JOURNAL OF VIROLOGY, May 1991, p. 2283-2289 0022-538X/91/052283-07$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 65, No. 5

African Swine Fever Virus Attachment Proteint ANGEL L. CARRASCOSA, ISABEL SASTRE, AND ELADIO VINUELA* Centro de Biologia Molecular (Consejo Superior de Investigaciones Cientificas-Universidad Aut6noma de Madrid), Facultad de Ciencias, Universidad Aut6noma, Canto Blanco, 28049 Madrid, Spain Received 31 October 1990/Accepted 24 January 1991

Treatment of African swine fever virus particles with nonionic detergents released proteins p35, p17, p14, and p12 from the virion. Of these proteins, only p12 bound to virus-sensitive Vero ceUs but not to virusresistant L or IBRS2 cells. The binding of p12 was abolished by whole African swine fever virus and not by similar concentrations of subviral particles that lacked the external proteins. A monoclonal antibody (24BB7) specific for p12 precipitated a protein that, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol, showed a molecular mass of 17 kDa (pl7*) instead of 12 kDa as found in the presence of 2-mercaptoethanol. The relationship between these two proteins was confirmed by the conversion of pl7* to p12 when the former was isolated from polyacrylamide gels in the absence of 2mercaptoethanol and subsequently treated with the reducing agent. The supernatant obtained after immunoprecipitation with the p12-specific antibody lacked the virus-binding protein.

African swine fever (ASF) virus is the causative agent of important disease of swine that has a wide variety of clinical forms (13). The structure of the ASF virus particle has been examined in detail by electron microscopy (5), and 34 structural proteins have been described 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 of a vaccine against ASF virus infection seems to be the absence of neutralizing antibodies in the infected animals, although proteins that react with antibodies from surviving or hyperimmunized pigs have been reported (7, 16, 23). Studies on the localization of seven ASF virus structural proteins in the virus particle by using virus-specific monoclonal antibodies (MAbs) (22) labeled with protein A-gold complexes showed that proteins p14 and p24 are present in the external region of the virion (6). To complete the identification of surface polypeptides in the ASF virus particle, we have analyzed the proteins released from purified virions by increasing concentrations of nonionic detergents. Assuming that proteins in the external envelope might play an important role in the immunological response, we have used the polypeptides released from ASF virus by treatment with n-octyl-p-D-glucopyranoside (OG) to obtain rat antiserum and to study the ability of this serum to neutralize ASF virus infectivity. We have previously shown the presence of cellular receptors that mediate ASF virus binding to susceptible Vero cells (1). This binding implies that virus attachment proteins exist in the virus particle. Here we describe experiments of binding and competition of OG-released viral proteins to ASF virus-sensitive or -resistant cells that lead to the identification of a viral protein that may mediate the attachment of ASF virus particles to cell receptors. This protein is recognized and efficiently sequestered by an MAb obtained after immunization of mice with the detergent-released virus proteins.

MATERIALS AND METHODS

an

Cells and virus. Vero, IBRS2, and L cells were obtained from the American Type Culture Collection. The strain of ASF virus (BA71V) adapted to grow in Vero cells and the conditions for plaque titration have been described elsewhere (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, and purified by Percoll sedimentation as described previously (4). Treatment of virus with detergents. Suspensions of purified ASF virus containing 1 to 3 jig of protein 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), or Triton X-100 (Hopkin and Williams), in the absence or presence of 0.5 M NaCl, for 1 h at 4°C in a final volume of about 30 ,ul. After treatment with the detergent, samples were taken for infectivity titration and for centrifugation over 20 ,ul of a sucrose (20% [wt/vol] in PBS) cushion in a Beckman Airfuge at 133,000 x g for 4 min at room temperature. Pellets were suspended in 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) and heated for 3 min in boiling water. Supernatants were mixed with an equal volume of 2 x dissociating buffer and heated in the same conditions. Radioiodination of ASF virus external proteins. The chloramine-T method of Davies and Stossel (8) was used to iodinate OG-released ASF virus proteins: 130 p.g of purified virus in 0.2 ml was treated with 1% OG as indicated above, and the proteins in the supernatant were iodinated with 1 mCi of 125I in the presence of chloramine-T (0.1 mg/ml) and 300 nM KI, at 4°C for 15 min, in a final volume of 0.4 ml. The reaction was stopped by addition of 1 mM NaI, and free 125I was eliminated by chromatography on a Sephadex G-25 spun column saturated with 5% bovine serum albumin (BSA) in PBS. The specific activity obtained for the iodinated virus external proteins was 15 x 106 cpm/,ug. Polyacrylamide gel electrophoresis. Electrophoresis was performed on 7 to 20% polyacrylamide gels by the method of Laemmli (14) in the presence of SDS, with '4C-labeled marker proteins (Bio-Rad) as standards. Radioactive protein

