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 withoutCa2+
and
Mg2+
(HBSS)to removeenzymesand reducepossiblecellaggregation. 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 fromPapayalatex (1 U perreaction; Sigma ChemicalCo., St. Louis,
Mo.)
preactivatedwith 0.38 ,ug ofL-cysteine hydrochloride
(Gibco
BRL, Grand Island,
N.Y.)
in 5RI
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
MannheimCanada, Laval,
Quebec)
andfrom Arthrobacterureafaciens (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 perreaction;
Sigma Chemical
Co.),
andLimuluspolyphemus
agglutinin
fromthehorseshoe crab
(0.5
mg perreaction;Sigma
ChemicalCo.).
Ascontrols, cellsampleswereincubated with PBS withouttest enzyme or lectin. In the case of protease
digestion,
which detaches theHeLacellmonolayerfromthe culturedish,
cellspredestinedforcontrolexperimentswereremovedby
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 cellspretreated
with 20jig
of trypsin for1hat37°Cwerewashed in severalchangesof HBSS and incubated in 2 ml of fresh culture mediumcontaining
2to12 ,ugofcycloheximide
(Sigma
ChemicalCo.)
for 8h ina5% CO2atmosphere
at37°C.
Control cellsweretreatedsimilarly
but without cycloheximide. Attachment of
3H-labeled
EMC virusto the cellswasdetermined aspresented below.Virus-bindingassay.In mostcircumstances, approximately5
,ug
(5,000
cpm)of3H-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 incubationonice,
unless otherwiseindicated,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 coldPBS,
andthe washwassaved.Finally,
the cellswerelysed
in 1 ml of 1% Triton X-100 at ambient temperature. The
radioactivity
insupernatants, washes,
and thelysed
cellsam-ples, constituting the total recovered
radioactivity,
wasmea-sured in a scintillation counter, after 10 ml of
Aquasol-2
(Dupont,
NENResearchProducts)
had been added. Attach-ment of virus to cells wasexpressed
as a percentage of virus-boundradioactivity
relativeto the totalrecoveredradio-activity.
To determine the time course of EMC virus
binding,
ap-proximately
5 ,ugof labeled viruswas incubated with 2 x 106HeLa or K562 cells for 2.5 to 60 min on ice.
Saturability
ofbinding
wasanalyzed by
incubation ofincreasing
amounts of3H-labeled
virus(0.4
to 50jig)
with a constantnumber(2
x106)
of cells for 30 min onice,
followedby
measurement of both bound and unbound virus.Specificity
ofbinding
wasdemonstrated in
competition experiments,
in which 2 x106
cells were
preincubated
on ice for 30 min with different amountsofunlabeled virus(approximately
50 to500,ug)
inafinal volume of 500
[lI,
thensupplemented
withlabeled virus(approximately
5pug),
and incubated for 2.5 to30 minonice.Preparation and solubilization of cell membranes.
Mem-braneswere
prepared
from cellsdisrupted by
hypotonic
shock andsubsequent
homogenization.
Nuclei and cellular debris wereremovedby
centrifugation
at1,000
xgfor 30 s, and cell membraneswerepelleted
at45,000
xgfor 60 min inaTiS0.1 rotor(Beckman Instruments, Inc.,
PaloAlto,
Calif.)
as de-scribedby
Atkinson and Summers(7).
Isolationwas done in the presence of2 mMphenylmethylsulfonyl
fluoride(PMSF)
and 2mM
N-ethylmaleimide
asprotease inhibitors. Membranepellets
wereresuspended
in PBSsupplemented
withproteaseinhibitors,
and theprotein
contentwasmeasuredby
theLowry
method with bovine serumalbumin
(BSA)
as astandard(43).
Cell membranes(1.5
mgofprotein
perml)
were solubilized inanequal
volumeof 12mMsodiumdeoxycholate
(DOC)
in 20 mM sodiumphosphate
buffer(pH 8.0)
on ice for 30 min. Insoluble membrane residueswereremovedby
centrifugation
at
16,000
xgfor 5 min. Soluble membraneproteins
contained in thesupernatant
were diluted with anequal
volume of sodiumphosphate
buffercontaining
protease inhibitors and storedat-20°C.
