African swine fever virus attachment protein.

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0022-538X/91/052283-07$02.00/0

Copyright © 1991,American Society for Microbiology

African Swine

Fever

Virus Attachment Proteint

ANGEL L. CARRASCOSA, ISABEL SASTRE,AND ELADIO VINUELA*

Centro deBiologia Molecular (Consejo Superior de Investigaciones Cientificas-UniversidadAut6noma de Madrid),

Facultadde

Ciencias,

Universidad

Aut6noma,

Canto

Blanco,

28049

Madrid,

Spain

Received 31October 1990/Accepted 24January 1991

Treatment of African swinefever virus particles with nonionic detergents released proteins p35, p17, p14,

and p12 from the virion. Ofthese proteins, only p12 bound to virus-sensitive Vero ceUs but not to

virus-resistantLorIBRS2cells. The bindingof p12wasabolished by whole African swine fever virus andnotby

similar concentrations of subviral particles thatlacked the external proteins. A monoclonalantibody(24BB7)

specific for p12 precipitated a protein that, when analyzed by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis in the absence of2-mercaptoethanol, showedamolecularmassof 17 kDa(pl7*) instead of12

kDaasfound in thepresenceof2-mercaptoethanol. Therelationshipbetweenthesetwoproteinswasconfirmed

by the conversion of pl7*top12 when theformerwasisolatedfrom polyacrylamidegelsin the absence of

2-mercaptoethanolandsubsequentlytreated withthe reducingagent.Thesupernatantobtainedafter

immuno-precipitation withthe p12-specific antibody lacked thevirus-bindingprotein.

African swine fever (ASF)virus is the causative agent of

an important disease of swine that has a wide variety of

clinical forms (13). The structure of the ASF virus particle has been examined indetail by electronmicroscopy(5),and 34structuralproteins have beendescribed in purified virions (4). Virus replication takes place mainly in mononuclear phagocytes (18, 19), and this phenomenon might be related

to the peculiar immune response to ASF virus. The main problem in the development ofavaccine against ASF virus

infectionseemstobe the absence of neutralizing antibodies in the infected animals, although proteins that react with antibodies from surviving or hyperimmunized pigs have

beenreported (7, 16,23).

Studieson the localization ofseven ASF virus structural

proteins in the virus particle by using virus-specific

mono-clonal antibodies (MAbs) (22) labeled with protein A-gold complexes showed that proteins p14 and p24arepresentin the external region of the virion (6). To complete the identification of surface polypeptides in the ASF virus

par-ticle, we have analyzed the proteins released from purified

virions byincreasingconcentrationsof nonionicdetergents. Assuming that proteins in the external envelope might play

animportant role in the immunological response, we have used thepolypeptides released from ASF virus bytreatment

with

n-octyl-p-D-glucopyranoside

(OG) toobtain rat

antise-rumandto study theability of this serumtoneutralize ASF virusinfectivity.

Wehavepreviouslyshownthepresenceof cellular

recep-torsthat mediateASF virusbindingtosusceptible Verocells

(1). Thisbinding implies that virus attachment proteinsexist

in the virus particle. Here we describe experiments of

binding and competition of OG-released viral proteins to

ASF virus-sensitiveor-resistantcells thatleadto the iden-tification ofaviralprotein that maymediatethe attachment of ASF virus particles to cell receptors. This protein is

recognized andefficiently sequesteredby an MAbobtained

after immunizationofmicewiththedetergent-releasedvirus

proteins.

*Correspondingauthor.

tDedicated to Severo Ochoaonthe occasion of his 85thbirthday.

MATERIALSANDMETHODS

Cells and virus. Vero, IBRS2, and L cellswere obtained

from the American Type Culture Collection. The strain of ASF virus (BA71V) adaptedto grow in Vero cells and the conditions for plaque titration have been described else-where(12).

Radioactive labeling and purification of virus. The stocks of ASF virus were produced in Vero cells cultured in roller

bottles,labeled with[35S]methionine,andpurifiedby Percoll sedimentation asdescribedpreviously (4).

