Identification of cell membrane proteins that bind visna virus.

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0022-538X/91/116137-07$02.00/0

Identification

of Cell

Membrane Proteins

That

Bind Visna Virus

SHARON E. CRANE,1 JEANINEBUZY,2 ANDJANICE E.

CLEMENTS'*

Division of Comparative Medicine, Department of Molecular Biology and Genetics1 and NeuroscienceDepartment,2

TheJohns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 16 April1990/Accepted 31 July 1991

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelopeglycoprotein. Theidentity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to targetcells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells ofapproximately 15, 30, and 50 kDa bound to visna virus. Thebinding ofvisna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. '251-labeled cell membranepreparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and had molecular masses of15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface ofbothliveSCP and GSMcells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of

[35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa

proteins did not block virus binding to cells. The blocking activity of antibody to the 50-kDa protein provided data that this protein is the molecule which visna virus recognizesand binds to on the surface of targetcells.

Visna virus is a sheep lentivirus which causes a

progres-sivepneumoencephalitis andarthritis in sheep (26). In vivo,

the virus infects cells of the monocyte-macrophage lineage (19). In primary cells from sheep and goats, visna virus binds toits receptor and probably enters the cell through fusion of the viral membrane with the cell membrane, as does a related lentivirus, human immunodeficiency virus (15). The

identity ofthe receptor for visna virus has not yet been

determined. Neutralizingantibodiestothevisna virusinhibit

thebinding of virus toits receptor on sheep cells in culture

(12). In contrast, neutralizing antibodies facilitate binding,

penetration, and uncoatinginmacrophages in culture,owing

tobinding ofthevirus-antibody complextoFc receptors (10,

11). The spread of virus from macrophage to macrophage

maybeamechanism forviral persistence in the host (5, 12).

Enveloped viruses such as visna virus attach to cells

through the binding of their envelope glycoprotein to a

specific cell surface molecule. Cells must have the virus

receptor on their surface to be susceptible to infection. However, the presence of the receptor on the cell surface

doesnotnecessarilyrender thecell susceptibletoinfection;

it has been shown in other viral systems that there are

additional species-specific and cell-specific factors which

allow the virus to continue its lifecycle and replicate in a

permissive cell (14). The receptor can be a ubiquitous

molecule such as a sialic acid-containing glycoprotein for

influenza virus (3) or a specific molecule such as the C3d

receptorfor Epstein-Barrvirus (8), the intercellular adhesion molecule ICAM-1for rhinovirus (9,29),ortheCD4 molecule

for human

immunodeficiency

virustype 1(14,

25).

Anumber

of the specific molecules found to be viral receptors are membersof theimmunoglobulinsuperfamily, asdetermined by theirconserved amino acids and domain structures (18).

These receptors may beimportant in the therapeutic treat-ment ofviraldiseases (13, 27).

To characterize the cell surface molecules which bind

visnavirus,weutilized theaffinity ofthevirusfor molecules

* Correspondingauthor.

onthe surfaceof sheepchoroidplexus(SCP) cells and goat

synovial membrane(GSM)cells. Virus overlay protein blot

assays(VOPBA)wereused to identify moleculeswhichthe virusrecognized (2). Three sizes of proteins wereidentified,

15,30, and 50kDa,towhich visna virus bound.Neutralizing

antibodiestovisnavirus reducedbindingof the virustothe

cellsurface molecules in the VOPBA. Affinitypurificationof theseproteinswasused to isolate them and raiseantibodies

against them. Antibody to the 50-kDaprotein reacted with

the surfaces ofboth live SCP and GSM cells in

immunoflu-orescence assays and blocked the binding of radiolabeled

virus to the surfaces ofintact cells. Therefore, the 50-kDa

protein isimportant in thebinding of visnavirus to

suscep-tible cells and may in fact be the virus receptor or a component ofthe receptor molecule.

MATERIALS ANDMETHODS

Cells and virus. SCP and GSM cells were obtained as

previouslydescribed(21,22, 30). L cellswereobtainedfrom

theAmericanType CultureCollectionand grown inminimal

essentialmedium (MEM) (GIBCO)

supplemented

with 10%

fetal bovine serum. Purified visna virus strain 1514 and

caprine

arthritis-encephalitis

virus strain COwere

prepared

aspreviouslydescribed (4, 23,24). Visna viruswas

radiola-beled with

[35S]methionine

as

previously

described

(12).

