the

Kdsof EMC virus binding were 1.1 and 2.7 nM for HeLa and K562 cells, respectively. It is noteworthy that EMC virus binding to another cell line, baby mouse pancreatic cells, exhibits a similar Kd value of 1.2 nM (8). These findings suggest that a similar binding force is involved in the attachment of EMC virus to all the different cell lines so far examined. Further, the straight line obtained by the Scatchard plots strongly suggests that a single class of receptor is involved in the binding of EMC virus to both HeLa and

K562

cells. Although other possibilities for having a straight line in the Scatchard plot exist, such as there being more than one class of receptor but all having the same affinity, this possibility is much

2 3 4 5 6 7

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(9)

7316 JIN ET AL.

a

1 2 3 4 5 6 7 8 9 10 11 12 13 14

b

1 2 3 4 6 6 7

,ws.f

....

s

t

_:_ji.

FIG. 8. Analysis of EMC virus-binding proteins by VOPBA. Cell membrane proteins (100 jig), receptor proteins purified on EMC virus-Sepharose columns (3 ,ug), and glycophorins (10 ,ug)wereseparatedonSDS-10%PAGEgels,electroblottedontonitrocellulose,and either directly probed with 125I-labeled EMC virus (a)orpreincubatedwithanexcessof cold EMC virus (b)orpoliovirus (c) and thenprobedwith 125I-EMC virus. Binding of viruswasvisualized byautoradiography. (a) Affinity-purifiedEMC virusreceptorproteins fromHeLa cells(lanes2 and 3), DOC-solubilized HeLa cell membranes (lanes4and5),affinity-purified receptorproteinsfromK562 cells(lanes 7and8),DOC-solubilized K562cellmembranes(lanes 9 and 10),glycophorins (lanes11 and 12), anderythrocytemembranes (lanes 13 and 14). (b) Affinity-purifiedEMC virusreceptorproteins from HeLa cells (lanes 2 and 3), DOC-solubilized HeLacellmembranes(lanes4and5),andglycophorins (lanes6 and7). (c) DOC-solubilized HeLa cell membranes (lane 2). "C-labeled protein molecularmassstandardsareshown in lanes 1 and 6(a)and lanes 1(b)

and(c).Molecularmasses(in kilodaltons[kD])areindicatedonthe left.

lesslikelyandno such instancehaseverbeenreportedinthe

bindingofpicornavirusestotargetedcells.

Mostpicornavirus receptors arebelieved to be cellsurface glycoproteins (39, 41, 53). ForEMCvirus, glycophorinAhas been definedasits receptoronnonpermissivehuman

erythro-cytes (2, 4). In this study, to characterize the biochemical natureofreceptors forEMC virus on HeLa and K562 cells, which readily support EMC virus infection, the effect of selected enzymes and lectins onvirus bindingwas examined.

The EMCvirus-binding moietywas sensitiveto all proteases used,whichincluded bothserineproteases,suchastrypsinand chymotrypsin, andathiolprotease, suchaspapain. However,

the susceptibility of viral binding to proteolysis does not necessarily mean that the protein itselfconstitutes the virus

receptor. Many proteases, including trypsin and papain, can

affect thestructure ofcell membranes andsurface molecules surroundingthe receptorsthat mayhold the receptor config-urationrequiredfor virusrecognition. Therefore,inourstudy, enzymaticanalysiswas performednotonlyonintact cells but

also on isolated cell membranes and affinity-purified virus

binding material. The results ofproteolytic treatment of all these preparations were consistent and demonstrated the

involvementofmembrane proteins in EMC virus attachment. The inhibition of recovery of the cell surface virus binding

capacity by cycloheximide furtherdemonstrated the

protein-aceous nature of the EMC virus-binding moiety and showed thatitsregeneration depends on active protein synthesis, but

not on recycling of preexisting internal proteins to the cell surface.

The drasticreduction ofvirusbindingtoeither intact cells, cell membranes, or purified virus-binding material by treat-ment with sialidases, which hydrolyze terminal sialic acid

residues from complex carbohydrates, demonstrated the

re-quirement of sialic acid for virus attachment to susceptible cells. Thiswasinagreementwith the inhibition of virusbinding topermissiblecells afterpretreatmentwith wheatgerm

agglu-tinin and L. polyphemus agglutinin, lectins which have an

affinity forsialic acids. The fact that free sialic acid residues released from HeLa and K562 cellsbyneuraminidase didnot demonstrate thebinding capacitysuggeststhat bothsialic acid and protein are necessary to maintain the integrity of a

functional binding site on the EMC viius receptor. This interpretation is inagreement with previous findings demon-strating thatEMC virus doesnotbind to pure sialic acids or

sialic acidresidues releasedbyneuraminidase treatmentfrom erythrocyte membranesorglycophorins (5).