Treatmentofvirus withdetergents.Suspensions of purified ASF virus containing 1 to 3 jig ofprotein and 15,000 to

20,000 cpm of acid-insoluble radioactivity were diluted in

phosphate-buffered saline (PBS) and incubated with OG (Calbiochem), Nonidet P-40(Sigma),orTriton X-100

(Hop-kin andWilliams), in the absenceorpresenceof 0.5 MNaCl,

for 1hat4°C inafinal volume of about 30,ul.Aftertreatment

with thedetergent, samples were taken forinfectivity titra-tion and for centrifugation over 20 ,ul of a sucrose (20%

[wt/vol] in PBS) cushion inaBeckmanAirfugeat133,000 x

gfor4minat roomtemperature. Pelletsweresuspendedin

50 ,ul of PBS or dissociating buffer (0.04 M Tris-HCI [pH

6.3], 5% 2-mercaptoethanol [2-ME], 2.3% sodium dodecyl sulfate [SDS], 10% glycerol) andheatedfor3mininboiling

water. Supernatantsweremixed withanequal volume of 2x

dissociating buffer and heated in the sameconditions.

Radioiodination of ASF virus external proteins. The chlo-ramine-T method of Davies and Stossel (8) was used to

iodinateOG-released ASFvirusproteins: 130 p.gofpurified virus in0.2 mlwastreated with 1% OGasindicatedabove,

and the proteins in the supernatant were iodinated with 1 mCi of125I in thepresence ofchloramine-T(0.1 mg/ml)and 300 nMKI,at4°C for15min,inafinal volume of 0.4 ml.The

reactionwasstoppedbyadditionof 1 mMNaI,and free125I

waseliminatedbychromatographyonaSephadexG-25spun column saturated with 5% bovine serumalbumin (BSA) in PBS. The specific activity obtained for the iodinated virus

externalproteinswas 15 x 106cpm/,ug.

Polyacrylamide gel electrophoresis. Electrophoresis was performedon7to20%polyacrylamide gels bythe methodof Laemmli (14) in the presence of SDS, with '4C-labeled

markerproteins(Bio-Rad)asstandards. Radioactiveprotein 2283

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bandsweredetected byautoradiography (for iodinated sam-ples) orfluorography(for 35S-labeled samples) (3) on preex-posed films (15).

Isolation of virus proteins from polyacrylamide gels. Sam-ples of 35S-labeled purified ASF virus (3.5 x 105 cpm), untreated or treated with 5% 2-ME, were subjected to standard polyacrylamide gel electrophoresis. After the un-fixed gelwasdried, bands correspondingtop12(obtained in the sample treated with 2-ME) and p17* (obtained in the untreated sample) were localized, cut, and rehydrated in distilled water. Then 150

RIl

of buffer containing 125 mM Tris-HCl (pH 5.8), 0.5% SDS, and 2.5% dextran T500 was added before heating for 3 min in boiling water. About one-sixth of the material was mixed with 1 volume of 2 x dissociating buffer with or without 5% 2-ME, boiled for 3 min, and subjected to electrophoresis in a 12 to 22.5% acrylamide gel.

Antiserum production. Antiserawere obtained from pairs ofrats immunized with one of two antigens: (i) UV-inacti-vated, purified whole ASF virus and (ii) external virus proteinsreleased from the virions by treatment with 2% OG and chromatographed on Sephadex G-25 spun columns (saturated with 10% calf serum in PBS) to remove the detergent.Rats were injected subdermallywith theantigens in Freund complete adjuvant, and after 21 days they re-ceived another dose of antigen in Freund incomplete adju-vant. One and two weeks later, rats were injected with antigens in PBS; 7 days after the last injection, serum was prepared from defibrinated blood. Each setofratsreceived, in each inoculation, anequal dose ofantigen corresponding to 0.2, 1.0, or 10.0 ,ugof protein for whole virus antigen or external proteins released from 50 ,ugof virus. Each serum was heat inactivated for 30 min at 56°C and titrated by enzyme-linked immunosorbent assay (25), using purified ASF virus as the antigen. The titers obtained were 2,150, 7,200, and 17,000 for the doses of0.2, 1.0 and 10.0

jIg

of whole virus, respectively, and 20,000 for that produced againsttheOG-released virus proteins.

Complement. As a source ofcomplement, we used non-immunized rabbit serum obtained after coagulation for 10 min at room temperature and 1 h at 4°C and stored under liquid nitrogen.

Virusneutralizationtest. Theneutralization capacity of all serawastestedby aplaque reductionassayofextracellular infectious ASF virus, diluted in Dulbecco modified Eagle mediumsupplementedwith20% fetal calfserum

(DME-20%

FCS). Asample of50 ,ulcontaining3,000 PFU ofvirus was mixed withanequal volumeof antiserumdiluted four times in

DME-20%

FCS. After overnightincubationat37°C, 50

RI

of rabbit complement (or medium alone) diluted twice in DME-20%FCS was added before incubation at 37°C for 1 h. Samples were titrated directly and after 1/10 dilution, on Vero cellmonolayers, by the standard plaque assay (12).