Viruswaspurifiedfrom the mediumbyclarifyingat10,000 x

gfor 15 min and centrifugingat 100,000 x gfor 2h. VOPBA. Virus binding assays were done as

previously

described (2) with the

following

modifications. A

1-mg

sample of cell

lysate

orcell membranewasrun on a5to20%

gradient sodium dodecyl sulfate

(SDS)-polyacrylamide

gel

and transferredtonitrocellulose membranes. Nitrocellulose

was stained with ponceau S (Sigma), cut in

strips,

and blocked in VOPBA buffer (50 mM Tris HCl

[pH

7.4],

150

mM NaCl, 1 mM EDTA, 0.05% Tween 20, 0.1% bovine

serum albumin [BSA])

containing

5% BSA and 5%chicken

serumfor 1h with

rocking.

Strips

werewashed three times for 5 min each in VOPBAbuffer. A2-mI

sample

of10 ,ugof

virus per mldiluted inMEM-10mM HEPES

(N-2-hydroxy-6137

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ethylpiperazine-N'-2-ethanesulfonic acid)-0.5% fetalbovine

serum was incubated with each strip for 1 h. For some assays,5 x 104 cpmof35S-labeled viruswereused, and the

strips were washed and autoradiographed. The strips were

washed three times, incubated with antivirus antibody for 1 h, and washed three times. The stripswereincubatedfor 1 h

in secondary antibody (peroxidase conjugated) which was

diluted 1:200 in VOPBAbuffer. Thestripswerethenwashed

andstained in 0.06%4-chloro-1-naphthol-0.014% H202-20% methanol in phosphate-buffered saline (PBS).

Cell lysates and cell membrane preparations. Twenty-five 150-cm2 flasks of SCP,GSM, or mouseLcellswerewashed

inPBS, and thecellswere scraped and pelleted at1,000 x g

for 10min.Thepelletwaswashed in PBS andpelleted again. The cells were lysed in lx lysate buffer (1% Zwittergent,

0.5% sodium deoxycholate, 0.15 M NaCl, 1 mM

phenyl-methanesulfonyl fluoride, 0.1% SDS, 1 mM EDTA, 25 mM Tris [pH 7.6], 0.02% sodium azide) and sonicated on ice.

Protein concentration was determined by modified Lowry assays (16) orbicinchoninic acid assays (Pierce).

Cell membranes were prepared by differential

centrifuga-tion ofSCP and GSM cellsgrownin25850-cm2 roller bottles

orLcellsin 25150-cm2flasks(7). Cellswerewashed inPBS,

scraped, andpelletedat 1,000 x gfor 10min. Thepelletwas

washed in PBS and repelleted. The pellet was then

sus-pended in 4 ml ofhomogenizing buffer (0.25 M sucrose, 5 mM TrisHCl [pH 7.4], 1 mMMgCI2, 0.1mgof

phenylmeth-ylsulfonyl fluoride per ml) (STM) per g of cell pellet and

homogenized in a Dounce homogenizer. The lysed cells werecentrifugedat 1,000 x gtopellet nuclei, and the pellet

was washed in STM and repelleted. The supernatant was

diluted to 40 ml in STM and centrifuged at 10,000 x g to

remove lysosomes and mitochondria, and the pellet was

washed and repelleted. This final supernatant was then

centrifugedat100,000 x gtopelletmembranes. Allfractions

were saved, and 5' nucleotidase (Sigma) assays were

per-formed to localize plasma membranes. Bicinchoninic acid

assays were doneto determine protein concentration. lodination ofcell membranes. Cell surface moleculeswere

iodinated as previously described (1). Intact SCPcells (107) wereresuspended in 10ml ofPBS (pH 7.4) which contained

10mCi of

Na1251,

20 p.goflactoperoxidase perml,and100,ul

of 0.03%H202.Thereactionmixturewasincubatedatroom

temperaturefor15min, and 100,ul offresh 0.03% H202was

added at 1, 5, and 10 min. At 15 min, the reaction mixture

wasdiluted in 15 ml of cold PBS containing 10 mMNal and the cellswere centrifugedat 1,000 x gfor 10min.