* Corresponding author. t Dedicated to Severo Ochoa on the occasion of his 85th birthday.

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bands were detected by autoradiography (for iodinated samples) or fluorography (for 35S-labeled samples) (3) on preexposed films (15). Isolation of virus proteins from polyacrylamide gels. Samples 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 unfixed gel was dried, bands corresponding to p12 (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. Antisera were obtained from pairs of rats immunized with one of two antigens: (i) UV-inactivated, purified whole ASF virus and (ii) external virus proteins released 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 subdermally with the antigens in Freund complete adjuvant, and after 21 days they received another dose of antigen in Freund incomplete adjuvant. 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 set of rats received, in each inoculation, an equal dose of antigen corresponding to 0.2, 1.0, or 10.0 ,ug of protein for whole virus antigen or external proteins released from 50 ,ug of 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 of 0.2, 1.0 and 10.0 jIg of whole virus, respectively, and 20,000 for that produced against the OG-released virus proteins. Complement. As a source of complement, we used nonimmunized rabbit serum obtained after coagulation for 10 min at room temperature and 1 h at 4°C and stored under liquid nitrogen. Virus neutralization test. The neutralization capacity of all sera was tested by a plaque reduction assay of extracellular infectious ASF virus, diluted in Dulbecco modified Eagle medium supplemented with 20% fetal calf serum (DME-20% FCS). A sample of 50 ,ul containing 3,000 PFU of virus was mixed with an equal volume of antiserum diluted four times in DME-20% FCS. After overnight incubation at 37°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 cell monolayers, by the standard plaque assay (12). Binding of ASF virus proteins to cell monolayers. Vero, L, or IBRS2 cells were grown in 24-well plates to about 150,000 cells per well and incubated with radioactively labeled ASF intact virions or OG-released virus proteins (50 to 100 IlI of each per well) obtained as described above. The incubation was performed at 37 or 4°C in culture medium buffered at pH 7.4 with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and supplemented with 2% calf serum. For the 4°C incubation, cells were precooled at 4°C for 10 min. After incubation for 2 h at 37°C or 4 h at 4°C, with shaking every 15 min, cultures were washed four times with 0.1 ml of PBS and lysed with 70 ,ul of dissociating buffer by heating for 3 min in boiling water before analysis by poly-