Binding of ASF virus proteins to cell monolayers. Vero, L, orIBRS2 cells weregrown in 24-well plates to about 150,000 cells per well andincubated with radioactively labeled ASF intact virions orOG-released virus proteins (50 to 100

IlI

of each perwell) obtained as described above. The incubation wasperformed at 37 or 4°C in culture medium buffered at pH 7.4 with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) and supplemented with

2%

calf serum. For the4°C incubation, cells were precooled at 4°C for10min.After incubation for2 h at37°Cor 4 h at 4°C, with shakingevery 15min, cultures were washed four times with 0.1 mlof PBSand

lysed

with 70 ,ul ofdissociatingbufferby heating for 3 min in boiling waterbefore analysis by

poly-acrylamide gel

electrophoresis.

In binding competition ex-periments, cells were

preincubated

with the competitor proteins(either BSAorunlabeled ASF virus

proteins)

in 150 ,ulofHEPES-buffered culture medium for4h at 4°C; then the cultures werefurtherincubated with the labeled material for4h at4°Cin the presenceof thecompetitor proteins and processed asabove.

Hybridoma production. The hybridomas secreting virus-specific MAbs were obtained and selected as previously described (22). The

antigen

used for immunization of mice was OG-released ASFvirus proteins (from 200 ,ug of total virusproteinin each inoculation), and the

specificity

ofthe MAbs obtainedwasdeterminedby

immunoprecipitation.

Immunoprecipitation and sequestration experiment. Puri-fied

[35S]methionine-labeled

ASF viruswastreatedfor1hat

4°Cwith2% OG. After

centrifugation

at133,000 x gfor30 minin aBeckman Airfuge,the extract was preimmunopre-cipitated for 2 h at room temperaturewith 50

,u1

ofa 10% suspension ofStaphylococcusaureus Cowan I (Pansorbin; Calbiochem) coated with normal rabbit serum. After the nonspecifically bound materialwas removed

by

centrifuga-tion,thereleasedproteinswereincubatedovernightwith 400 ,ul of hybridoma supernatant, and then 50

RI

of a 10% suspension of S. aureus coated with anti-murine immuno-globulin rabbit serumwas added. After4 hof incubationat 4°C, the immune complex was collected and washed four times with0.5% Nonidet P-40in PBS. The

immunoprecipi-tatedlabeled proteinsweredissociatedbyheating for 3 min in boiling water with 200

IL1

ofdissociating buffer without 2-ME, 5% 2-ME wasadded to halfofthe sample, and the samplewasanalyzedby

polyacrylamide

gel

electrophoresis.

Forthe

sequestration

experiment, OG-released virus pro-teinslabeled with

[35S]methionine

wereincubatedovernight at4°C with400,ul ofhybridomasupernatant. Then 100

p.l

of a 10% suspension of S. aureus coated with rabbit anti-murine immunoglobulin serum was added, and incubation wascontinued for4h.Theimmunoprecipitate wascollected

by

centrifugation, and the supernatant was assayed for binding to Vero and L cells for 2 h at

37°C

as described above.

RESULTS

Differential release of viralproteinswithOG.Theeffectsof different concentrationsof Nonidet P-40, Triton X-100, and OGonASF virus

infectivity

weretested. Thevirus titerwas

reducedto alesser extentbyOG thanbythe othernonionic detergents at the same final concentration (e.g., a 90% reduction of virus infectivitywasachievedwith about0.3% OGorwith0.003% NonidetP-40orTritonX-100).Thus,OG was chosenforfurther studies.

Whensuspensions ofpurifiedASFvirusweretreated with OG at a concentration of 0.5 or 2% in the absence or

presence of0.5 M NaCl(Fig. 1), several structuralproteins werereleasedfromthevirions.The firstproteinsdetected in thesupernatant after treatment with0.5%OG,a concentra-tion that reduces the initial virus infectivity to about 2%, werep35and

p12

(Fig.1, lanelb). Proteins

p17

and

p14

were also partially released when 2% OG was used (lane 3b). Other proteins, such as

p150,

p37, p34, and plO, were

partiallyremovedfromthevirusparticle when the samples wereincubated with2%OG in the presence of 0.5 M

NaCl

(lane 4b). Detergent treatment during which about 5% or

moreoftheinitialinfectivitywasretained didnotrelease any detectable viral protein. Essentially the same results were obtainedwhenothernonionicdetergents (NonidetP-40and

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0.5 %

V 1

a b

2 3

a b a b

-150

2 % 4

a b

a - 150

TABLE 2. Binding of"S-labeledASF virusproteins todifferent cells

%of radioactivity bound to cell monolayersa

Inoculum

Vero L IBRS2

Whole virus 25.1 (8) 2.4 (8) 1.9(5)

OG-supernatant 16.4(5) 2.7(5) 0.9(2)

OG-sediment 3.2 (1) 1.0 (1) ND

" Percentage of the total radioactivity (recovered in medium and cells) bound to cell monolayers after incubation for 2 h at 37'C. Number of experiments is given in parentheses. ND, Not done.