The 125I-labeled SCP cells(107cellsin10ml ofPBS)were

incubated with 500 ,ug ofpurified unlabeled visna virus for 1 h at 37°C. The virus was cross-linked to any bound

pro-teins (see below), and then one-half was incubated with

preimmune serum or serum against purified gpl35. The

immune complexes were isolated with protein A-Sepharose

(Pharmacia) and analyzed by SDS-polyacrylamide gel elec-trophoresis (PAGE). The labeled proteinsweredetected by

autoradiography.

Affinity purification. Cell surface virus-binding molecules

wereaffinity purifiedaspreviously described(17).Cells from 25 flasks of SCP cells were suspended in 12.5 ml ofMEM containing 50 mM BES (N,N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid) (pH 6.5) and 1 mg of BSAper ml and

incubated with1.25mgofpurified visna virus for1hat37°C.

Cells were pelleted, washed, and resuspended in 25 ml of

PBS (pH 8.3) containing 50 p.g of dithiobis(succinimidyl)

propionateperml, 1 mM MgCl2,and0.02%NaN3for 1 hat room temperature. This cross-linked the virus to the cell

proteins it bound. Cells were again pelleted, washed, and

thenlysed in 25 ml of 0.02 M Tris (pH 8.0)-0.2 M NaCl-5 ,ug

of aprotinin per ml-0.2 mM EGTA-0.2 mM NaF-0.2%

sodium deoxycholate-0.5% Nonidet P-40. Nuclei were

re-moved by centrifugation at 1,000 x g for 10 min. Serum

raisedagainstvisna virusgp135, wasadded to the superna-tant at adilution of 1:400 for 1 hat 37°C. Protein A-Sepha-rosebeads(125 mg)wereaddedovernightat4°C. The beads

were centrifuged, washed three times in lysate buffer, and

applied to a 5 to 20% gradient SDS gel. Labeled proteins

werevisualizedbyautoradiography, and unlabeledproteins

were visualizedby staininginCoomassie blue.

Immune serum. Neutralizing antibodies to visna virus were obtained from a hyperimmune sheep as previously

described (20).

Preparationand purificationof antibodiestovirus-binding

protein. Antibodies to purified virus-binding proteins from

cell membranes were made by immunizing rabbits with

proteins affinity purified by cross-linkingtovirus (described

above) andseparated by SDS-PAGE (6). A 1-mg sample of

purified proteinfrom cellmembraneswas run on anSDSgel

and stained with Coomassie blue. Bands at the molecular

weightindicated inFig.3wereexcised andequilibratedwith

150 mMNaCI-10mMNa2PO4 (pH 7.4) andhomogenizedin 5 mlofsaline inaDouncehomogenizer.Thecrushedgelwas

lyophilized and resuspended in 1 to 5 ml of saline and

emulsified in Freund's complete adjuvant, and 2 ml was

injected intradermally into rabbits. Rabbits were boosted

every 4 weeks with an intramuscular injection of 2 ml of

crushed gel emulsified in Freund's incomplete adjuvant.

Immunoglobulins from the sera of the rabbits (20 mg of

proteinperml)were purified by precipitationwith saturated

ammoniumsulfate and resuspended at aconcentration of5

mg ofprotein per ml. Sera were tested by

enzyme-linked

immunosorbent assay(ELISA)(28)againstSCP membranes (200 ng perwell) purifiedas describedabove.

Immunofluorescence. GSM and SCP cells were plated in

35-mm2tissue culturedishesandallowedto growto

conflu-ency. Live-cell immunofluorescence was done, using no fixation or permeabilization of the cells. Primary antibody

from rabbit 2-21 pre- andpostimmunization (15-kDa protein)

andrabbit 2-23(50-kDaprotein)wasdilutedto1:20in MEM

supplementedwith2%lambserumand addedtoslidesfor20

min at 25°C. The cells were washed three times in PBS.

Fluorescein-conjugated swine anti-rabbit antibody was

added in MEM-1% lamb serumfor 30 min at a dilution of 1:20 inPBS, and the cells were washed threetimes in PBS andexaminedby fluorescence microscopy.

