Binding characteristics of Rauscher leukemia virus envelope glycoprotein gp71 to murine lymphoid cells.

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Binding Characteristics of

Rauscher Leukemia Virus

Envelope

Glycoprotein gp7l

to

Murine Lymphoid

Cells

A. K. FOWLER,l* D. R.TWARDZIK,1 C. D. REED,1 0. S. WEISLOW,2 AND A. HELLMAN'

National CancerInstitute'andLitton Bionetics,Inc.,2Frederick Cancer Research Center, Frederick,

Maryland21701

Receivedforpublication 15 July 1977

The major envelope glycoprotein (gp71) purified from Rauscher leukemia

virus (R-MuLV) binds efficiently to murine lymphoid cells but not to either murinenonlymphoid cellsorlymphoidcells from other species. Binding of 125I-labeledR-MuLVgp7lwascompetitively inhibitedbyunlabeled glycoprotein, as wellasby whole R-MuLV, butnotbymurine xenotropic viruses, R-MuLVp30, and several unrelated proteins. Polyacrylamide gel electrophoresis profiles of

iodinatedgp7lafter bindingtolymphoid cellswere similar to prebound profiles. Antibodyto R-MuLV gp7l prevented binding, whereas normal serum had no effect. Adsorption of the glycoprotein to murine lymphoid cells occurs rapidly and is time and temperature dependent. The procedure described is sensitive fordetecting thebindingactivity of approximately104cells.Binding was propor-tional up to 2.5 X 105cellsperml andplateaued above 107 cells perml. In the

presence of excess R-MuLV gp7l, BALB/c thymocytes bound approximately

2.4 X 10 moleculespercell.

Oncornavirus infection of mice results in the

depressionofboth humoral and cellular immune reactions in vivo (for review,see5,22).

Further-more, evidence that in vitro cellular immunity

is impairedin virus-infected mice has been

re-ported (10, 12). These observations have more

recently been extended by the demonstration that freeze-thawed extracts of Rauscher leuke-mia virus (R-MuLV) suppress in vitro cell-me-diated immune reactions of normalmouse

lym-phocytes (A. K. Fowleretal.,in press).Similarly, UV-inactivated feline leukemia virus also

sup-pressesin vitroblastogenic responsesofnormal

cat lymphocytes (L. L. Hebebrand et al., in press). Sincemost, ifnotall,mammals contain

genetic information for related type C viruses-xenotropic viruses-thatare inducible in vivo in the host byhormonal (6, 7, 11) and

immunological (14) stimulation, we have

sug-gested that virion components endogenous to the host function as regulators in normal

im-munological processes (13). Indeed, xenotropic

viruses are actively expressed in maternal (8)

and fetal (15, 17) tissues during pregnancy, a

periodofintricate hormonal andimmunological interaction.

Theprimaryeventinvirus-cellinteraction is the adsorption ofthe virion,

presumably

via a

viral envelope component, to specific cellular

receptors. Recent progress in

identifying

and

isolating oncornaviral proteins now permits a

more detailed elucidation of virus-cell

interac-tion. Accordingly, we have undertaken studies

todeterminetheeffect ofpurified virion proteins on in vitro murine lymphocyte transformation andtoexamine theirbinding characteristics, the

receptorsinvolved, and the mechanisms of viral

protein-cell membrane interaction that lead to modification of cell behavior.During these stud-ies, wehave observed that the major envelope

glycoprotein(gp7l)ofR-MuLV, like AKRgp7l

(18),induces transformation ofnormal

lympho-cytes, whereas alower-molecular-weightvirion

protein(s) appears to depress T-cell function (Fowleretal.,inpress). Inthis communication,

weextendourprevious findingsby characteriz-ingthebindingof R-MuLVgp7ltolymphocytes

and othercells derived from various organsof several strains of mice.

MATERIALS AND MERTHODS

Cellpreparation. Thymuses, spleens,or

epididy-mides were excised and minced in 20 ml of RPMI 1640 medium and gently sieved through a 60-mesh

stainless-steel filter. Thymic cell preparations were

thenfurther washed three timesbyrepeated centrif-ugation (250 x g)andresuspensionwith fresh medium.