J. VIROL.

acrylamide gel electrophoresis. In binding competition experiments, cells were preincubated with the competitor proteins (either BSA or unlabeled ASF virus proteins) in 150 ,ul of HEPES-buffered culture medium for 4 h at 4°C; then the cultures were further incubated with the labeled material for 4 h at 4°C in the presence of the competitor proteins and processed as above. Hybridoma production. The hybridomas secreting virusspecific MAbs were obtained and selected as previously described (22). The antigen used for immunization of mice was OG-released ASF virus proteins (from 200 ,ug of total virus protein in each inoculation), and the specificity of the MAbs obtained was determined by immunoprecipitation. Immunoprecipitation and sequestration experiment. Purified [35S]methionine-labeled ASF virus was treated for 1 h at 4°C with 2% OG. After centrifugation at 133,000 x g for 30 min in a Beckman Airfuge, the extract was preimmunoprecipitated for 2 h at room temperature with 50 ,u1 of a 10% suspension of Staphylococcus aureus Cowan I (Pansorbin; Calbiochem) coated with normal rabbit serum. After the nonspecifically bound material was removed by centrifugation, the released proteins were incubated overnight with 400 ,ul of hybridoma supernatant, and then 50 RI of a 10% suspension of S. aureus coated with anti-murine immunoglobulin rabbit serum was added. After 4 h of incubation at 4°C, the immune complex was collected and washed four times with 0.5% Nonidet P-40 in PBS. The immunoprecipitated labeled proteins were dissociated by heating for 3 min in boiling water with 200 IL1 of dissociating buffer without 2-ME, 5% 2-ME was added to half of the sample, and the sample was analyzed by polyacrylamide gel electrophoresis. For the sequestration experiment, OG-released virus proteins labeled with [35S]methionine were incubated overnight at 4°C with 400 ,ul of hybridoma supernatant. Then 100 p.l of a 10% suspension of S. aureus coated with rabbit antimurine immunoglobulin serum was added, and incubation was continued for 4 h. The immunoprecipitate was collected 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 viral proteins with OG. The effects of different concentrations of Nonidet P-40, Triton X-100, and OG on ASF virus infectivity were tested. The virus titer was reduced to a lesser extent by OG than by the other nonionic detergents at the same final concentration (e.g., a 90% reduction of virus infectivity was achieved with about 0.3% OG or with 0.003% Nonidet P-40 or Triton X-100). Thus, OG was chosen for further studies. When suspensions of purified ASF virus were treated with OG at a concentration of 0.5 or 2% in the absence or presence of 0.5 M NaCl (Fig. 1), several structural proteins were released from the virions. The first proteins detected in the supernatant after treatment with 0.5% OG, a concentration that reduces the initial virus infectivity to about 2%, were p35 and p12 (Fig. 1, lane lb). 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 partially removed from the virus particle when the samples were incubated with 2% OG in the presence of 0.5 M NaCl (lane 4b). Detergent treatment during which about 5% or more of the initial infectivity was retained did not release any detectable viral protein. Essentially the same results were obtained when other nonionic detergents (Nonidet P-40 and

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0.5 % V

2 %

1

2

a b

a b

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TABLE 2. Binding of "S-labeled ASF virus proteins to different cells

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% of radioactivity bound to cell monolayersa

Inoculum

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-150

2285

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IBRS2

25.1 (8) 16.4 (5) 3.2 (1)

2.4 (8) 2.7 (5) 1.0 (1)

1.9 (5) 0.9 (2) 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

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FIG. 1. Polypeptides released from ASF virus particles by incubation at 4°C for 1 h with different concentrations of OG in the, absence (1 and 3) or presence (2 and 4) of 0.5 M NaCl. The 35Slabeled polypeptides present in the sediment (lanes a) and supernatant (lanes b) resulting from centrifugation in an Airfuge were resolved by electrophoresis on a 7 to 20% polyacrylamide gel and detected by fluorography. The standard profile of "'S-labeled ASF virus structural proteins is shown on the left (V), with their molecular masses indicated in kilodaltons.

Triton X-100 at concentrations of 0.02 to 0.5%) were used to solubilize the viral proteins (not shown). Neutralization of ASF virus by rat antiserum against whole virus or external virus proteins. A pool of rat antisera against whole virus (titer of -8,500) was tested for ASF virus neutralization by a plaque reduction assay in the absence or presence of complement (Table 1). The rat antiserum produced against whole ASF virus was able to eliminate, in the presence of complement, more than 97% of the infectivity present in virus samples. When a mixture of OG-released ASF virus proteins (containing p35, p17, p14, and p12), obtained by treatment with 2% OG and chromatographed on Sephadex G-25 columns to remove the detergent, was used TABLE 1. ASF virus neutralization by specific rat antisera

Nonimmune Whole ASF virus OG-released virus proteins

Neutralization (% survival)'

-Complement 101.5 ± 13 (19) 12.8 ± 4.7 (5) 40.4 ± 7.3 (7)

a Number of experiments is given in parentheses.

b c

a

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Serum specificity

induced antiserum was low in the absence of complement but similar to that obtained by the antiserum against whole virus in the presence of complement (Table 1). The neutralizing titers (reciprocal of highest dilution inactivating >90% of virus infectivity) were about 5,000 for the rat antiserum specific against whole virus and 300 for that specific against the OG-released proteins. Binding of ASF virus proteins to different cells. Different cell monolayers (-150,000 cells) were incubated with [35S]