-72 *

to inject rats, the neutralization capacity exhibited by the induced antiserum was low in the absence of complement but similar to that obtained bytheantiserum againstwhole virus in the presence of complement (Table 1). The neutral-izing titers (reciprocal ofhighestdilutioninactivating >90%

37 of virus

infectivity)

wereabout5,000for theratantiserum

34 specificagainstwholevirus and 300forthatspecificagainst the OG-releasedproteins.

Binding of ASF virus proteins to different cells. Different cell monolayers (-150,000cells) were incubated with [35S]

a -- _17

a b c d e f g h j k

150-9

t

FIG. 1. Polypeptidesreleased fromASF virusparticles by incu-bation at 4°C for 1 h with different concentrations of OG in the,

absence(1 and 3) orpresence (2and4)of 0.5 M NaCl. The 35S-labeledpolypeptidespresentin thesediment(lanesa) and

superna-tant (lanes b) resulting from centrifugation in an Airfuge were resolvedby electrophoresis on a7to20% polyacrylamide geland detected byfluorography. The standard profileof "'S-labeled ASF virus structural proteins is shown on the left (V), with their molecularmassesindicated in kilodaltons.

72-4

37-a 35-.

-66-s

35-.

Triton X-100atconcentrations of0.02 to0.5%)wereused to solubilizethe viralproteins (not shown).

Neutralization of ASF virus by rat antiserum against whole virus or external virusproteins. Apool ofratantiseraagainst whole virus (titer of -8,500) was tested for ASF virus neutralizationbyaplaque reductionassay in theabsenceor presence ofcomplement (Table 1). The ratantiserum pro-ducedagainst wholeASFviruswasable toeliminate, in the presence ofcomplement, more than 97% ofthe infectivity present in virus samples. When a mixture of OG-released ASF virus proteins (containing p35, p17, p14, and p12), obtained

by

treatmentwith2% OGandchromatographedon

Sephadex G-25 columnsto remove the

detergent,

was used

TABLE 1. ASF virus neutralizationby specificratantisera

Neutralization(%survival)'

Serum specificity

-Complement +Complement

Nonimmune 101.5 ± 13(19) 69.8 ± 16(19) Whole ASF virus 12.8± 4.7 (5) 2.7 ± 1.6 (5) OG-released virusproteins 40.4 ± 7.3 (7) 4.0± 2.2 (7)

a Number ofexperimentsisgiveninparentheses.

17-Vs*o

12-l

a

10-s .

12-l

I

35s

125

FIG. 2. Bindingto sensitive and resistant cells of OG-released ASF virus proteins labeled with either [35S]methionine or 125i. Proteins in whole virus(lane a) and in the sediment (lane b)and

supernatant(lane c) resultingfromcentrifugationinanAirfugeafter

treatment of35S-labeledASF virus with 2% OG for 1 hat4°Care shownascontrols.About150,000VeroorL cellswereincubated for

2 hat37°Cwith 7 x 104(35S)or2 x 106(1251)cpmof OG-released

virusproteins (lanescandh), washed withPBS, andsubjectedto

polyacrylamide gel electrophoresis.Also shownareproteinsinthe

washingsof Vero cells(lanesd andi)and boundtoVero cells(lanes

eandj)and Lcells(lanesf andk)andproteinboundtoVero cells

analyzed in the absence of 2-ME (lane g). Molecular masses in

kilodaltonsareindicated. --37

'* -35 -'34

a -14

*-12

lb- 10 a.

--w -09

Olk---K 4 Ns-.a

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a

b

c

d

ef

g h

12-

-:

i

j

k I m

n

o pq r

i!'