Virus binding assays.

[35S]methionine-labeled

virus was bound to SCP cells in 96-well plates. Cells werecooled to

4°C

and washed in cold Hanks' balanced salt solution.

Labeled viruswasdilutedto50ng/,ulin cold MEM

contain-ing0.5%fetal bovineserumand 25 mM HEPES andserially

diluted, and 100 ,ul was added per well. The plates were

incubatedat4°Covernight.Supernatants were removed, and

the cellswere washed twice with PBS (pH 7.4). Atotal of 100,ul of1%SDSwas added toeachwell, lysed cells were removed, and the plates were washed twice with PBS. The

35S-labeled

virus in the supernatants and cells was counted

separately in Liquiscint (National Diagnostics) on a

scintil-lation counter. Unlabeled virus competitions were done in the same manner, butplates were preincubated with unla-beled visna virusfor2hat4°C. Antibody competitionswere

done by preincubating plates for 2 h at 4°C with 5 mg of

purified immunoglobulinperml from rabbits.

Western (immunoblot) analysis. Cell membrane (1 mg

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a b c d ef

92"-I

691

i-46

I

9 h i

Lcells SCP GSM GSM

FIG. 1. VOPBA with 2 mg of protein from L cells (lanes a to d), SCP cells (lanes e to h), and GSM cells (lanes i to r). Visna virus was bound tolanes c, d, g, h, k,1, and o to r and was detected with either preimmune serum (negative control) (lanes b, d, f, h, j,1,n, p, and r) orpostimmune serum (lanes a, c, e, g, i, k, m, o, and q). Lanes a, b, e, f, i, and j were incubated in the absence of virus. Lanes q and r were preincubated with hyperimmune serum at a dilution of 1:100 before visna virus was added. Numbers on the left show size standards (in kilodaltons).

prepared as described above) from SCP, GSM, and L cells wasdiluted in 2x protein buffer (20% glycerol, 50 mM Tris

[pH 7.4], 2%SDS, 0.01%bromophenol blue), run on a 5 to

20% gradient SDS-polyacrylamide gel, and transferred to

nitrocellulose. The nitrocellulose was stained with ponceau

S, and strips were cut and incubated in blocking buffer containing5%BSA, 10 mM Tris (pH 7.4), and 0.15 M NaCl.

The strips were incubated with purified immunoglobulin

from pre-and postimmune rabbits for 2 h at concentrations

of0.25, 0.5,and1.0mg/mlinbinding buffer containing0.25%

gelatin, 20% chick serum,0.05% NonidetP-40,50 mMTris

(pH 7.4),5 mM EDTA, and0.15%NaCl. The nitrocellulose

strips werewashed three times in buffercontaining 0.15 M

NaCl,10 mMTris(pH 7.4),0.005%Tween 20,and1% BSA.

Thestrips were thenincubated with peroxidase-conjugated

swine anti-rabbit antibody (Dako)at adilution of1:200for 2

h and washed three times. Substrate containing

4-chloro-1-naphtholandhydrogenperoxide in PBS was added, and the

reaction was stopped by rinsinginwater. RESULTS

Identification of visna virus-binding proteins on cells. To

identify the molecules on susceptible cells which bind to

visna virus, the virus was bound to cell lysates and

mem-braneswhichhadbeenrunonSDS-PAGE and transferredto

nitrocellulose. Virus wasbound directly to the transferred

proteins, antivirus antibody was added, and a

peroxidase-conjugated secondary antibodywasaddedtovisualize bands

on thenitrocellulose which bound virus. Visna virus bound

to molecules of

approximately

15and 30kDain cell

lysates

from both SCP and GSM cells

(Fig.

1). To control for

nonspecific binding of

antibodies,

we ran a control in the

absence of virus. Preimmune serum was also used as a

control forserumfrom virus-immunizedanimals. No bands were seeninthe absenceofvirusorin the presenceofvirus

plus preimmune serum. In

addition,

no

protein

bandswere

visualized in celllysates frommouse L

cells,

whichare not

susceptibletoinfection

by

visnavirus. Proteinbandswhich

bound to visna virus were diminished in

intensity

when

strips of nitrocellulosewere

preincubated

with

hyperimmune

serumfromasheepatadilutionof 1:100 for1h

(Fig. 1,

lanes

qandr). Proteins

corresponding

tothebands bound

by

virus

donotappeartobedisulfide-linked

subunits,

as

nonreducing

gel conditions giverisetoprotein bands of the same

molec-ularweight(data not shown).