Ourexperimentsdemonstrated, byseveralcriteria,thatthe EMC virus-binding sites on HeLa and K562 cell lines are

sialoglycoproteins. However,unlike typicalsialoglycoproteins, which usually have a plof less than 4.0 (15), the membrane

proteins mediating attachment of EMC virus to HeLa and K562 cellsappearedtobe lesssialylated,astheyhadhigherpls

(6.4and4.8 for HeLa and K562cell-binding sites, respective-ly). Nevertheless,the shift ofthepIsof theseproteinstoward neutrality by neuraminidase treatment indicates that sialic acids contributetothe acidicnatureof the EMC virus

attach-mentproteinsonbothcell lines.

The cell membrane proteins to which EMC virus was

bindingwassuccessfullyisolated from HeLa andK562 cellsby

virus affinity chromatography. The virus-binding activity had been enriched about 60-fold in the affinity-purified material comparedwith that in the membranes for both cell lines, as

determinedbydot blotprotein-bindingassay(datanotshown). Further, identicalchromatographic profiles andthe detection

mol wt(kD)

200

--97.4 --69

--c

1 2

..:.: .: ....

... ..

.!:

Ps ,S

a

l.*.

!: >-; <,,5W >

*. ::.:.;:.

... ..

: :

....< r>< :;i

w..,.,-i.gge

s. ...

*: ..

..S

::..

:::: .:

t;

.s;<<<>.;.

e

...:7::6:: ... sn_s .:::

.:

'.-;

*:.:..

46

30

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5

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on November 9, 2019 by guest

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[image:9.612.108.520.72.296.2]
(10)

E a.

U

a

HOLa

. ... 7

6

4

2 I

5

~~~~~~~~K562

6

3

~~~~5.64

2 2

U F I 0 0 5

0 10 20 30 40 50 60a 7

[image:10.612.67.276.75.291.2]

FractionNumber

FIG. 9. Chromatofocusingof EMCvirusreceptorproteins purified by virusaffinitychromatography fromHeLa and K562 cellmembranes. Affinity-purified

1251I-labeled

EMC virusreceptorproteins fromHeLa andK562cells(continuous lines)orreceptor proteinsfrom the same cells desialylated by treatment for 1 h at 37°C with 80 mU of neuraminidase from V cholerae (dashed lines)werechromatofocused

onaPBE 94ion-exchange column,and the proteins were eluted with Polybuffer74 atflowrateof 0.4ml/min.Theradioactivity (countsper minute)and pH(dotted lines) of each1-ml fractionwasmeasuredand plotted againstthefraction number.

ofasingleproteinspecieswithamolecularmassof 70kDa in

the material purified from either unlabeled or surface-radio-labeled HeLa and K562 cells demonstrated that the 70-kDa proteins were located on the cell surface. Proof of specific

bindingofEMC virusto the 70-kDa membrane proteins was

obtained byusing VOPBA.This techniquewasfirstdescribed

by Co etal. (17) toidentifythe receptor onmurine thymoma R1.1cellsforreovirus type 3.Subsequently,the technique has

beenusedtoidentifyputative cell receptor proteins forseveral

viruses,includingSendai virus(25),mousehepatitisvirus (11),

cytomegalovirus (1, 57), Theiler's virus

(38),

visnavirus(19),

and lymphocytic choriomeningitis virus (10). Using VOPBA,

wedemonstratedthatEMC virus recognized the same 70-kDa

proteinbands both in the

affinity-purified

material and in the

membranes of HeLa and K562 cells. The specificityof EMC

virus bindingtothe 70-kDa proteins wasconfirmedby inhibi-tion of theinteractionofradiolabeledEMCvirusbyunlabeled EMC virus but not by poliovirus, another member of the

picornavirus family (Fig. 8), and by the fact that membrane

constituents of nonpermissive K562 D clone cells failed to

interact with EMC virus(Fig.5 and7). Moreover,the VOPBA

techniquerevealed that no molecularmass equivalent of the

70-kDa protein exists in human erythrocyte membranes or purified glycophorins, although both of these preparations were able to bind EMC virus in VOPBAs.Collectively, these

findings suggest that the candidate 70-kDa receptor protein

mightbe specifically expressed by cells susceptible for EMC

virus infection.However,whether the sameprotein is usedby

EMC virustoattachtootherhumanpermissive cellsorcells of otherspecies remainstobe established. Inthecontext ofthe recentidentification of VCAM-1 asareceptor forDstrainof EMCvirus onmurine vascularendothelial cells (30) andour

1 2 3 4 5 6

a.

mol wt

(kD)

-- 200 --97.4 --69

--46

--

30

-- 14.3

FIG. 10. Analysis ofEMC virus receptorproteinsand cell

mem-branes from K562 cells

by

Western blottingwithanti-glycophorin A

antibody. Cell membrane proteins (100 ,ug), purified EMC virus

receptorproteins(3 ,ug),andaglycophorin preparation (10 ,ug)were

separated

by

SDS-PAGE,electrotransferred ontonitrocellulose,and

incubatedwithmonoclonalanti-glycophorinAantibody.Eachreaction

was visualized by '25I-labeled sheep anti-mouse immunoglobulin G

antibody

and autoradiography. Lanes: 1, K562 cell membranes; 2,

erythrocytemembranes;3 and4,affinity-purifiedEMC virusreceptor

protein from K562 cell membranes; 5, glycophorins; 6, 14C-labeled

proteinmolecularmassstandards(inkilodaltons[kD],asindicatedon

theright).

findings,

it isreasonabletoexpect that EMC virusmayutilize different molecules ondifferent cell types orcellsofdifferent

species

toinitiateinfection.

