Differential Inhibition of Attachment and Eclipse Activities of HeLa Cells for Enteroviruses

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Differential

Inhibition

of

Attachment

and

Eclipse

Activities of HeLa

Cells for Enteroviruses1

IHOR ZAJAC AND RICHARD L. CROWELL'

DepartmentofMicrobiology, JeffersonMedicalCollege,Philadelphia, Pennsylvania19107,and

LangbordVirusLaboratory,Departmentof Microbiology,HahnemannMedical

College,

Philadelphia, Pennsylvania19102

Receivedforpublication4February 1969

Receptor activities ofHeLa cells were evaluated for ability to both attach and eclipseenteroviruses after exposureofcells toacid or heat. A modified procedure ofacid(pH1.5) elution of cell-associatedvirus, ascompared with otherprocedures, provided a general method for the optimal recovery of receptor-bound entero-viruses. With this procedure,eclipse ofvirusoperationallywas considered to be that amountofvirusinfectivity whichwasdeterminedinitially to becell-associated and whichwas notdissociable from thecells.HeLacellskilledbyheatingat56 C for 30 min could not attach or eclipse poliovirus TI, but they attached and eclipsed coxsackieviruses B1 and B3, and they attached echovirus 6 but did not eclipse it. HeLacells treatedatpH2.5for 10 minat 2 C could not attach or eclipsepoliovirus TI, but they attachedcoxsackievirusesB1 and B3 and echovirus 6, although these viruseswere noteclipsed. These resultsshowed that, within the operationaldefinition ofviruseclipse, the eclipse activity ofHeLa cells for some viruses can beirreversibly inactivated withoutimpairing the activityof thereceptorsforattachingthese viruses. The dataprovided additional evidence thatHeLa cells possess specific receptors for the different enteroviruses.

Initiation of infection of

susceptible

cells

by

enteroviruses has been shown to be

dependent

uponthe presenceof

specific

receptors to attach virus (14, 21, 22) and anefficient mechanism for viral eclipse (5). Extensive

studies

have been

performed

in an attempt to characterize

entero-virusreceptorsby theuseof cellularhomogenates (8, 9,

21),

cellextracts

(16, 19,

21),

redblood cells

(18),

and

living

HeLa cells

(1-4, 10, 23, 24)

as a

source

of

receptor

activity.

These studies have

emphasized

the diverse characteristics of the

cellu-larreceptors

responsible

for attachment of

polio-viruses,

coxsackieviruses,

and echoviruses. This paper describes the

stability

of the different enterovirus receptors of HeLa cells to acid and heat.The receptor activities of treatedcells were

evaluated for

capacity

both to attach and to

eclipse virus.Inthese

studies,

theterm

eclipse (7)

wasused

operationally

todenote thatamountof

virus

infectivity

whichwasdetermined

initially

to

becell-associated and whichwas not dissociable fromthecellsbytreatmentwith

glycine

buffer

(6)

atpH 1.5.

'Presented inpartatthe 66thAnnualMeetingof theAmerican Society forMicrobiology,LosAngeles,Calif., May,1966.

2ResearchCareerDevelopment Awardgranteeof the Public HealthService,National InstituteofAllergyandInfectious Dis-eases.

MATERIALS AND METHODS

HeLa cells. Cells designated HeLa (JJH) were culturedasmonolayers in growthmediumcontaining

calf serum (GMCaSlO), asdescribedelsewhere (23). Virus strains. Coxsackieviruses Bl and B3,

polio-virusTl,andthe method of plaque assay used in this

investigation were described previously (4). Echo-virus 6, kindly supplied by M. Klein of Temple

University Medical School, Philadelphia, Pa., was

propagated and assayed in a fashion similar to the other enteroviruses.

Determination of receptor activity to attach and eclipse enteroviruses. Determination of the receptor

activityof HeLa cells forattachingviruseswascarried out at 37 Cor at 2C, as described elsewhere (23).