To demonstrate direct binding of virus to the protein

bandson nitrocellulose,we used an assay with

[35S]methio-nine-labeled visna virus. Labeled virus bound to molecules

of 15 and 50 kDa in SCP cell lysates (Fig. 2, lane b). No directbindingof labeled virustoLcells was detected(Fig.2, lane a). The detectionof the 50-kDa protein may bedue to the increased sensitivity of the labeled virus procedure,

whichincludes fewerstepsand therefore appears to be more

sensitive.

Affinitypurification and antibody response to

purffied

mol-ecules.Thecell membranes of SCP cellswereiodinatedwith

lactoperoxidaseto labelsurface molecules. Visna viruswas

allowed to bind to the iodinated cells, and the virus was

cross-linkedtotheboundproteinsasdescribed inMaterials

a b

200-921m

6g10

FIG. 2. VOPBA with [35S]methionine-labeled visna virus (105 cpm)on 2mgofproteinfromLcells(lanea)and SCP cells(lane b).

Numbersonthe left show size standards(inkilodaltons).

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a

b

(<50

>

<~--30

^ <15

-FIG. 3. SDS-PAGE of '25I-labeled surface proteins recognized by visna virus. Iodinated cell membranes from SCP cells were incubated with visna virus and preimmune serum (lane a) or antiserumto visna virusgp135(lane b). The arrowheads indicate the 15-,30-,and50-kDa bands. The bands above and below the 50-kDa band arerecognizedby preimmune serum.

and Methods. The labeled cells and virus were pelleted at 1,000 x g,and unbound virus was removed by washing and

repelleting the cells. The cells were lysed (described in

Materials and Methods, "Affinity purification"), and

anti-bodiesto thevisna virusgp135wereused to

immunoprecip-itate the virus-protein complexes. The complexes were analyzed on SDS-polyacrylamidegels, and125I-labeled pro-teins were detected by autoradiography. Three species of

125I-labeled

proteins with molecular masses of approxi-mately 15, 30, and 50 kDa were identified. These proteins were not observed when preimmune serum was used (Fig. 3). Preimmune serum did recognize bands above and below the 50-kDa protein. Noniodinated cells were then used to

purify larger quantities of the three protein species (see

Materials and Methods for details) for injection into rabbits. The rabbit immunized with the 15-kDa band (rabbit 2-21) developeda high titer of antibody after the first inoculation when its serum was tested against SCP membranes in an

ELISA.This titer diminished after the next boosterinjection

and did not increase after subsequent inoculations. Rabbit 2-22did not developappreciableamountsofantibodytothe 30-kDa protein, and rabbit 2-23 responded weakly to the initial inoculation of the 50-kDa protein. The ELISA is

againstall the components ofthe SCP membranes and thus

the relative amount of any protein in the membrane will

affect the assay.Although rabbit 2-23respondedonlyweakly

in the ELISA, these antibodies were found to recognize a component of the SCP cell surface in an

immunofluores-cence assay (seebelow).

Immunofluorescence. To determine whether the immune rabbit serarecognized molecules on the surface oftheSCP

cells,wedidlive-cellimmunofluorescenceonSCP and GSM

cells. Antibodies to the 50-kDa protein

recognized

surface

moleculesonboth GSM and SCP cells (datashownforSCP

cells,Fig. 4b),and preimmuneserum wasnegative (Fig.4a).

The antibodies to the 15-kDa protein werealso

positive

in

thelive-cell immunofluorescence assay (datanot shown).