The EMC

virus-binding proteins

purified

from K562 and HeLa cells had similar molecularmassesof around 70

kDa,

as

estimated

by

SDS-PAGE under

reducing

conditions.

However,

since the

degree

of

glycosylation

can affect protein

electro-phoretic mobility,

themolecularmassofthesame

protein

may vary.

Here,

we observed that the appearance of the EMC

virus-binding protein

band

purified

frommembranes of K562 cellswasmorediffuse than theappearanceofthose from HeLa

cells,

adifference which could be relatedtodifferent

oligosac-charide contents. So

far,

our attempts to obtain

completely

carbohydrate-free polypeptides

havenotbeensuccessful;

thus,

it isunclear whether thereceptor

proteins

onHeLa and K562 cell lines are indeed the same molecules. Our

preliminary

attemptstoobtain amino acidsequencesoftheseproteinshave

also metwithlittlesuccess. Itappearsthat the N terminus of these

proteins

maybeblocked and thatglycosylationmayalso interfere with sequence determination. Further work on the amino acidsequenceandcDNAcloningof thereceptor genes will

help

to

provide

answerstothisissue. Proof that

polyclonal

ormonoclonal antibodies

prepared

against

thepurified recep-tors can indeed prevent infection of the virus in HeLa and

sm,

0

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http://jvi.asm.org/

[image:10.612.341.529.80.355.2]
(11)

K562 cells will provide direct evidence that the purified proteinsplayafunctional role in EMC virus infection.

Ourresults show that the70-kDaproteinsbound by EMC virus did not match any glycophorin components in SDS-PAGEgels (Fig. 7) and that themonoclonal anti-glycophorin

A antibodies, which block EMC virus attachment to

glyco-phorin A onerythrocytes (16), did notrecognize the 70-kDa protein from K562 cells byWestern blotting (Fig. 10). It has been found previously that transfection of K562 cells with antisense glycophorin AcDNA, to block synthesis of glyco-phorin A, or saturation of cells with anti-glycophorin A antibodiesdoesnotaffectEMCvirusbindingtoand infection ofK562 cells(27). Thus, results obtained inthisstudysupport previous indications that glycophorin Aisnot a receptorfor EMC virus onK562cells. It mustbe added, however, thatwe do notexclude thepossibilitythatglycophorinAcan serve as a receptor for EMC virus in cells under suitable conditions. Forexample,Madin-Darbybovinekidneycellsdonotexpress

glycophorin A and are resistant to EMC virus infection.

However, transfection of the cells with glycophorinA cDNA

results inexpression ofglycophorinAonthecellsurface,and cells become susceptible to infection with EMC virus (26),

suggestingthatglycophorinAcan serve as afunctionallyactive

receptor for EMCvirus in an appropriate cell line. It isnot

completelyclearwhyEMC virus bindstothe70-kDa

sialogly-coproteinbutnottoglycophorinAonK562 cells. It has been

reported, however, that glycophorin A on these cells is less

glycosylatedandcontains fewer sialic acid residues than it does

onhumanerythrocytes(54).This mayexplain,to someextent,

theinabilityof EMCvirustointeract withglycophorinA, since

weknow that sialic acid residues are involved in thebinding of virus toglycoproteinA(56).Ontheotherhand,the

noneryth-roidoriginof HeLacells, excluding glycophorinAinvolvement

in EMC virus binding, provides strong evidence that 70-kDa

sialoglycoproteinsrepresentanovel, not-yet-describedclass of

cell surface molecules mediating EMC virus attachment to permissive human cells.

ACKNOWLEDGMENTS

This work was supported by grants from the Medical Research Council ofCanada and Canadian Diabetes Association.Y.-M.J.was

the recipient of a student fellowship from Memorial University of Newfoundland.

We thank M. Pah Baldeh for generous advice, especially on the EMCvirus-Sepharoseaffinity chromatography;D.J. Anstee(Radcliffe Infirmary, Oxford, United Kingdom)for thegiftofanti-glycophorinA monoclonal antibody; and Y. Svitkin and N. Sonenberg (McGill University, Montreal, Canada) for providing poliovirus.

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Figure

FIG. 1.withvirusHeLapurified3H-EMCrepresentsdeterminedand Time course and competition of EMC virus binding to cells
FIG. 2.viruscreasednumberparticlespointpoints(Insert)1.4 Saturability of binding of EMC virus to HeLa cells
FIG. 3.andHeLaalone)treatment(value Effect of trypsin and neuraminidase treatment on binding of EMC virus to HeLa and K562 cell membranes
FIG. 6.bindingofHeLaDOC-solubilizedcollectedcolumnsimatelyticalControldetermined neuraminidase Effect of trypsin and neuraminidase treatment on the of HeLa cell membrane components to EMC virus-Sepharose
+4

References

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