Cell membranes from human type 0
erythrocytes
wereprepared
fromrecently
outdatedpacked erythrocytes
provided
by
the Canadian Red Cross Blood Transfusion Service(St.
John's,
Newfoundland,
Canada), by
hypotonic
lysis
(23).
Gly-cophorins
werepurified
fromerythrocyte
membranesby
the lithium3,5-diiodosalicylate-phenol
procedure
of Marchesi and Andrews(45).
Radioiodination. EMC
virus,
isolated cellmembranes,
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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 gelfiltrationonSephadexG-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 conditionsrecommended 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 EMCvirus-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,
andlyophilizedforfurther analysis.
Enzyme treatment ofcell membranes and affinity-purified EMC virus-binding proteins. Membrane protein
(about
280jig)
orDOC-solubilized membranes derived from1'I-labeledcells (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 enzymewasinactivatedby
theaddition of 100 jil of 0.1 M EDTA. The membranes were pelletedby
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 3.5x105
cpm) were treated with V cholerae neuraminidase under the conditions described above. The enzyme was inactivatedbyboiling for 5min,
and the sample was analyzed by chromatography on an EMC virus-Sepharose column. Neuraminidase treatment of the affinity-purified moiety was done by using105
cpm of
125I-labeled
protein and 80 mU of enzyme under the condi-tions presented above. The enzyme was inactivatedbyboilingand 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 2x106
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 at4°C
or for 2 h at ambient temperature on a rocking platform. In competition experi-ments designed to determine the specificity of1251I-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 with1251I-labeled
virus by using the conditions described above. The blot was washed threeon November 9, 2019 by guest
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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 ReferenceLaboratory,
Radcliffe Infirmary, Oxford, UnitedKingdom)
diluted 1:10 with PBS containing 1% BSA and 0.05% Tween 20. Theantibody
recognizes amino acids inpositions
34to39onglycophorin
A(6)
andblocks EMC virus attachment to humanerythrocytes
(16).
After being washed,the membranes were incubated with
12I1-labeled
sheepanti-mouse
immunoglobulins (Amersham
Canada) diluted 1:1,000 in PBScontaining
1% BSA and 0.05% Tween 20 for 2 h at ambient temperature orovernight
at 4°C. Subsequently, theblots werewashedas described for theVOPBA, andbinding
was visualized
by autoradiography
after 3 days of exposure. Humanerythrocyte
membranes and purified glycophorins were usedaspositive
controls. Substitution of theanti-glyco-phorin
Aantibody
withPBSsupplemented
with1% BSA and0.05% Tween 20wasusedtoinsure that anti-mouse antibody
didnot reactwith cellmembraneproteins.
Chromatofocusing. Chromatofocusing
ofaffinity-purified
vi-rus-binding proteins
derived from both HeLa and K562 cells wasperformed
on columns(220 by
9mm)
ofPolybuffer
exchanger
PBE 94(Pharmacia).
About 0.5 ,ug(105 cpm)
of125I-labeled proteins purified by affinity chromatography
on EMC virus columns and radiolabeledpreparationsof thesamepurified
proteins desialylated by
treatment with V choleraeneuraminidase were
separately applied
ontoexchanger
col-umns and allowed to adsorb for 5 min. The columns were washed with
Polybuffer
74(Pharmacia),
diluted 1:8 with deion-ized water, andadjusted
topH
4.0 with 0.1 NHCl,
at aflow rateof 0.4ml/min.
Fractions of1mlwerecollected,
and theirpHs
and radioactivitieswere measured. RESULTSKineticsof EMCvirus
binding.