More than 90%of thethymocyteswereviablebythe trypan dye exclusion test. Splenic cell suspensions

werewashedoncewith fresh medium and thenlayered

on a Ficoll-Hypaque gradient. The

lymphocyte-en-richedlayerwascollected and washedthreeadditional timeswithfresh medium. Theviabilityof thesplenic

lymphocyteswasconsistently above90%. Forsperm

cellpurification,theepididymalcellpreparationswere

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730 FOWLER ET AL.

initially low-speed centrifuged (200xgfor3 min)to

remove particulate material. The cells remaining in thesupernatant werethen concentrated by

centrifu-gation (1,000xgfor20min) and furtherpurified by

discontinuous sucrose gradient (20:50:80%)

centrifu-gation (approximately 80,000xgfor60min)at40C. Thespermcells collectedatthe50:80%sucrose inter-facewerewashedanadditional threetimes with fresh

medium. The finalspermpreparationcontained fewer

than 5% contaminating cells. No estimate ofsperm viabilitywasmade. Peripheral lymphocyteswere pre-pared from heparinized blood by Ficoll-Hypaque sep-aration andwereprocessed identicallytothe splenic

lymphocyte preparations. Greater than 90% of the

cellswere viable. Blood used for thepreparation of

peripheral lymphocyteswascollected from rodentsby

cardiacpunctureand fromprimatesbyvenipuncture. Erythrocytes used in these studieswerealso washed threetimeswith fresh medium.

Glycoprotein purification and

characteriza-tion. BandedR-MuLV (1012 virus particlesperml),

produced in JLS-V9 cells and having an infectious titer of2x108focus-forming unitsperml,was

freeze-thawed twiceandcentrifugedat105,000xgfor90mi at40C. Thesupernatantwaslyophilized and dialyzed

againsta solution containing 0.01 M

NN-bis-(2-hy-droxyethyl)-2-aminoethanesulfonic acid (BES) (pH

6.5),0.001MEDTA, and 1.0M NaCland appliedto

aSephadex G-100 column (1.5 by 90 cm)equilibrated

withthesamebuffer. Peak fractionscontaining

enve-lopeglycoproteinwere identified bysodium dodecyl

sulfate-polyacrylamide gel electrophoresis stained with either Coomassie blue orSchiffreagentand, if

contanatedwithbovineserumalbumin, further

pu-rifiedbyphosphocellulose chromatography as previ-ously described (25). The purified glycoprotein was

labeled with'25I(1x10Wto5x1iO cpm/ngofprotein),

using the chloramine-T method(9). Afteriodination,

95 to98% ofthe acid(10% trichloroacetic acid)-precip-itableglycoproteinwasprecipitable with specific

an-tiserumprepared against purified R-MuLV gp7l and demonstrated less than a 2%immune precipitation with anti-bovineserumalbumin. Sodiumdodecyl

sul-fate-polyacrylamide gel electrophoresis of the iodi-nated glycoprotein demonstrated a single band

mi-gratinginthe71,000-dalton region (see Fig. 3A) and hereafter will be referredtoasgp7l.

Binding assay. Cells were incubated at 370C in

thepresenceof['MI]gp7lin1.0 ml ofmedium

contain-ing1%bovineserumalbumin.Cellconcentrationwas

maintainedat 10i cellsperml unlessotherwise indi-cated.All bindingassayswereperformedin polysty-renetubes (12 by75 mm) that had beenprewashed withmedium containing bovineserumalbumin.

Dur-ing incubation, cells were gently mixed on a roller

drum(10 rpm). Afterincubation, the cellswere

cen-trifugedat400xgfor 10minat40C.Thepelletwas

resuspended in2.5ml ofcold mediumcontaining bo-vineserumalbumin andrecentrifuged. This procedure wasrepeatedtwoadditionaltimes.Theradioactivity associated with the final cell pellet was measured

directly in a Nuclear-Chicago gamma counter. To

estimatenonspecific binding of the radiolabeled gp7l

toreaction vessels, control tubes,towhich no cells were added, were processed identically. This value,

which was consistently less than 300 cpm per tube, wassubtracted from theradioactivity associated with

correspondingtestculturestoderiveacorrected

spe-cificbindingvalue.

RESULTS

The binding of

[1"I]gp71

to murine splenic andthymiccellswasinitiallylinearand temper-ature dependent (Fig. 1). At 370C, binding

oc-curred rapidly, and within 15 min 50% of the maximum bound level was attained. The rate ofbinding subsequentlydecreased, and after100 min therewasminimalincrease intotalbinding level. After prolonged incubation (24 h) total

cell-associated label decreased approximately 20%,presumably duetodegradationofthe gly-coprotein. Incomparison, at00C (wet ice) the rateofbindingwasmuchslower, with 50% sat-urationoccurringatapproximately75min. For

maximum binding at 00C, nearly 6 h was

re-quired, but the total level boundwassimilarto that observed at370C, 3,693 versus 3,607 cpm per

101

thymic cells,respectively.