Binding ofvisna virus to SCP and GSM cells. 35S-labeled visna virus binds to the surface of both SCP and GSM cells (Fig. 5aandb).Competitionexperiments in whichunlabeled

visna virus was preincubated with SCP cells before the

addition oflabeled visna virus showed thata150-foldexcess

ofunlabeled visna virus competes for35%of thebindingof labeled virus (Fig. Sc). To examine which of the three

affinity-purified moleculesweremostimportantinbindingto

visnavirus, wepreincubated SCP cells withpurified

immu-noglobulinfrom rabbits 2-21 (15-kDaprotein), 2-22 (30-kDa

protein), and 2-23(50-kDa protein)at5mg/ml before adding

labeled virus. Only immunoglobulins fromrabbit 2-23 inhib-ited the binding of visna virus. Although the antibody

responseof this rabbit to SCP membranes was low,purified antibody blocked 34.7%of labeled virus binding or 99% of the inhibition by cold virus (Fig. Sd and e). An equal concentration ofimmunoglobulins directed against the 30-kDa banddid not block binding. However, since there was such a low antibody response against this protein, no con-clusionscan be made about theroleof this protein in virus

binding. Further, antibodies directed against the 15-kDa

band did not block binding despite the good response of

serum from the animal to SCP membranes in the ELISA.

FIG. 4. Immunofluorescenceassay ofSCP cells using preimmune serum from rabbit 2-23 (50-kDa protein) (a) or postimmune serum bleed 5(b), eachat adilution of1:20.

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a

0.20 ,

0.15-C

0

a

C

a

30 0.10

IIL

a.W I , . . ..

0

C 10007

x

0

E

a 0

er

CS

la

.5

is 900

-800

-

700-0

c

0

E

30

0

0-I ,

200 400

BvirmcpmonSCP

600

100 200

unWlbd vIu viru (ug)

1:80 1:40 1:20 1:10

SwumDiktlon

ba

0

E a

U.

800

d

X

c

0

E

IL 0 S C

i

.5

0

a

'0

Therefore, the 50-kDa protein appears to be an important

molecule inthe binding of visna virus tocells.

Western analysis of antibodies to the 50- and 15-kDa membrane proteins. Purified SCP and L-cell membranes wereprepared to determine whether the antibodies to the 50-and15-kDaproteinsbind to theappropriate proteins. West-ern blots containing purified cell membranes were reacted

with purified immunoglobulin from the rabbits. L cells

showednospecific reactivitytopostimmune rabbit

antibod-ies from rabbits immunized with the 15- or50-kDaprotein. Immuneserumfrom rabbit2-21, immunized with the 15-kDa

band, recognizedalargegroup of moleculesat a molecular

massrange of200 kDa(datanotshown). This couldbe due to the fact that the 15-kDa molecule is actually a cleavage

product of a larger molecule important in virus binding.

Antibodies tothe 50-kDaprotein (postimmune serumfrom

0.05'