+Complement

69.8 ± 16 (19) 2.7 ± 1.6 (5) 4.0 ± 2.2 (7)

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FIG. 2. Binding to 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) resulting from centrifugation in an Airfuge after treatment of 35S-labeled ASF virus with 2% OG for 1 h at 4°C are shown as controls. About 150,000 Vero or L cells were incubated for 2 h at 37°C with 7 x 104 (35S) or 2 x 106 (1251) cpm of OG-released virus proteins (lanes c and h), washed with PBS, and subjected to polyacrylamide gel electrophoresis. Also shown are proteins in the washings of Vero cells (lanes d and i) and bound to Vero cells (lanes e and j) and L cells (lanes f and k) and protein bound to Vero cells analyzed in the absence of 2-ME (lane g). Molecular masses in kilodaltons are indicated.

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a b

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FIG. 3. Competition of binding of '25I-labeled OG-released ASF virus proteins to Vero cells. About 200,000 cells precooled at 4°C for 10 min were incubated with increasing concentrations of different competitor proteins for 4 h at 4°C, and then -2.5 x 105 cpm of 125I-labeled OG-supernatant was added to each well. Cultures were further incubated for 4 h at 4°C, washed with PBS, and analyzed by polyacrylamide gel electrophoresis. Shown are proteins bound to L cells (lane b) or to Vero cells incubated in the absence (lanes d and o) or presence of 5, 15, 40, or 120 p.g of whole ASF virus (lanes e to h), 5, 15, 40, or 120 ,ug of OG-sediment (lanes j to m), or 15, 40, or 120 ,ug of BSA (lanes p to r). Representative profiles of the labeled proteins present in the washings of each set of samples are shown in lanes a, c, i, and n. Proteins from 35S-labeled ASF virus were run in the same gel (lane s). Molecular masses in kilodaltons are indicated.

methionine-labeled whole virus or OG-released virus proteins (OG-supernatant) for 2 h at 37°C. There was a specific binding of the labeled proteins to ASF virus-sensitive Vero cells but not to nonsensitive L or IBRS2 cells (Table 2). When the incubation with whole ASF virus was done at 4°C for 4 h, the percentage of radioactivity bound to cell monolayers (in three experiments) was 23.4 and 6.2 for Vero and L-cell cultures, respectively (data not shown). The subviral particles generated after the OG treatment (OG-sediment), which lacked the external proteins, had lost most of their capacity to bind to Vero cells, and no binding to L cells was seen (Table 2). The material that remained bound to the cell monolayers after four washings was disrupted and analyzed by polyacrylamide gel electrophoresis. There was no binding of any virus protein to L-cell monolayers (Fig. 2, lane f), while a 12-kDa polypeptide (p12) was retained in Vero cells after incubation at 37°C with the 35S-labeled OG-released virus proteins (lane e). The protein that bound to the sensitive cell monolayers had a mobility corresponding to 17 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); most of the label in p35 and in the serum albumin (66 kDa), which became iodinated during the chromatography of the labeling mixture through Sephadex G-25, was detected in the washings of both Vero cells (lane i) and L cells (not shown), while there was no binding of any labeled protein to the nonsensitive L-cell (lane k) or IBRS2 cell (not shown) monolayers. The specificity of the binding of the 125I-labeled OGreleased virus proteins to Vero cells was studied by competition with increasing concentrations of unlabeled ASF virus,

BSA, or OG-sediment. Electrophoretic analysis of the labeled material bound to Vero cells (Fig. 3) showed that the binding of viral protein p12 was unaffected by the presence of different concentrations of BSA (lanes o to r) or OGsediment (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 e to h) even at the lowest protein concentration used (5 ,ug; lane e). The absence of binding of any viral protein to nonsensitive L cells is shown in lane b as a negative control. The electrophoretic profile of the labeled material in the washings was analyzed in all cases, yielding similar results to those presented 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 virus proteins, was used to immunoprecipitate the ASF virus proteins released by OG; the electrophoretic profile of the immune complex analyzed either with or without 5% 2-ME in the dissociating 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, we used a parental mouse myeloma cell supernatant (NP3; lane d). The same results were obtained when the immunoprecipitation was done with the ASF virus proteins dissociated with 0.3% SDS and 1% Nonidet P-40 (not shown). To ensure that the protein recognized by MAb 24BB7 was the same protein as that involved in the interaction of the 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 medium from 24BB7 or NP3 and sedimented with S. aureus. After removal of the immune complex, the supernatant was assayed for