To further investigate binding specificity, thymiccellswerepreincubatedfor 1 hat370C withvaryinglevels ofunlabeled R-MuLVgp7l

andp30,as wellaswithseveral unrelated

pro-teins (bovine senun albumin V, ovalbumin, myoglobin, and cytochrome c), before

adding

['25I]gp71.

Afteranadditional 30min of incuba-tion, only theunlabeled gp7l

competitively

in-hibited binding (Fig. 2). The

preincubation

of cells with as little as 3 ng of unlabeled gp71 reducedbindingby 10%,andincreasingthis level to 6 and 13 ng reduced binding 20 and

50%,

respectively. Total competition, however,

re-quired approximately a50-fold excess of

unla-beled to labeled

glycoprotein

(200 ng per 106

>100 10 0030 00 20

, looS~~~~~in t

860- K <

WE E m 37°C

!~~~~~~~~~~~~~~~OC

100 200 300 1000 2000

Minutes

FIG. 1. Effect oftemperature onthe binding rate

ofR-MuL V gp71 byBALB/c thymic cells.A3.5-ng

amountof125I-labeled R-MuLVgp7l (102,000 cpm)

wasaddedto106thymocytes and incubated at either

0 or370C. Cell-boundradiolabel wasdetermined as described in the text. Maximum levels boundwere 3,693 and3,607 cpmat0and

370C,

respectively. Each

point represents the mean value of quadruplicate

cultures.

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100

'

O , 80

_ rL

, o60

E>

0

40

c2 !

ILCM 20

10-1 100 101 102 103 104

Unlabeled RLVgp7lAdded(ng)

FIG. 2. Competitive inhibition by unlabeled R-MuLVgp7l of '25I-labeled R-R-MuLVgp7l binding by BALB/c thymic cells. Thymocytes (106/ml) were

in-cubatedfor60minat37C withvarying amountsof unlabeledgp71 before3ngof

I5IIgp71

(30,200 cpm) wasadded.Afteranadditional 30minof incubation,

cell-bound radiolabelwasdetermined asdescribed inthetext.Eachpointrepresentsthemeanvalueof

duplicatecultures. Thepreincubation of thymocytes with 6ngofunlabeled gp71 reduced binding20%.

cells). Similarly (data not shown), antibody to R-MuLV gp7lprevented binding,whereas

nor-mal serum and antimurine immunoglobulin G and immunoglobulin Mserahadnoeffect.

Ad-ditionalevidenceofspecificitywasalsoobtained

by thepretreatmentof thymic cells with

infec-tiousR-MuLVaswellastwomurinexenotropic

viruses, NZB-C135 (21) andM-55 (1). At

equiv-alentconcentration, 1010 virus particlesperml,

R-MuLV reduced binding 80%, whereas no

ef-fect was observed with the xenotropic viruses.

The observationthatunlabeledgp7l

compet-itivelyinhibitedbinding indicates thatthe iodi-nated and unlabeled R-MuLV gp7l molecules

are biologicallyverysimilar and thatthe

mea-sured binding is not an artifactresulting from

thenonspecific binding of by-productsof

iodin-izationorfromtheuptake ofsmalllevels offree

"I.

Tofurtherexamine this possibility,the

so-diumdodecylsulfate-polyacrylamide gel electro-phoresis profile of

['"I]gp71

bound to splenic

cells after a 60-min incubation period at370C

wascomparedwith nonreacted labeled

glycopro-tein (Fig. 3). Although several minorpeaks of radioactivitywerenoted in thecellularextract,

themajorpeakofactivitycorresponded tothe

standard labeled R-MuLVgp7l, indicatingthat

no significant change in molecular weight

oc-curred duringthebindingassayprocedures.The

minorpeaks undoubtedlyrepresentaggregation

and degradation products of the glycoprotein

andare consistent with theinterpretationthat

the observed reduction in cell-bound label after prolonged incubation is largelyattributable to

glycoprotein degradation.