Theamountof virus whichwasattachedtocellsat a

given time was determined by the difference between theamounts of free virus found and theinputvirus. In addition, in experiments conducted at 2 C, the

amountof virus whichwasattachedtocellswas deter-mined directly byelution of virus from the cells by use ofglycine buffer at pH 1.5. A close correlation

wasfound between thetwo methods used for deter-mining the amount of attached virus provided the system waskept cold. Determinations ofthe ability

of HeLa cellstoeclipseviruswerecarriedout at37C. Eclipsed virus (7) wasconsidered to be thatamount

of virusinfectivitywhichwasdeterminedinitiallytobe cell-associated (by difference between free virus and

inputvirus) and whichwas not dissociable from the

422

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cell by treatment with 0.05 M glycine buffer (6)

ad-justedtopH 1.5.

Preparation of cell-associated virus (CAV). A

sample of5 X 107 washed HeLa cellswascentrifuged

at225 X gfor 5 min, andthe sedimentedcellswere resuspended in 5 ml of virus at 2 C. [Input virus

multiplicity usually was approximately 10 plaque-forming units (PFU) forcoxsackievirus B3, 50 PFU

for coxsackievirusBi,and 100 PFU forpoliovirusTi and echovirus 6.] The cell-virus mixturewasincubated

at2 C for1 hr withfrequent agitation. Virus

attach-ment was stopped by diluting the cell-virus mixture

10-fold with cold balanced salt solution (BSS)-CaS3

solution and centrifuging thecells at225 X gfor 5

minat2 C. Thecellswerereadied foruseby washing

them four more times with cold BSS-CaS3 solution

(50ml eachwash)toreduce freevirus. The CAVwas

determined by the acid reversal method described below.

Reactivationof CAVbyacidpH.Theprocedureof

Fenwick and Cooper wasmodified and used (6). A

known number of cells with associated virus was diluted 100-fold in cold (2 C) 0.05 M glycine buffer

(pH 1.5). Aftertreatmentfor 1 min, thesample was

diluted 10-fold further intophosphate-buffered saline

(PBS), devoid of calcium andmagnesium,containing 3% calf-serum (PBSA-CaS3), which was adjusted topH10.9sothat the finalpHof theviruspreparation

was neutral. Theamountoftotal virusrecovered, as

determined by plaque assay and corrected for the

amount of virus remaining free prior to acid treat-ment, was consideredto be theamount ofinfectious viruswhichwascell-associated and boundtoreceptors atthe time of acid treatment.Fromacomparison of

several methods used for dissociation of

coxsackie-viruses from cell receptors, this method was found

to be themosteffective (seeTable2).

Chemicals and enzymes.Allchemicalswere

analyt-ical reagent grade.Chymotrypsin (3 Xcrystalline)was purchasedfromWorthingtonBiochemicalCorp.,

Free-hold,N.J. Freshenzymesolutionwaspreparedforeach

experimentand usedatafinal concentration of1%. RESULTS

To study effectively the receptor activity of HeLa cells for attachment and eclipse of

entero-viruses, we considered it important that the

following preliminary conditions be met: (i) the

viruses should attach andremain at the cell

sur-face for extended periods of time; and (ii) a

method should be used that will measure most accurately theamount of CAVpriortoinitiation of viraleclipse.The use oflow temperature has proved to be the only efficient method for main-taining the attached viruses on the cell surface,

and, aspresented below, the method of acid

re-versalof CAVappeared tofulfillthesecond

con-dition. Accordingly,experimentswere performed

in an attempt to follow the distribution of the total amount ofinput virus addedto HeLacells afterincubation for1hrat2C.

Six separate experiments with coxsackievirus

B3andthreeseparateexperiments with poliovirus

TI were performed in which the total PFU of virus was determined in each of five consecutive wash fluids and in the CAV fraction (Table 1). The resultsshowed that the major portion of the input virus remained in the fluid phase. The find-ing of almost complete recovery of the input virus inoculum attested to the efficacy of acid pH for

recovery of total CAV under conditions which prevented viral eclipse.

Capacity of assorted reagentstodissociate CAV. Interest in the fate of cell-associated virus has produced many procedures fordissociating virus-cellcomplexes (6, 7, 12, 15, 18). These procedures were compared for their capacity to dissociate CAV under conditions which precluded viral eclipse. Earlier experiments had shown that the acid pH method used todissociatevirus-antibody complexes (11, 20) and CAV (6, 15) effectively released coxsackievirus B3 from the surface of HeLacells (3). Todetermine the pHwhichwould optimally reverse CAV at 2 C, a wide range of acidpH was tested.