VOL. 65, 1991

ASF VIRUS ATTACHMENT PROTEIN

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10- a FIG. 4. Immunoprecipitation of ASF virus proteins by MAb 24BB7. I'S-labeled purified ASF virus (105 cpm) was treated with 2% OG and then immunoprecipitated by supernatant of MAb 24BB7. The immune complex was washed and resuspended in dissociating buffer with (lane b) or without (lane c) 2-ME. Proteins immunoprecipitated by supernatant of NP3, washed, and analyzed in the presence of 2-ME (lane d). Also shown are proteins of whole virus (lane v) and OG-released virus proteins (lane a) analyzed in the presence of 2-ME. Molecular masses in kilodaltons are indicated.

FIG. 5. Sequestration of ASF virus-binding proteins by MAb 24BB7. OG-released virus proteins labeled with [35S]methionine (4.7 x 105 cpm; 1.6 jig of protein; lane b) were incubated with 400 pAl of culture medium from MAb 24BB7 or NP3. After immunoprecipitation with S. aureus, proteins in the sediment (24BB7, lane g; NP3, lane h) and in the supernatant (24BB7, lane i; NP3, lane j) were analyzed. The supernatant of the immunoprecipitation was assayed for binding to Vero cells (24BB7, lane e; NP3, lane f) and compared with the binding of the OG-released virus proteins to Vero (lane d) or L (lane c) cells. Proteins of 35S-labeled whole virus are also shown (lane a). Molecular masses in kilodaltons are indicated.

treatment with the reducing agent and that this reduction was not reversible.

DISCUSSION binding to Vero or L cells and the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The binding of p12 to Vero cells was similar before (Fig. 5, lane d) and after (lane f) immunoprecipitation with NP3, whereas there was no binding after immunoprecipitation with 24BB7 (lane e) or in the negative control of binding of OG-released virus proteins to L cells (lane c). Protein p12 was present only in the sediment (lane g) or in the supernatant (lane j) of the samples immunoprecipitated with 24BB7 or NP3, respectively. A similar experiment with the OG-released virus proteins, labeled in this case with 125I, was run in parallel, yielding the same results (not shown), although the viral protein bound to Vero cells and immunoprecipitated by MAb 24BB7 appeared as a broad band around 12 kDa (Fig. 3). The relationship between the viral proteins p17* and p12 was confirmed by isolation of the corresponding bands after polyacrylamide gel electrophoresis of 35S-labeled purified ASF virus under reducing and nonreducing conditions (Fig. 6, lanes a and f, respectively) and subsequent treatment with 2-ME and electrophoresis of the rehydrated bands (lanes b and d); the untreated controls are shown in lanes c and e. It can be seen that protein p17* was totally converted to p12 by

The reason for the absence of neutralizing antibodies against ASF virus remains obscure. Several possible explanations have been suggested (24): (i) existence of antigenic competition, either inter- or intramolecular, in which a dominant antigen suppresses the response to a critical one; (ii) existence of both neutralizing and blocking antibodies; and (iii) existence of antigenic variability. In any case, identification of the virus component carrying the critical antigenic determinant that might induce the synthesis of neutralizing antibodies is one of the most important problems in ASF research. It seems likely that this putative critical antigen will be found among the external proteins of the ASF virus particle. The identification of surface polypeptides of ASF virions has been carried out by differential release of viral proteins with several nonionic detergents. We have chosen OG, the mildest of them, to solubilize the viral proteins since it has a high critical micellar concentration and is easily removed (2). The proteins first released from the virions by OG treatment were p35, p17, p14, and p12. These proteins were also labeled when ASF virus particles were radioiodinated (21) in the presence of lactoperoxidase (26) or chloramine-T at 4°C (8). These results are consistent with the localization of