The effect of celldensityonbindingwas

stud-ied by adding 3 ng of

['II]gp71

to BALB/c thymic cells. As showninFig. 4,the assaywas

sensitive for detecting the receptor activity of 104 cells, and binding was proportional up to a cell density of 105 cells per ml. At higher cell concentrations, the relative binding level grad-ually decreased andeventually plateaued above concentrations of107/ml. Aconsistent observa-tion made throughout these experiments was that only a portion of the input radiolabeled

glycoprotein, rangingfrom 15 to40%,depending ontheprobepreparation,wasboundby murine lymphoid cells despite a large excess of cells (107). Although receptor site masking or inter-ference may beinvolved, it is reasonable that a

significant portion of the iodinated gp7l main-taining imnmunological reactivity is not

biologi-callyactive basedonitsbinding properties. Data favoring thisinterpretationhave been obtained by multiple reincubation of reactant superna-tantsremoved from thymus cells (107/ml) pre-viously pulsed and incubated with

['5I]gp71

withfreshthymus cells. Fresh cells, during the

second and third incubations of the reactant supernatants, boundonly 6.7and 3.1%,

respec-tively, of the residualimmunologically reactive

[1251]gp71.

In comparison, 29.8% of the imnmu-nologicallyreactiveglycoproteinwasbound dur-ing the first incubationperiod. The total

[1"I]-gp7lbound during four reincubationcycles

rep-resented only41.7% of theimmunologically re-activeglycoproteininput.The observed variable

bindingefficiencyprobablyreflectsprocedurally

related degradation of the glycoprotein; how-ever,anaturalheterogeneityof the gp7l mole-culemustalso be considered.

Toestimate the number of receptor sites per

cell, BALB/cthymiccellswereincubated with an excess of

"2I-labeled

glycoprotein. As may benoted inFig.5, the total iodinatedgp71bound per 106 cellsincreased upto

approximately

2.7 ng (38.5 x 10-15 mol) as the level of

supple-mentedglycoproteinwaselevatedto107

ng/ml.

Extrapolating from this value, the number of R-MuLV

gp71

molecules bound per cell is

ap-proximately2.5x 104.

Cellspecificity ofR-MuLVgp7l bindingwas examinedtwoways and is summarized in Table 1.The firstcomparedthebindinglevelof murine

lymphoid cells to nonlymphoid cells

(erythro-cytes and sperm), and the second determined the relativebindinglevel of

lymphoid

cellsfrom several species. In general, murine lymphoid

cells exhibited thehighest

potential

forbinding

R-MuLV

gp71,

and, in most strains

studied,

thymus-derivedcells boundmoreviralenvelope glycoproteinthanspleniccells.Anotable excep-tiontotheobserved highrelative bindinglevel oflymphoidcellsforR-MuLV

gp7l

was

consist-ently noted in the AKR mouse, in which the binding level of both

thymic

and

splenic

cells

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732 FOWLER ET AL.

A Bovine

Serum Ovalbumin Albumin

Cyctochrome-C

B

10 2

10 20 30 40 50 60 70 80

FractionNumber

FIG. 3. Polyacrylamidegelelectrophoresis of lu2I-labeled R-MuLVgp71run on75to25% linear gradients

(cylindricalgels10cminlength) inthepresenceof0.1%sodiumdodecyl sulfate. Sampleswereprepared by

heating for2minat1XfCinasolutioncontaining50mMTris-hydrochloride(pH 6.7),2%sodiumdodecyl

sulfate,0.5%,8-mercaptoethanol,and0.01%bromophenolblue.After16 hofelectrophoresis (9 mApergel, 75

pulsesper s,0.5 ,uF), gelsweresliced into 1.3-mmfractions.Arrows indicatepositions of marker proteins. (A) ControlR-MuLVgp71; (B) BALBIcspleniccellextractafter a 60-min incubation at37°C with R-MuLV gp71.Before extraction,cellswerewashedasdescribedinthetext.

was approximately 20% of that noted for the

otherstrains. Of the murinenonlymphoid cells

examined, erythrocytes exhibited the lowest

bindinglevel(<2%ofreferencecontrol), whereas

the binding level of sperm cells was slightly

higher (10to15% of referencecontrol).

Except for thebinding level of the

Sprague-Dawleyrat(20%ofreferencecontrol), the

bind-ing of R-MuLV gp7ltolymphoid cellsfromthe

other species tested (rabbit, baboon, human)

wasconsistentlylow(<10%).