Preparation of CAV by the use ofpoliovirus TI, echovirus 6, and coxsackieviruses Bi and B3

TABLE 1. Distributioni ofiniput virus in the cellular andfluidphases of the HeLa cell suspensions

after incubationforIhrat2C

Amt ofinput virus recoveredb Phase'

Coxsackie B3 Polio Ti

Wash 1 82 i 6 86 i 3

Wash2 3±-0.5 6± 3

Wash 3 <1 <1

Wash 4 <1 <1

Wash 5 <1 <1

CAV(pH1.5) 13 ± 4 9-± 5

CAV lost 1 <1

Total recovery 99 ± 10 101 i 3

a The amount of free virus remaining in the fluid phase of the cell suspension used to deter-mine CAV was <1 for bothcoxsackievirusB3 and

poliovirusTi.

IPresented as the mean from six experiments with coxsackievirus B3 and three experiments with poliovirus T1 with the standard error of the mean, respectively. Washed HeLa cells (5 X 107)

were suspended in 5 ml of virus at 2 C. Input

multiplicity ofviruswas 10 PFUand 100PFUfor coxsackievirus B3 and poliovirus TI, respectively. After 1 hr of incubation at 2 C, the cell-virus

suspension was diluted to 50 ml with BSS-CaS3, and centrifuged at225 X g; the supernatant fluid (wash no. 1) was assayed for virus infectivity. The cells were washed accordingly four more

times, and thefluidswere assayedforvirus. After

the fifth wash, the CAV was determined, as indicated in Materials and Methods.

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was described in Materials and Methods. Cells with associated virus, kept at 2 C, were diluted 1:100 intocold0.05 M glycine bufferatdifferent pHvalues and held for1 minat2C,after which the pH wasbroughttoneutrality byafurther1:10 dilution into PBSA-CaS3. The cells were sedi-mented by centrifugation and the supernatant fluidswerecollectedandassayedtodetermine the amountof dissociated virus. The results of these experiments (Fig. 1) showed that optimal

re-covery of coxsackievirusesBi and B3 and

polio-virusTl occurredatpH1.5, whereas therecovery ofechovirus 6wasaccomplished

equally

wellatall pHvalues tested. Determination of virus stability inthemediumused showed that all of the viruses remainedcompletelystableatpH1.5at2Cforat

least 3 min. At pH 1.0,

poliovirus

Ti was in-activated within1min,andcoxsackievirusBi and echovirus6lost close to 50% of their

infectivity,

whereas coxsackievirus B3 remained stable. The recovery ofCAVremained constant

irrespective

of whether the durationof the celltreatment at

pH

1.5at 2C wasfor30 sec orfor 5min. Treatment ofcellsfor 1minat2CatpH2.0or2.5 recovered only small amounts of the totalCAV.

However,

byincreasing the duration oftreatment,at

pH

2.0

or2.5,to 5min,

progressive

elution of CAVwas

found to approach the

quantity

of virus disso-ciated atpH 1.5. It can beconcluded from these results and from those presented inTable 1 that, within the limits ofthe accuracy of the

plaque

assay,pH1.5

yields

totalrecoveryofthe receptor-bound virus.

In the next series ofexperiments,theefficacyto

7106

ILI \

a.0/ ^

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2.0 pH

FIG. 1. Effect of pHon the dissociation of entero-virusesfromHeLa cells.

elute CAV at pH 1.5, pH 2.5 or with 8 M urea, 6 M lithium chloride (LiCl), 4% sodiumdodecyl sul-fate (SDS), 1 % chymotrypsin, and alternate freezing and thawing was compared for each of the four viruses used previously. The cell-asso-ciated virus was prepared as described in Materials and Methods. The washed cells were taken up in a small volume of cold BSS-CaS3, and counted with a Coulter electronic cell counter. The cell samples were diluted in cold BSS-CaS3 to givea finalconcentration of 3 x 106cells/mlfor coxsackievirus B3,and 107cells/ml for the other three virus preparations. The CAV was deter-mined byfurther diluting each sample into acid solution, used for elution ofCAV, as described previously. The reversal of CAV with 8 M urea, 6 MLiCl, and 4% SDS wasperformedbymakinga 10-folddilution ofeachcell sample into the rea-gent followed bya 100-folddilution into PBSA-CaS3 . The amountof freevirus obtainedby each of the methods tested was determined by the plaque assay. In these experiments, no attempt wasmadetoaccount forthetotal amount of the input virus aswas donein the experiments pre-sented in Table 1.