-'

IIi

FIG. 3. Competitionofbindingof '25I-labeledOG-released ASF virus proteinstoVerocells. About200,000cellsprecooledat4°Cfor 10 minwereincubated with increasingconcentrationsof differentcompetitor proteinsfor 4 hat4°C,and then -2.5 x 105cpmof125I-labeled OG-supernatantwasaddedtoeach well. Cultures werefurther incubated for 4 hat4°C,washed withPBS,andanalyzed by polyacrylamide gelelectrophoresis. Shownareproteins boundtoL cells(laneb)ortoVerocells incubatedinthe absence(lanesdando)orpresenceof5, 15,40,or120 p.gof wholeASF virus (lanese toh), 5, 15, 40,or120,ugof OG-sediment (lanes jtom),or15, 40,or120,ugof BSA(lanes ptor).Representative profiles of thelabeledproteinspresentinthewashingsof eachsetofsamplesareshown in lanesa,c,i,andn.Proteins from35S-labeled ASFviruswere runin thesamegel (lanes). Molecular massesinkilodaltonsareindicated.

methionine-labeled whole virus or OG-released virus

pro-teins (OG-supernatant) for 2 hat 37°C. Therewasaspecific

binding of the labeled proteins toASF virus-sensitive Vero cells but not to nonsensitive L or IBRS2 cells (Table 2).

When theincubation with whole ASF viruswasdoneat4°C for 4h, thepercentage ofradioactivity boundtocell

mono-layers (in three experiments)was 23.4 and 6.2 for Vero and

L-cellcultures, respectively (datanot shown). The subviral particles generated after the OG treatment (OG-sediment), which lacked the external proteins, had lost most of their capacitytobindtoVerocells, andnobindingtoL cellswas seen(Table 2). The material that remained boundtothe cell monolayers after four washingswasdisrupted andanalyzed

bypolyacrylamide gel electrophoresis. Therewasnobinding

ofany virus protein to L-cell monolayers (Fig. 2, lane f), while a 12-kDapolypeptide (p12)wasretained in Vero cells

after incubation at 37°C with the 35S-labeled OG-released virus proteins (lane e). The protein that bound to the sensitivecellmonolayers hadamobility correspondingto17 kDa when the electrophoresis was done in the absence of

2-ME (lane g). The binding of p12 to Vero cells was

confirmed by incubation with radioiodinated OG-released virus proteins. In this case a broad band (around 12 kDa),

corresponding to the protein bound to Vero cell cultures, appeared (lane j); mostof the label in p35 and in the serum albumin (66 kDa), which became iodinated during the chro-matography of thelabeling mixture through Sephadex G-25,

was detected in the washings of bothVerocells (lane i) and

L cells (not shown), while there was no binding of any

labeledproteinto the nonsensitive L-cell (lane k)orIBRS2 cell (not shown) monolayers.

The specificity of the binding of the

125I-labeled

OG-released virusproteinstoVero cellswas studiedby

compe-titionwithincreasingconcentrations of unlabeled ASFvirus,

BSA, or OG-sediment. Electrophoretic analysis of the

la-beled material boundto Vero cells(Fig. 3) showed that the binding of viral protein p12 wasunaffected by the presence

of different concentrations of BSA (lanes o to r) or

OG-sediment (lanes j to m), whereas a dramatic decrease of

binding of this protein was observed in the cell cultures incubated with unlabeled whole virus (lanes etoh) evenat

the lowest protein concentration used (5 ,ug; lane e). The absence of binding ofany viral protein to nonsensitive L cells is shown in lane bas a negative control. The

electro-phoretic profile ofthe labeled materialin thewashings was

analyzed in all cases, yielding similar results to those pre-sented in lanes a, c, i, and n.

Relationship between viral proteins pl7* and p12. The

MAb 24BB7, obtained after immunization of mice with

OG-released virusproteins, was usedtoimmunoprecipitate the ASF virus proteins released byOG; theelectrophoretic profile of the immune complex analyzed either with or

without 5% 2-ME in thedissociating buffer is shown in Fig.

4. MAb 24BB7 immunoprecipitated the viral protein p12 (lane b) or p17* (lane c), depending on the presence or

absence of 2-ME in the dissociation buffer. As a negative

control,weusedaparentalmousemyeloma cellsupernatant

(NP3; lane d). The same results were obtained when the

immunoprecipitation was donewith the ASF virusproteins

dissociated with 0.3% SDS and 1% Nonidet P-40 (not shown).

Toensurethat theprotein recognized byMAb 24BB7was

the same protein as that involved in the interaction ofthe

virus with the sensitive cells, a sequestration experiment was carried out in which the OG-released virus proteins labeled with 35S were incubated with culture mediumfrom

24BB7orNP3 and sedimentedwithS.aureus.Afterremoval

of the immune complex, the supernatant was assayed for

66

-s

35- a

-72

-37

-35

-17

-14

12

-10

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v a b c d

150--.0.

a

b

c d e f g h i

i

150 -

-72-

i

72

-37- . 35- .