DISCUSSION

The results presented demonstrate that the

major envelope glycoprotein purified from

R-MuLV binds efficiently to murine lymphoid

cells. The rate of R-MuLV gp7l binding to

splenic and thymic cells is rapid and initially

linear but, in contrast to that noted for

fibro-blastic cells(4), is temperaturedependent. The

rateofbindingat370Cwasapproximately

five-fold that observed at 0°C (wet ice), although

the maximum extents ofbinding were similar

atboth temperatures. These findings are

con-sistentwith earlier data showing that the rate

ofpoliovirus adsorption to cells in vitro is

de-creasedbyareductionintemperature (16)and

mayreflectachangeinthe randomprobability

ofvirus-cell interaction.

The adsorption of gp71 tolymphoid cells is

highlyspecific. This is indicated by the

inhibi-tion ofbinding by antiserum toR-MuLV gp7l

x

C,

r._ 0. cm -J

-j z

CM

i-8

10

x

a

0L

6

4

21

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and the competition for available membrane

receptors by either murine ecotropic virus (R-MuLV) orits purified viral envelope

glycopro-tein(R-MuLV gp7l). Murine xenotropic viruses

a

i

a

0

i3

Iol

a2

4 5 6 7

CellNumbe (LOgic)

FIG. 4. Effect of celldensityonthebindingof

R-MuLV gp71 by BALBIc thymocytes. Thymocytes at

varyingconcentration(104to107/ml)wereincubated

for45min at370C in the presence of3 ngof

125I-labeledR-MuLVgp7l (25,000 cpm). Cell-bound

ra-diolabel was determined as described in the text.

Eachpointrepresents themeanvalue of triplicate

cultures.

U',

20 40 60 50 100

112511 gpil1Added(ng)

FIG. 5. Effect ofR-MuLVgp7l concentration on

the level boundbyBALB/cthymiccells. Thymocytes

(106/Ml) were incubatedfor 60 min at370C in the presence of increasing amounts of '251-labeled R-MuL V gp71. Maximum input(11I0 ngofgp71) con

[image:5.491.292.413.66.225.2]

tained525,000) cpm.Cell- boundradiolabelwas deter-minedasdescribed in thetext.Eachpointrepresents themeanvalueofduplicatecultures.

TABLE 1. Relativebinding of'25I-labeledR-MuLVgp71 to different cells

No.of Relative

[fnI]gp71-binding

expts efficiency(mean%,range)

Mouse BALB/c 21days Thymic 8 100.0

65days Thymic 2 139.2(130.5-143.2)

Splenic 3 65.7(61.7-69.9)

Sperm 2 12.4(10.8-14.0)

RBCb 5 0.7(0.5-1.2)

NIHSwiss 65days Thymic 2 105.8(93.8-118.5)

Splenic 3 94.2(85.0-107.0)

RBC 2 1.2(0.6-1.7)

NZB 65days Thymic 2 158.3(149.4-167.2)

Splenic 3 88.1 (72.7-99.5)

Sperm 2 8.2(6.6-9.8)

RBC 2 0.6(0.5-0.8)

AKR 65days Thymic 2 16.8(15.1-18.6)

Splenic 3 21.7 (14.4-30.0)

Sperm 2 11.9(11.0-12.8)

RBC 2 1.2(0.7-1.6)

Rat Sprague-Dawley 6mo Thymic 2 20.2(19.9-20.4)

Splenic 2 28.3(18.1-38.5)

Rabbit NZW 7days Thymic 2 6.6(4.6-8.7)

Splenic 2 9.3(9.2-9.4)

6 mo Peripheral 2 4.2(3.2-5.2)

Baboon Cynocephalus 5yr Peripheral 1 8.4

Man Caucasian 20-30yr Peripheral 2 5.6(3.6-7.7)

aAU

values are relative to the binding level ofthymic cells from 21-day-old BALB/c mice assayed in

parallel. Cells (106n/ml)wereincubated for 60min at370C in the presence of 3 to4ngofR-MuLV[I251]gp7l.

Cell-boundradiolabelwasdeterminedasdescribed in thetext.The number ofreplicateculturesperexperiment

rangedfromtwo tofour. RBC,Erythrocytes.

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734 FOWLER ET AL.

(NZB and M-55), on the other hand, failed to interfere with R-MuLV gp7l adsorption to lymphoidcells, indicating that mouse lymphoid cells, like mouse fibroblastic cells (4), contain a population of membrane receptors for ecotropic virus that have no demonstrable affinity for mouse xenotropic virus. This is further evi-denced by the highbinding affinity for R-MuLV gp7l exhibited by lymphoid cells from NZB mice, a strain known to produce high titers of xenotropic virus (20) and whose thymocytes ex-pressconsiderable endogenous xenotropic viral envelopeglycoproteinontheir cell surface (19). Similarly, thymocytes from BALB/c mice, re-centlyreported tonaturally express endogenous xenotropicviral envelope glycoprotein (3), also bindhighlevels of R-MuLVgp71.