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FIG. 6. Conversion of protein p17* to p12 by treatment with 2-ME. 35S-labeled purified ASF virus (3.5 x 105 cpm) was dissociated in the presence (lane a) or absence (lane f) of 5% 2-ME and subjected to polyacrylamide gel electrophoresis. Bands corresponding to p12 (lane a) and pl7* (lane f) were subsequently treated or not with 2-ME and further analyzed by electrophoresis. Protein p12 treated (lane b) or not (lane c) with 2-ME and pl7* treated (lane d) or not (lane e) with 2-ME are shown. Molecular masses in kilodaltons are indicated.

antigenic determinants in the virus particle by immunoelectron microscopy with MAbs specific for ASF virus proteins (6), except in the case of protein p17, which was considered an external component in our study but localized in a more internal region than p72, the major capsid protein, in the immunoelectron microscopy analysis. This apparent contradiction may be due to the presence of at least two polypeptides of 17 kDa in the virus particle upon analysis in two-dimensional gels (unpublished results). Previous reports have studied the existence of neutralizing antibodies against ASF virus. Tabares et al. (23) showed that antiserum against purified vp73 does not neutralize the virus infectivity. Ruiz Gonzalvo et al. (20) have reported that pigs infected with ASF virus may recover and resist challenge exposure with virulent homologous viruses, and they found partial protection by sera from ASF-resistant pigs. Partial neutralization of ASF virus by polyvalent rabbit, mouse, and swine immune sera as well as by MAbs directed against p24, a virus protein of probable cellular origin, has been obtained (11). In none of these cases was the degree of neutralization higher than that obtained in our study by rat antiserum against whole virus or against a mixture of OG-released virus proteins, and neutralization was also 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 mean that there are no neutralization sites, since the virions 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 with the mildest possible treatment), could induce the synthesis of neutralizing antibodies. Although there are multiple mechanisms of neutralization of animal viruses, the most common involves antibody blocking of the binding of the virus attachment protein to a cell receptor unit (10). We have previously described the existence of ASF virus-specific receptors on the plasma membrane of Vero cells and the absence of saturable binding sites on the surface of L cells (1) as a factor that determines the sensitivity of cells to the infection. In the study reported here, we found that the external ASF virus protein p12, extracted from 35S-labeled virus by OG treatment, was able to bind at 37°C to sensitive Vero cells but not to nonsensitive L cells. When the OG-released virus proteins were iodinated before incubation with the cell monolayers, we detected the binding to Vero cells of protein that, by SDS-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 the fact that it was abolished only by the presence of whole ASF virus particles and not by similar concentrations of BSA or even by noninfectious subviral particles that lacked the more accessible proteins. Thus, viral protein p12 is a good candidate to be the ASF virus attachment protein. Protein p12 showed an apparent molecular mass of 17 kDa when analyzed in the absence of 2-ME; the conversion of pl7* to p12 has been observed in immunoprecipitation experiments and by isolation of the corresponding band from polyacrylamide gels and subsequent treatment with 2-ME. Protein pl7* was totally converted to p12; this could be clearly detected because there was no overlap between p17* and the 17-kDa protein (p17) visualized in the presence of 2-ME, since p17 changed to 30 kDa in nonreducing conditions (not shown). The N-terminal amino acid sequences of proteins p17* and p12, obtained after separation on an SDS-polyacrylamide gel and electroblotting onto an Immobilon 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 detected 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 from the site of interaction of p17* with the cellular receptor. It is also clear that protein p12 isolated from polyacrylamide gels in the presence of SDS could have lost the critical epitope. It will be necessary to obtain larger quantities of 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 the possibility of induction of an effective immune response.