The results of these experiments, compiled in Table 2, show that maximal recovery of cox-sackievirusesBi and B3 wasobtainedonly bythe method involving pH 1.5 and, therefore, the amounts obtained by this method were assigned values of 100%. The

high

salt

concentration

re-versed onlyasmallproportion,and SDS approxi-mately

50%,

of the

coxsackieviruses

available at the cell surface. In contrast, the poliovirus

Ti

receptorcomplexwasdissociated equally wellby the

high

saltconcentration and pH 1.5, withSDS againbeing less efficient. Theamountofrecovery of echovirus6 was

approximately

thesamefor all of the methodstested.Ingeneral,theprocedureof alternate

freezing

andthawingwasfoundtobean ineffective method for recoveringCAV.

Effectof acidpHonthecapacity ofHeLa cells to attach and eclipse enteroviruses. The effective use ofacidpHfordissociation ofreceptor-virus complexespromptedastudyof thestabilityatlow pH of theviralreceptors of HeLacells.HeLacells were suspended in 5 ml of cold 0.05 M glycine buffer atpH2.5 toa finalconcentration of 107/ ml. At intervals during incubationat 2 C,

1-ml

samples were withdrawn and diluted 10-fold in PBSA-CaS3 to bring the pH to neutrality. The cellsweresedimented by centrifugation for5min at225 X g,washed, counted,andresuspendedin

BSS-CaS3

toa concentration of 107

cells/ml.

The

treated cells were tested for their capacity to attachpoliovirus Ti andcoxsackievirusB3during a60-min

period

at37 C. Asa

control,

cellstreated atneutralpHweretested

concurrently.

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TABLE 2. Capacity of assorted reagents to dissociate enterovirusesfrom HeLa cells

Amt ofvirusrecoveredafter 1 minat 2Ca

Reagents CoxsackieBi Coxsackie B3 PolioTi Echo 6

PFU/ml Percent PFU/ml Perc PFU/ml PerPcentPFUml Percent (108) Pe et (107) Pe ent (i01) Pe et (10'1) Pern

0.05 M Glycine (pH 1.5) 1.07 100 1.34 100 3.9 100 1.26 100

0.05 M Glycine (pH2.5) 0.058 5.4 0.03 2.2 0.05 1.3 1.34 106

8 M Urea 0.028 2.6 0.05 3.7 4.6 118 1.48 117

6M LiCl 0.022 2.0 0.008 0.6 3.5 90 1.56 124

4% SDSin BSSb 0.57 53 0.4 30 2.7 69 1.38 109

1% Chymotrypsinc

NDd ND 0.02 1.5 ND ND ND ND

Freezing andthawing (5times) 0.16 15 0.11 8.2 0.18 4.6 1.30 103

None' 0.0078 0.7 0.002 0.2 0.01 0.2 0.24 19

aWashed HeLacells (5 X

107)

were suspended in5ml of virusat 2 C. Input multiplicities of virus

were 10, 50, and 100 PFU for coxsackievirus B3, coxsackievirus Bi, andpoliovirusTi and echovirus 6, respectively. After incubation for 1 hr at 2 C, the cell-virus suspensionwas dilutedto 50ml with

BSS-CaS3andcentrifugedat225 X g. Thecellswerewashedaccordinglyfourmoretimes. After the fifth wash, the cellswereadjustedto3X 106cells/mlforcoxsackievirus B3 andto10'cells/mlfor the other

viruses.CAVwasdetermined,asindicated in Materials and Methods. In theseexperiments,noattempt wasmadeto account for the totalamountofinputvirus.

b Duetotheformation ofaprecipitateat2C,the temperature of the SDSsolutionwas15C. cCells were treatedfor 30 minat37Cat aconcentrationof 3 X

106/ml.

dNot done.

eTheamountof virusin the fluidphaseofthe cellsuspensionpriorto treatment.

Theresultspresentedin

Fig.

2demonstrate that the receptor activity for coxsackievirus B3 re-mainedstableto treatmentatpH2.5for 10min, whereas the receptor activity for poliovirus Ti wasrapidlyinactivated.