37-m

17-u

12-0

4.

10-f

17- UP

i2-

w

S

a

10- a

FIG. 4. Immunoprecipitation of ASF virus proteins by MAb 24BB7.

I'S-labeled

purified ASF virus (105 cpm) wastreated with 2% OG and then immunoprecipitated by supernatant of MAb 24BB7. The immune complex was washed and resuspended in dissociating buffer with (lane b)orwithout (lanec) 2-ME. Proteins immunoprecipitated bysupernatantofNP3, washed, andanalyzed in thepresenceof 2-ME (lane d). Also shownareproteins ofwhole virus(lane v) and OG-released virus proteins (lanea)analyzed inthe presenceof 2-ME. Molecularmassesinkilodaltons areindicated.

$

FIG. 5. Sequestration of ASF virus-binding proteins by MAb 24BB7.OG-released virusproteinslabeled with[35S]methionine(4.7 x 105cpm; 1.6

jig

of protein; laneb) wereincubatedwith 400pAl of culture medium from MAb24BB7 or NP3.After immunoprecipita-tion withS.aureus, proteinsinthe sediment (24BB7, laneg; NP3, lane h)and in the supernatant (24BB7, lane i; NP3, lane j) were analyzed. Thesupernatantoftheimmunoprecipitationwasassayed forbindingtoVero cells(24BB7,lane e;NP3,lane f)andcompared withthebinding oftheOG-releasedvirus proteins to Vero (lane d) orL(lanec)cells. Proteins of35S-labeledwholevirusarealso shown (lanea).Molecularmassesinkilodaltonsareindicated.

treatment with the reducing agent and that this reduction

was not reversible.

binding to Vero or L cells and the bound proteins were

analyzed by SDS-polyacrylamide gel electrophoresis. The bindingofp12to Vero cellswassimilarbefore(Fig. 5,lane

d)and after(lanef) immunoprecipitationwithNP3,whereas therewas nobindingafterimmunoprecipitationwith 24BB7

(lane e)orin thenegative controlofbindingofOG-released

virus proteins to L cells (lane c). Protein p12 was present

onlyinthe sediment(lane g)orinthe supernatant(lane j)of the samples immunoprecipitated with 24BB7 or NP3,

re-spectively.

A similarexperimentwith theOG-releasedvirusproteins,

labeled in thiscasewith125I,was runinparallel, yieldingthe

sameresults(not shown), althoughthe viralproteinboundto Vero cells andimmunoprecipitated byMAb 24BB7appeared

as abroad band around 12 kDa(Fig. 3).

Therelationshipbetween the viral proteinsp17* andp12

wasconfirmed byisolation of thecorrespondingbands after

polyacrylamide gel electrophoresis of 35S-labeled purified

ASF virus underreducing andnonreducingconditions (Fig. 6,lanesaandf,respectively)andsubsequenttreatmentwith 2-MEand electrophoresis of therehydrated bands (lanesb andd);the untreated controlsareshown in lanescande. It canbeseenthatproteinp17*wastotallyconvertedtop12 by

DISCUSSION

The reason for the absence of neutralizing antibodies againstASF virus remains obscure. Severalpossible expla-nations have been suggested (24): (i) existence ofantigenic competition, either inter- or intramolecular, in which a

dominant antigen suppresses the responseto acritical one; (ii) existence of both neutralizing and blocking antibodies; and (iii) existence of antigenic variability. In any case, identification ofthe virus component carrying the critical antigenic determinant that might induce the synthesis of neutralizing antibodies is one of the most important prob-lems in ASF research. It seems likely that this putative criticalantigen will be found among the externalproteinsof

the ASF virusparticle.

The identification of surfacepolypeptides of ASF virions hasbeen carried outby differential release of viralproteins with severalnonionic detergents. We have chosen OG, the mildestofthem,tosolubilize the viralproteinssince it hasa

high criticalmicellar concentration and iseasilyremoved(2). Theproteinsfirst released from thevirionsbyOGtreatment

were p35, p17, p14, and p12. These proteins were also labeled when ASF virusparticleswereradioiodinated(21)in the presenceoflactoperoxidase (26) orchloramine-Tat4°C (8). These results are consistent with the localization of

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:s:

71~1

4::-37:4

bc

d

e

f

30-17 **

12-.:

10 t -4

FIG. 6. Conversion of protein p17* to p12 by treatment with 2-ME. 35S-labeled purified ASF virus (3.5 x 105cpm) was dissoci-ated in the presence (lane a) orabsence (lane f) of5% 2-MEand subjectedtopolyacrylamide gelelectrophoresis.Bands correspond-ingtop12(lane a) and pl7* (lanef)weresubsequentlytreatedornot

with 2-ME and further analyzed by electrophoresis. Protein p12 treated (lane b)ornot(lane c) with2-ME and pl7*treated(lane d) ornot(lanee)with 2-MEareshown. Molecularmasses in

kilodal-tonsare indicated.

antigenic determinants in the virus particle by

immunoelec-tronmicroscopy with MAbs specific forASF virusproteins (6), exceptin thecaseof protein p17, which wasconsidered an external component in ourstudy but localized in a more

internal region than p72, the major capsid protein, in the

immunoelectron microscopy analysis. This apparent

contra-diction may be duetothepresence ofatleasttwo polypep-tides of 17 kDa in the virus particle upon analysis in

two-dimensional gels (unpublished results).

Previousreportshave studied the existence of neutralizing antibodies against ASFvirus.Tabaresetal.(23)showed that

antiserum against purified vp73does notneutralize the virus

infectivity. Ruiz Gonzalvoetal. (20) have reported that pigs infected with ASF virus may recover and resist challenge

exposure with virulent homologous viruses, and theyfound

partial protection by sera from ASF-resistant pigs. Partial

neutralization of ASF virus bypolyvalent rabbit,mouse,and

swineimmune seraaswellasby MAbs directed against p24, avirusprotein of probable cellular origin, has been obtained

(11). Innone ofthese cases wasthe degree of neutralization

higher than that obtained in our study by rat antiserum

against whole virusoragainstamixture ofOG-released virus

proteins, and neutralization wasalso complement mediated.

When each of 22 structural virus proteins, isolated from SDS-polyacrylamide gels, was used to immunize rats, the

monospecific antisera obtained were not able to neutralize

ASF virus, either alone or in mixtures, including those

specific for the external and major viral proteins (data not

shown). Although most animal viruses bear among their external proteins specific sites for neutralization, some

vi-ruses, such as frog virus 3 and ASF virus, have been included in a different category by their inability to elicit a neutralizing response (9). However, this does not meanthat there are no neutralization sites, since thevirions may have such sites but in a nonimmunogenic state (10); thus, it is still reasonable to look for a viral component that, in a certain state(perhaps obtained withthe mildestpossible treatment), could induce the synthesis of neutralizing antibodies.

Although there aremultiple mechanisms of neutralization of animal viruses, the most common involves antibody blocking ofthebinding of the virus attachment protein toa

cell receptor unit (10). We have previously described the existence of ASF virus-specific receptors on the plasma membrane of Verocells and theabsence of saturable binding sites on thesurface of Lcells (1) as a factor that determines the sensitivity of cells to the infection. In thestudy reported here, we found that the external ASF virus protein p12, extractedfrom 35S-labeled virus by OG treatment, was able tobind at37°C to sensitive Vero cells but not to nonsensitive Lcells. When theOG-releasedvirus proteins wereiodinated beforeincubation with thecell monolayers, wedetected the binding to Vero cellsof proteinthat, bySDS-polyacrylamide gel electrophoresis, appeared as a broad band of about 12 kDa. None of the labeled virus proteins associated with L-cell monolayers or with other ASF virus-resistant cells such as IBRS2. The biological relevance of the binding of p12 to sensitive cells was supported by thefact that it was abolishedonly by the presence of whole ASF virus particles and not by similar concentrations of BSA or even by noninfectious subviral particles that lackedthe more acces-sible proteins. Thus, viralproteinp12isagood candidate to be the ASF virus attachment protein.

Proteinp12 showed an apparentmolecular mass of 17kDa when analyzed in the absence of 2-ME; the conversion of pl7* to p12 has been observed in immunoprecipitation experiments and byisolationofthecorrespondingband from polyacrylamide gels and subsequent treatment with 2-ME. Protein pl7* was totally converted to p12; this could be clearly detected because therewasnooverlapbetweenp17* and the 17-kDa protein (p17) visualized in the presence of 2-ME, since p17 changed to 30 kDa in nonreducing condi-tions (not shown). The N-terminal amino acidsequences of proteins p17* and p12, obtained after separation on an

SDS-polyacrylamide gel and electroblotting onto an Immo-bilon polyvinylidene difluoride transfer membrane, were

identical (17). Thus, it seems that p12 is released from the virus particle by OG treatment as a dimer of about 17 kDa and that the binding to sensitive cells occurs in the pl7* form.