Thedynamics
of the EMC virus interaction with HeLa and K562cellswasanalyzed by
avirus-binding
assay in which 3H-labeledpurified
virionswere incubated on ice with intact cells. The results showed that EMC virusbinding
to HeLacells increased almostlinearly
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 thebinding
wascomplete
inapproximately
30 min.Thetimecourseof EMC virusbinding
toK562 cells(data
not
shown)
wasclosely comparable
tothatpresented
for HeLa cells.Nonpermissive
K562Dclone cellswereresistanttoEMC virusbinding
under all testedconditions.Competition
assayswereusedtodemonstrate thespecificity
ofthe EMC virus interaction with the cell surface. Preincuba-tion of HeLa cells withanexcessof unlabeled
virus,
followedby
measurement of the 3H-labeled virusbinding
at differenttime
points,
showed that a 10-fold excess ofunlabeled virus(approximately
50,ug)
inhibitedbinding
by approximately
70% within 20 min, whereas a 30-fold excess of unlabeled virus(approximately
150,ug)
blocked about95% of thebinding
in thesametimeperiod
(Fig.
1).
Nofurther inhibitionof3H-virus
binding
wasobservedby
preincubation
of cells witha100-foldexcess of unlabeled virus
(approximately
500,ug) (data
notshown).
Increasing
the incubation time with labeled virus did notaffect theinhibition rates,suggesting
that thecompetitive
displacement
of unlabeled virionsby
labeled virus from the cell surfacewasveryslowand,
thus the virusbinding
wasrelatively
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 equilibriumdissociationconstant(Kd)wascalculated from theslopeofthe Scatchardplottobeapproximately1.1 nM. These resultswere
comparable to those obtained for K562
cells,
which gave anestimated 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, asdetermined 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 thatacellsurfaceprotein is an essential component of the EMC
virus-binding
moietyoneither cell type. Incontrast,
phospholipase C,
whichon November 9, 2019 by guest
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[image:4.612.327.531.77.291.2]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 ofasialoglycoprotein
in thevirus-binding
site.Effect ofenzymatictreatmentof isolatedcellmembraneson
EMC virus recognition.To be moreconfident that the virus attachment ismediated
by
cell surfacesialoglycoproteins,
cell membranes isolated from HeLa and K562 cellswere treatedwith trypsin or V cholerae
neuraminidase,
and thevirus-binding activity
was determined in adot blotprotein-binding
assay.
Trypsin
treatmentdecreased thebinding activity by
8-to 16-fold both in HeLa and K562 cell membranescompared
with that in untreatedmembranes,
whereasdigestion
withneur-aminidase resulted in 8- and64-fold reduction of EMC virus
bindingtoHeLa and K562 cell
membranes,
respectively
(Fig.
3). Although
virusbinding
to cell membranes wasgreatly
reduced aftera
single digestion
withneuraminidase,
itwasnottotally
abolished. Double neuraminidasedigestion
was per-formed to test whether theremaining
activity
could bede-stroyed.
The results showed that the seconddigestion
of the same HeLa or K562 cell membranes further reduced virusbinding
by
two-toeightfold
toalmostbackground
levels(Fig.
3).
Moreover, supernatantsresulting
from eithersingle
ordouble neuraminidase
digestion
of the membranes did notshow EMC
virus-binding
ability
(Fig. 3).
As determinedby
high-performance
liquid chromatography
(data
notshown),
these supernatantscontained released sialic acids. This obser-vation may suggest that free sialic acid alone doesnotserve asthe virus receptor
moiety.
Overall,
the data obtained fromdigestion
of cell membranes with protease and sialidasewereconsistent with thoseobtained from the
enzymatic
treatment of intact HeLa and K562cells and confirmed that the EMCvirus-binding
site isacell surface-associatedsialoglycoprotein.
Blockage of EMC virus binding to the cell surface by
cycloheximide. It was found that both
trypsin-treated
HeLaand K562 cells were able to
fully
restore their EMCvirus-binding capacity
within6to8 h of incubation in fresh medium aftertrypsin
had been removed(data
notshown).
To deter-mine whether such recovery was due tosynthesis
of newproteins
or toreplacement
of receptorprotein
from apreex-isting
intracellularpool
to thecellsurface,
various concentra-tions ofcycloheximide
wereaddedtothe culture medium and cellswereincubated for 8 h aftertrypsin
treatment.Asshown inFig.