To determine the pH at which the cellular receptorsforeachoffourenteroviruses would be inactivated,replicatesamplesof HeLacells (2 x

107 cells) were resuspended in 10-ml volumes of cold 0.05 M glycine buffer

adjusted

to different pH levels. After a 10-min treatment at 2 C, the cells werewashed,resuspended in asmallvolume

of

BSS-CaS3,

and

assayed

for

capacity

to attach

and to eclipse each ofthe viruses at neutralpH

during

a 1-hr

period

at 37 C. The amount of

attached virus which could not be recovered by 0.05Mglycine bufferatpH 1.5 wasconsidered to be eclipsed (7). The resultspresented inTable 3 show that the receptors for

attaching

coxsackie-viruses B1 and B3 and echovirus 6 were stable overthepHrange 1.0 to7.0,whereas thereceptors forpoliovirus TI were inactivated at a pH below 3.0. In contrast, theresults presented in Table 4 demonstrate thattheeclipse activityof HeLacells for each ofthe viruses tested was inactivatedby treatmentbelowpH 3.0. It should be noted that such treatmenthadnoeffectonthe attachment to HeLa cells of coxsackieviruses Bi and B3 and echovirus 6. Since HeLa-cellreceptors for polio-virus

Ti

were inactivated below pH3.0, the

in-loor

2 50

Id10

Iq 2

QC ZIN 10

5

A - COX B3

F

PTI

0 2 4 6 8 10

MINUTES FOR PRETREATMENTOFCELLSatpH2.5 FIG. 2. Comparative rates of inactivation, at pH

2.5, ofthe ability of HeLa cells to attach coxsackie-virus B3 andpoliovirus Ti.

activationofeclipse below that level could not be demonstrated.

Effect oftemperature on the capacity of HeLa

cells to attach and eclipse enteroviruses. The

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TABLE3. Comparative stability ofenterovirus receptors ofHeLa cellsto acidpH

Virusattachedb Pretreatment of

cellsa Coxsackie Coxsackie Echo 6 PolioTi

Bi B3 Eco6PloT

pH % %

N

%

1.0 99 97 74 2

2.0 96 97 86 7

3.0 99 99 80 54

4.0 98 99 82 65

5.0 98 97 76 55

6.0 99 97 77 57

7.0 98 97 80 64

BSS-CaS3 98 99 75 67

aReplicate samples of2 X 107 cells were

sus-pended in 10 ml ofcold 0.05 M glycine buffer at

thepH indicated and treated for 10 min at 2 C. BSS-CaS3atpH 7.0 served as acontrol.

bTreated cellswere sedimented, washed, resus-pended in 1 ml of BSS-CaS3, and counted. A

sample ofcellsuspension containing 107 cells/ml wasadjustedtovolumeof0.9ml,and 0.1ml of re-spective virus was added. Theinput PFU/0.1 ml was6.7 X 105 for coxsackieBi,2.9 X 105 for cox-sackie B3,4.2 X 105 for echo6, and5.5 X 105for

polioTi.Thecapacityof thecellsto attach each of theviruseswas determined at neutral pH for 1 hr at37 C.

porteddifferences in theheatstabilityofreceptors for enteroviruses (18, 21) suggested temperature as a means for the selective inactivation ofthe capacity of HeLa cells to attach and to

eclipse

enteroviruses. Replicate samples of 2 X 107 HeLa cellsweresuspendedin1.9 mlofBSS-CaS3 and heated at selectedtemperatures for 30 min. The cellsuspensions werecooledpriorto receiv-ingvirusinoculum, andthecapacityof the heat-treated cells to attach and to eclipse coxsackie-virusesBi andB3, poliovirus

TI,

andechovirus6 was determined at 37 C for 60 min.

Replicate

samplesofcells which were notheatedservedas

controls.

The results presented in Table 5 indicate that the receptors for

attaching

poliovirus

Ti were

inactivated at 56C, revealingthat theywerethe mostlabileof thereceptors studied. The receptors for echo 6 were

partially

inactivated at 60

C,

whereas a

slightly

greater temperature was

re-quired for inactivation of the receptors for

cox-sackieviruses Bi and B3. The results in Table 6 present the data

pertaining

tothe

thermostability

ofthe capacityofHeLa cells toeclipsethefour enteroviruses tested. These results demonstrated that, when HeLacellswereheatedat56 Cfor 30 min,theylost their

capacity

bothtoattach andto

eclipse

poliovirus

TI. On the other

hand,

these

cells attached but did not eclipse echovirus 6, whereas the attachment and eclipse mechanisms for coxsackieviruses Bi and B3 were affected

mninimally.