Although a partially neutralizing activity has been de-tected in rat antisera specific for the OG-released virus proteins, neither MAb 24BB7 nor monospecific antibodies against p12 have been shown to neutralize the ASF virus infectivity in culture cell assays. These results indicate that the epitope recognized by MAb 24BB7 must be different fromthe siteof interactionofp17*with the cellular receptor. It is also clear thatproteinp12isolatedfrompolyacrylamide gels in the presence of SDS could have lost the critical epitope. Itwill be necessary toobtainlargerquantitiesof the viral protein p17* in the less denatured state and as pure as possible (from virus particles, from infected cells, or as a

recombinant DNA product) to allow further study of the ASF virus attachment protein and thepossibility of induc-tion ofan effective immune response.

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(7)

ACKNOWLEDGMENTS

We thank M. L. Nogal for skillful assistance in hybridoma production and M. Salas for critical reading of themanuscript.

This research was supported by grants from the Comisi6n Ase-sora para la Investigaci6n Cientffica y Tecnica, the Consejeria de Agricultura de la Junta de Extremadura, and the European Eco-nomic Community and by an institutional grant from Fundaci6n Ram6n Areces.

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Figure

FIG.1.detectedabsencemolecularresolvedtantlabeledvirusbation Polypeptides released from ASF virus particles by incu- at 4°C for 1 h with different concentrations of OG in the,(1 and 3) or presence (2 and 4) of 0.5 M NaCl
FIG.1.detectedabsencemolecularresolvedtantlabeledvirusbation Polypeptides released from ASF virus particles by incu- at 4°C for 1 h with different concentrations of OG in the,(1 and 3) or presence (2 and 4) of 0.5 M NaCl p.3
TABLE 1. ASF virus neutralization by specific rat antisera

TABLE 1.

ASF virus neutralization by specific rat antisera p.3
TABLE 2. Binding of "S-labeled ASF virus proteinsto different cells

TABLE 2.

Binding of "S-labeled ASF virus proteinsto different cells p.3
FIG. 2.eASFtreatment2kilodaltonsanalyzedshownwashingsProteinssupernatantpolyacrylamidevirus and h Binding to sensitive and resistant cells of OG-released virus proteins labeled with either [35S]methionine or 125i
FIG. 2.eASFtreatment2kilodaltonsanalyzedshownwashingsProteinssupernatantpolyacrylamidevirus and h Binding to sensitive and resistant cells of OG-released virus proteins labeled with either [35S]methionine or 125i p.3
FIG. 3.fromgelOG-supernatantminp15, to Competition of binding of '25I-labeled OG-released ASF virus proteins to Vero cells
FIG. 3.fromgelOG-supernatantminp15, to Competition of binding of '25I-labeled OG-released ASF virus proteins to Vero cells p.4
FIG. 5.fororwith(laneanalyzed.culturetion24BB7.lanex 105 L Sequestration of ASF virus-binding proteins by MAb OG-released virus proteins labeled with [35S]methionine (4.7 cpm; 1.6 jig of protein; lane b) were incubated with 400 pAl of medium from MAb 24BB7
FIG. 5.fororwith(laneanalyzed.culturetion24BB7.lanex 105 L Sequestration of ASF virus-binding proteins by MAb OG-released virus proteins labeled with [35S]methionine (4.7 cpm; 1.6 jig of protein; lane b) were incubated with 400 pAl of medium from MAb 24BB7 p.5
FIG. apresencedissociatinginvirusimmunoprecipitated24BB7.24BB7.2% the MAb OG (lane presence by The I'S-labeled and of Immunoprecipitation with v) buffer 2-ME
FIG. apresencedissociatinginvirusimmunoprecipitated24BB7.24BB7.2% the MAb OG (lane presence by The I'S-labeled and of Immunoprecipitation with v) buffer 2-ME p.5
FIG. 6.treatedtonsoratedingwith2-ME.subjected not Conversion of protein p17* to p12 by treatment with 35S-labeled purified ASF virus (3.5 x 105 cpm) was dissoci- in the presence (lane a) or absence (lane f) of 5% 2-ME and to polyacrylamide gel electrophore
FIG. 6.treatedtonsoratedingwith2-ME.subjected not Conversion of protein p17* to p12 by treatment with 35S-labeled purified ASF virus (3.5 x 105 cpm) was dissoci- in the presence (lane a) or absence (lane f) of 5% 2-ME and to polyacrylamide gel electrophore p.6

References