4,thevirus-binding capacity,
whichfully
recovered aftertrypsin
treatment inthe absence ofcycloheximide,
was inhib-ited in adose-dependent
mannerinthe presence ofcyclohex-imide,
producing
almostcomplete
inhibition of receptorrecov-ery at aconcentration of 8 to 10
,ug/ml.
Cellviability
was notimpaired by trypsin
andcycloheximide
under the conditionsused,
as determinedby
trypan blue exclusion. These data indicate thatregeneration
of the EMC virusbinding
depends
on active
protein synthesis
butnot onrecycling
ofthepreex-isting protein.
Isolation and biochemical characterization of cellularEMC
virus-binding moiety by
affinity
chromatography. Inprelimi-nary
experiments,
it was established that the bond between EMC virus and itsbinding moiety
onHeLa andK562cells isarelatively
weak ionic interaction whichcanbe brokenby using
0.2 M
NaCl,
similar to the bond between EMC virus andglycophorin
A, as determinedpreviously
(3). Thus,
it wasfound that treatment of cell membranes derived from HeLa and
K562
cells with 0.2MNaCl in 20mM sodiumphosphate
buffer
(pH 8.0)
for 15 min at ambient temperature released morethan90% of the bound3H-labeled
virus. Onthe basis of theprotocol
forglycophorin
Apurification
from humaneryth-rocytemembranes
by
EMC virusaffinity chromatography
(3),
DOC-solubilized membranes
(approximately
6mg ofprotein)
derived fromHeLa,
K562,
and controlK562Dclone cellswereon November 9, 2019 by guest
http://jvi.asm.org/
[image:5.612.66.308.523.673.2]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 recoveredwith 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
on November 9, 2019 by guest
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[image:6.612.122.489.79.266.2] [image:6.612.76.272.427.614.2]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
.
AjNeurami~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.
on November 9, 2019 by guest
http://jvi.asm.org/
[image:7.612.367.517.74.453.2] [image:7.612.80.285.76.390.2]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 theK;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 ofpI
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-solubilizedK562
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 isolatedK562
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 fromK562
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
to106
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 much2 3 4 5 6 7
on November 9, 2019 by guest
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[image:8.612.61.290.77.268.2]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
--14.3
--4E
J.VIROL.
5
..., O.:--1
on November 9, 2019 by guest
http://jvi.asm.org/
[image:9.612.108.520.72.296.2]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)werechromatofocusedonaPBE 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 themembranes 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 Aantibody. Cell membrane proteins (100 ,ug), purified EMC virus
receptorproteins(3 ,ug),andaglycophorin preparation (10 ,ug)were
separated
by
SDS-PAGE,electrotransferred ontonitrocellulose,andincubatedwithmonoclonalanti-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 orcellsofdifferentspecies
toinitiateinfection.The EMC
virus-binding proteins
purified
from K562 and HeLa cells had similar molecularmassesof around 70kDa,
asestimated
by
SDS-PAGE underreducing
conditions.However,
since the
degree
ofglycosylation
can affect proteinelectro-phoretic mobility,
themolecularmassofthesameprotein
may vary.Here,
we observed that the appearance of the EMCvirus-binding protein
bandpurified
frommembranes of K562 cellswasmorediffuse than theappearanceofthose from HeLacells,
adifference which could be relatedtodifferentoligosac-charide contents. So
far,
our attempts to obtaincompletely
carbohydrate-free polypeptides
havenotbeensuccessful;thus,
it isunclear whether thereceptor
proteins
onHeLa and K562 cell lines are indeed the same molecules. Ourpreliminary
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 willhelp
toprovide
answerstothisissue. Proof thatpolyclonal
ormonoclonal antibodies
prepared
against
thepurified recep-tors can indeed prevent infection of the virus in HeLa andsm,
0
on November 9, 2019 by guest
http://jvi.asm.org/
[image:10.612.341.529.80.355.2]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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