To confirm the observation that HeLa cells have a mechanism for eclipse of coxsackievirus

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Bi

whichcouldbe separated from that for echo-virus 6, anadditional experimentwasperformed. HeLacellsweresuspendedin BSS-CaS3 at a con-centration of107cellsperml and heated at 56 C. Atintervals, samples ofcells were removed and testedfortheircapacity to attach and eclipse each virus as described previously. The results of this experiment (Table 7) confirmedthe previous ob-servationthatattachment ofbothviruses occurred normallytotheheatedcells. However, the eclipse mechanism for echovirus6 wasrapidly inactivated whereasthecellswerestillcapable ofeclipsing at least 60% of the attached coxsackievirus Bi (Table 7). These results reinforce the previous observations and suggest that the mechanism which is involved in the eclipse of coxsackievirus Bi is distinct fromthatfor echovirus 6.

TABLE 4. Effect of acid pHon the capacity of

HeLacells toeclipse

eniteroviruses

Viruseclipsedb

Pretreatment

ofcellsa coxsackie Coxsackie Echo 6

Polio Ti

Bi B3

pH N S % %

1.0 <10 <10 <10 NDC

2.0 <10 <10 <10 ND

3.0 76 31 56 80

4.0 89 45 97 94

5.0 85 62 100 98

6.0 93 62 100 92

7.0 91 70 95 97

a For conditions, see the footnotes in Table 3. AftertreatingthecellsatthepHindicated,the

capacityof the cellstoattach (seeTable3)andto eclipse each of the viruses was determined at

neutral pH for 1 hr at 37 C. Eclipsed virus was

consideredtobe thatamountofinput virus

infec-tivity (seeTable3)whichwasdeterminedinitially tobecell-associatedandwhichwasnotdissociable from the cells by treatment with 0.05 M glycine

buffer (pH 1.5). The values of viruseclipsed were

calculated from thefollowing equations: (i) virus

attached (PFU) = input virus - unattached

virus; (ii) virus not eclipsed (PFU) = acid

re-covered virus - unattached virus; (iii) virus eclipsed (PFU) virus attached - virus not

eclipsed; (iv) viruseclipsed (%) = virus eclipsed

(PFU) X 100/virus attached (PFU).

Not done; the pretreatment inactivated the

capacity of the cellsto attach poliovirus Ti.

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TABLE5. Thermostability ofenterovirus receptors of HeLacells

Virusattachedb Pretreatment

of cellsa Coxsackie Coxsackie Echo6 PolioTi

Bi B3 Eco6PloT

C % No No %

37 98 96 77 81

40 NDc ND 80 74

45 ND ND 74 78

50 ND ND 67 62

56 99 98 69 15

60 98 97 42 <10

65 <10 <10 25 <10

70 <10 <10 <10 <10

aReplicate samples of 2 X 107 cells were sus-pended in 1.9 ml ofBSS-CaS3 and heated at the temperatureindicated for 30 min.

bCellsuspensionswerecooledpriortoreceiving the respective virus. The input PFU/0.1 ml was

1.98 X 106 for coxsackieBi,2.5X 107forcoxsackie

B3, 1.38 X 106 for echo 6, and 1.16 X 106 for

polioTI. Thecapacityof the cellsto attach each of the viruseswasdetermined at37Cfor 1hr.

c Not done.

TABLE 6. Thermostability of the capacityof HeLa cells toeclipse enteroviruses

Virus eclipsedb Pretreatment

ofcellsa Coxsackie Coxsackie Echo 6 Polio1

Bi B3 Eh oi

C % N % %

37 72 73 68 83

40 NDC ND 68 68

45 ND ND 61 82

50 ND ND 28 56

56 65 43 13 <10

60 63 39 <10 d

aFor conditions, see the footnotes in Table 5. bAfter treatment, the capacity of the cells to attach (see Table 5) and to eclipse each of the

viruseswas determined at neutralpH for 1 hrat

37 C. Eclipsed virus was considered to be that

amount of input virus infectivity (see Table 5)

which was determined initially to be cell-asso-ciated and which was not dissociable from the cells by treatment with 0.05 M glycine buffer (pH 1.5). The values of virus eclipse were

cal-culated from the following equations: (i) virus attached (PFU) = input virus-unattachedvirus; (ii) virus not eclipsed (PFU) = acid recovered virus - unattached virus; (iii) virus eclipsed (PFU) = virus attached - virus not eclipsed; (iv) virus eclipsed (%) = virus eclipsed (PFU) X 100/virus attached (PFU).

e Not done.

dPretreatment inactivated the capacity of the cellstoattach poliovirusTi.