Several differenceswereapparent in the rela-tivebinding capacityofcells from different or-gans and different strains of mice. Lymphoid

cellsderived from thespleen, the primary target organ of R-MuLV-induced erythrocytopoietic

disease, consistently exhibited a much greater capacity for binding R-MuLV gp7l than did either sperm cellsorerythrocytes.Thymic cells, by comparison, bound as much viral envelope glycoprotein as splenic cells, and thymic cells fromsomestrains bound

significantly

morethan spleniccells.Thehigh degreeofthymocyte bind-ing of R-MuLV gp7l is best explained by a cross-reactivity of murineecotropic viruses for the same cell surface receptors. Thus, thymo-cytes, the target cells for certain murine

eco-tropic viruses,suchasMoloneyleukemiavirus,

contain receptors in vivo thatprobablybind R-MuLV and other murine ecotropic viruses

equally well. Indeed, murine ecotropic viruses havebeen shown touse thesame receptors on mousefibroblast cellsinvitro(2, 4, 24). Further-more, the observed reduced binding of R-MuLV

gp7lby AKRlymphoid cells,astrainknown to

synthesize endogenous ecotropic virus, is con-sistentwiththisinterpretation, since viral syn-thesis would result in in vivo saturation of the available cellular receptor sites forecotropic

vi-ruses.Inpreliminarystudies,wehave alsonoted

asignificant reductionof R-MuLVgp7l binding tolymphoid cells of BALB/c mice after exoge-nous infection with either R-MuLV or Friend leukemia virus(unpublished data).

The existenceof cross-reactive bindingsites

forR-MuLVenvelope glycoproteinoncells from

at least one other species, the rat, is also

sug-gestedfrom these data. Theextentof R-MuLV

gp7l bound byrat lymphoid cells, though low

compared with mouse lymphoid cells (20 to 30%), was consistently observed. This binding

appeared to be specific by competition

experi-mentsand isconsistent with the earlier

obser-vation that newbornrats arehighlysusceptible

toR-MuLV infection (23).

In thepresence of excess iodinated

glycopro-tein, thymocytes from BALB/c mice bind

ap-proximately2.7ng(38.5 x 1015mol)of R-MuLV per 106 cells. This isequivalentto anestimated binding level ofapproximately 2.4 x

10W

mole-cules perlymphoidcell.Bycomparison, murine

fibroblasts-cellsofdifferent derivation and

ex-hibiting morphological characteristics widely

different fromlymphoidcells, includingalarger cellsurface area-bind5.3 X

10'

molecules per

cell(4), orapproximately20-fold that observed for thymocytes. Such estimates, however, are based on the assumption that all cells within thepopulation examined contain the same fam-ily orfamilies of receptors atequivalent numbers and thattheybind the sameamountof glyco-protein. These assumptions may be more valid with certain murine cell populations thanwith

others;certainly,the datapresentedhere do not rule out thepossibilitythat thebinding efficien-cies of specific lymphoid cell subpopulations vary, that the numbers of receptors per cell

differ,orthatmultiple receptorforms exist on

individualcells.Indeed,thedifference noted be-tween the relative binding level of thymocytes andsplenic cells from NZB and BALB/c mice suggests that one ormore of thesepossibilities islikelyin vivo. It shouldalso be notedthat,in addition to lymphoid cells serving as vehicles for virus adsorption and replication, immuno-cytes may also bind virus via antigen-specific receptors as part of the normal events leading toantiviralimmunity.Althoughantigen-binding cells are present in only low numbers before immunization, we cannot completely rule out their involvement in the binding studies per-formedhere;however,preliminary blocking ex-periments with anti-mouse immunoglobulins in-dicate that their participation is minor. Addi-tional studiesareneededto

clarify

these points andtodetermine the extent of cell type specific-ity and maturation level in the regulation of virus-cell receptor activity in vivo. Such infor-mation mayprovide insights into the mechanism

of viralprotein-cell membrane interaction that

leads to the modification of cell behavior in immunity andoncogenesis.

ACKNOWLEDGMENT

Thisworkwassupported by Public Health Service contract

NO1-CO-254-23from theNational Cancer Institute.

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