0.07

0.08

0.05

0.04I

0

~~~~10000

20000

avim

cpm

an

GSM

400

300

200

100

-1:80 1:40 1:20

SwumDikhtlon

1:10

FIG. 5. Binding of35S-labeledvisna virus(specific activity,7 x

104cpm/,ug)tothesurface of cells. (a) Scatchard plot of visna virus boundtoSCP cells.(b)Scatchard plot of visna virus boundtoGSM cells. (c) Competition of unlabeled visna virus preincubated with SCP cells for2 hbeforetheaddition of5 x 104 cpmof35S-labeled visna virus. (d)Inhibition of1 x 104cpmof35S-labeledvisna virus bindingtoSCP cellsbyserumfrompreimmune rabbit2-23(50-kDa protein).

E,

preimmune; *, postimmune. (e) Inhibition of labeled visnavirus binding toSCPcellsby postimmunerabbit2-23bleed4 (50-kDa protein) serum divided by inhibition of labeled virus by unlabeled virus x 100. B/F, bound/free.

rabbit 2-23) specifically recognized molecules at 50 and 30 kDa in the SCP membranes (Fig. 6, lane d). This demon-stratesthat theantibodiestothe50-kDaproteinwereagainst

cell membrane proteins and also that the antibodies to the 50-kDaproteinrecognized aprotein oftheappropriate size.

DISCUSSION

Virus overlay assays have been used successfully to

identifythe receptormolecule whichbindsto murine

hepa-titisvirus(2). Thismoleculewasfoundtobe presentonlyon

cellswhich weresusceptibletoviral infection. The VOPBA

was utilized in this study to identify molecules which bind

visnavirus.UsingtheVOPBA,itwasshown that visna virus

binds to molecules of approximately 15 and 30 kDa on

susceptible SCP and GSM cells. Reduced binding ofvisna

virus was demonstrated by preincubating the virus with

hyperimmune serumbeforebinding.

Bindingof visna virustoLcellswas not

observed,

except

when radioactively labeled virus was used in the assay,

which appears to increase the sensitivity of the assay.

Although visna virus does appear to bind L cells at a low

0

-.- I .

-lq

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a b c d

200:-

9210-

46_-303t 14

FIG. 6. Western blot with 1 mg of protein from cell membrane preparation ofL cells (lanes a and b) and SCP cells (lanes c and d). Purified immunoglobulin was diluted to 0.5 mg/ml. Lanes a and c contain immunoglobulin from preimmune rabbit 2-23 (50-kDa protein), and lanes b and d contain immunoglobulin from postim-munerabbit 2-23 (bleed 4). Numbers on left show molecular mass markers (in kilodaltons).Arrowheads on right show 50- and 30-kDa proteins.

antibody were made to the 15-kDa band; these antibodies reacted in immunofluorescence assays with surface mole-cules on SCP and GSM cells; however, they were not active ininhibiting virus binding to cells. Therefore, this molecule or portion of a molecule does not seem to be required for virus binding. However, antibodies to the 50-kDa band, which never reached high titers against SCP cell membranes,

blocked bindingof visna virus to the surface of SCP cells and

did so to the same extent as unlabeled virus. In addition, these antibodies recognized cell surface molecules in an

immunofluorescence assay. This result demonstrates that

thebinding of the antibody to the cells is highly specific and blocks the virus binding to the cell. Thus, the 50-kDa protein appears tobe the molecule or an essential component of the molecule which serves as the cellular receptor for visna virus.

ACKNOWLEDGMENTS

Wethank MaryannBrooks for invaluable assistance in prepara-tion of the manuscript.

This work was supported by Public Health Service grants NS16145, NS23039, and A128748 from the National Institutes of Health.

level, L cells are not susceptibleto infection. This may be

duetothefact that L cells have only low levels of expression ofthereceptormolecule for visna virus.Morelikely, there is

an intracellular species-specific block which prevents the completion of the life cycle of visna virus in a murine cell

(14).

The sizes of the virus-binding proteins were verified by affinity purification of purified cell membranes.Inadditionto the 15- and 30-kDa proteins identified in the VOPBA, a

50-kDabandwasobserved. Itwas thoughtthat the 15-kDa

molecule mightbe a subunit ofthe 30-kDa molecule; how-ever, no disulfide linkages could be demonstrated, since nonreducing gels showed identical 15- and 30-kDa bands in theVOPBA. It ispossiblethat the 15- and30-kDamolecules

are cleavage products of the larger 50-kDa protein. This is supported by the fact that antibody to the 50-kDa band recognizes not only the 50-kDa band but also the 30-kDa bandonnitrocellulosetowhich cell membraneproteinswere

bound.However,thisantibodydidnotrecognizethe 15-kDa band. Thiscould be due tomany reasons. Two possibilities

are(i)that theantibodydirectedtothe 50-kDa band bindsto

anepitopeorepitopespresenton the 30-kDaportionof the larger50-kDa molecule andnot tothe 15-kDaportionand(ii) that the 15-kDa molecule is a portion of another larger molecule which is associated with the virus-binding mole-cule but isnotactuallythe high-affinity virus-binding mole-cule or receptor. The notion that the 15-kDa band is a

cleavage productofalargermoleculewassupported bythe fact that antibody to the 15-kDaprotein recognized larger molecules onthe Western blot.

Protein moleculespurified by gel electrophoresisand used to immunize rabbits may not be optimal for inducing anti-bodies to the biologically active molecule. These proteins have been denatured and therefore maynotinduce antibod-ies against epitopes whichdepend on secondary structure. Fewor noantibodiesweremade to the 30-kDa bandinjected into rabbits and were therefore not efficient in any of the

assays performed. No conclusions can be made about the role of the 30-kDa protein in virus binding. High levels of

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