TABLE7. Comparative ratesofinactivation of

the ability of HeLa cells toattach and eclipse coxsackievirusBland

echovirus 6

Coxsackie Bi Echo 6 Pretreatment of

cellsat 56CG

Attached Eclipsedb Attached Eclipsedb

min % % % %

None 96 78 88 68

5 97 61 81 5

15 96 61 72 <1

30 97 59 69 <1

aReplicate samples of 107 cells/0.9 ml of

BSS-CaS3 were heated. At time intervals indicated,

the cellswere removed andcooled prior to addi-tion ofvirus.Theinput PFU/0.1mlwas8.7 X 105 and 7.9 X 106for coxsackieBl andecho 6,

respec-tively. The capacity of the cells to attach and eclipse each of the viruseswasdetermined at 37C for 1 hr.

bValues were calculated from the equations given in Table 4.

DISCUSSION

Thecomparative experimentsdescribed inthis report represent butonephase of ourcontinuing

investigations

directed toward characterizing the

properties and functions of enterovirus receptors onintactHeLacells

(1-4, 10, 23, 24).

The results of these

experiments

showed

clearly

that the receptor

activity

ofHeLacellsforattaching polio-virus Ti wasmorelabiletoelevatedtemperatures andtolowpHthanwerethe receptoractivitiesfor

binding

echovirus6and coxsackieviruses Bi and

B3. These

findings,

in part, confirmed and ex-tended the results of

Quersin-Thiry

and Nihoul (21), who showed a difference between the

thermostability

of receptor

activity

ofHeLa cell

extractsfor

poliovirus

Ti and coxsackievirus B4. Thus, additional evidence is provided which serves to reemphasize the differences among the receptorsofHeLacells forattachingthedifferent enteroviruses.

To determine the thermostability and acid

stability

ofHeLacellsto

eclipse enteroviruses,

we

considered it important first to obtain a method tomeasure accuratelytheamount ofviruswhich

becomes cell-associated under conditions pre-cluding viral eclipse. Thus, of all the methods tested, a brief exposure ofCAV to buffer at pH

1.5 at 2Cprovidedageneralmethod for recovery of maximal amounts of each of the viruses studied. Bydetermining the amount of receptor-bound virus at a given

time,

it was possible to

assessthecapacityof HeLacellstoeclipse entero-virusesat37 Caftertreatmentof cells with acid or heat. Theresults showed that theeclipse activities

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of cells (as defined in this paper) for coxsackie-viruses Bi and B3 and echovirus 6 were inacti-vated irreversibly at pH2.0 without impairing the activity of the receptors for attaching these viruses. In contrast, thefinding that both of these functions were inseparable for poliovirus Ti suggests that different events may be operative during theearlystagesof infectionby the different enteroviruses.

It was observed that even though heating of HeLacellsat56Cresulted in lossof cellviability, as determined by trypan blue staining (un-publisheddata),such "dead" cells, which did not attachpoliovirus Ti, attachedbutdidnoteclipse echovirus 6; yet these cells both attached and eclipsedcoxsackieviruses Bi and B3. Thefinding thatheat-killedcells remained capable of attach-ing and eclipsing coxsackieviruses Bi and B3

raises again the question whether the uncoating of coxsackieviruses occurs within phagocytic vesicles (13) oratthe receptors on thecell surface (3, 17). The improbability that heat-killed cells retainedtheability of engulfing virussupportsthe latter possibility. Resolution of this question, however, requires a better understanding of the relationship between the various operational de-finitions of viral eclipse and ofthe uncoating of the RNA genome ofeachofthe different

entero-viruses.

ACKNOWLEDGMENT

Thisinvestigation wassupported by Public Health Research grantAI-03771 toR. L. Crowellfromthe National Institute of Allergy andInfectiousDiseases.

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