VOL. 65, 1991

ACKNOWLEDGMENTS We thank M. L. Nogal for skillful assistance in hybridoma production and M. Salas for critical reading of the manuscript. This research was supported by grants from the Comisi6n Asesora para la Investigaci6n Cientffica y Tecnica, the Consejeria de Agricultura de la Junta de Extremadura, and the European Economic Community and by an institutional grant from Fundaci6n Ram6n Areces. REFERENCES 1. Alcami, A., A. L. Carrascosa, and E. Vinuela. 1989. Saturable binding sites mediate the entry of African swine fever virus into Vero cells. Virology 168:393-398. 2. Baron, C., and T. E. Thompson. 1975. Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. Biochim. Biophys. Acta 383:276-285. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Carrascosa, A. L., M. del Val, J. F. Santaren, and E. Vinuela. 1985. Purification and properties of African swine fever virus. J. Virol. 54:337-344. 5. Carrascosa, J. L., J. M. Carazo, A. L. Carrascosa, N. Santisteban, and E. Vinuela. 1984. General morphology and capsid fine structure of African swine fever virus particles. Virology 132:

160-172. 6. Carrascosa, J. L., P. Gonzalez, A. L. Carrascosa, B. GarciaBarreno, L. Enjuanes, and E. Vinuela. 1986. Localization of structural proteins in African swine fever virus particles by immunoelectron microscopy. J. Virol. 58:377-384. 7. Dalsgaard, K., E. Overby, and C. Sfinchez-Botija. 1977. Crossed immunoelectrophoretic characterization of virus-specific antigens in cells infected with African swine fever (ASF) virus. J. Gen. Virol. 36:203-206. 8. Davies, W. A., and S. P. Stossel. 1981. External membrane proteins of rabbit lung macrophages. Arch. Biochem. Biophys. 206:190-197. 9. Dimmock, N. J. 1984. Mechanisms of neutralization of animal viruses. J. Gen. Virol. 65:1015-1022. 10. Dimmock, N. J. 1987. Multiple mechanisms of neutralization of animal viruses. Trends Biochem. Sci. 12:70-75. 11. Enjuanes, L. Personal communication.

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12. Enjuanes, L., A. L. Carrascosa, M. A. Moreno, and E. Vinuela. 1976. Titration of African swine fever (ASF) virus. J. Gen. Virol. 32:471-477. 13. Hess, W. R. 1981. African swine fever: a reassessment. Adv. Vet. Sci. Comp. Med. 25:39-69. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 15. Laskey, R. A., and A. D. Mills. 1975. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335-341. 16. Letchworth, G. J., and T. C. Whyard. 1984. Characterization of African swine fever virus antigenic proteins by immunoprecipitation. Arch. Virol. 80:265-274. 17. Lopez-Otin, C. Personal communication. 18. Malmquist, W. A., and D. Hay. 1960. Hemadsorption and cytopathic effect produced by African swine fever virus in swine bone marrow and buffy coat cultures. Am. J. Vet. Res. 21:104108. 19. Plowright, W., J. Parker, and R. F. Staple. 1968. The growth of a virulent strain of African swine fever virus in domestic pigs. J. Hyg. (Cambridge) 66:117-134. 20. Ruiz Gonzalvo, F., C. Caballero, J. Martinez, and M. E. Carnero. 1986. Neutralization of African swine fever virus by sera from African swine fever resistant pigs. Am. J. Vet. Res. 47:1858-1862. 21. Santaren, J. F. Personal communication. 22. Sanz, A., B. Garcia-Barreno, M. L. Nogal, E. Vinuela, and L. Enjuanes. 1985. Monoclonal antibodies specific for African swine fever virus proteins. J. Virol. 54:199-206. 23. Tabares, E., J. Martinez, F. Ruiz Gonzalvo, and C. SanchezBotija. 1980. Proteins specified by African swine fever virus. II. Analysis of proteins in infected cells and antigenic properties. Arch. Virol. 66:119-132. 24. Vifiuela, E. 1985. African swine fever virus. Curr. Top. Microbiol. Immunol. 116:151-170. 25. Voller, A., D. Bidwell, and A. Bartlett. 1980. Enzyme-linked immunosorbent assay, p. 359-371. In N. Rose and H. Friedman (ed.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, Washington, D.C. 26. Yin, H. L., S. Aley, C. Bianco, and Z. A. Cohn. 1980. Plasma membrane polypeptides of resident and activated mouse peritoneal macrophages. Proc. Natl. Acad. Sci. USA 77:2188-2191.

African swine fever virus attachment protein.

Treatment of African swine fever virus particles with nonionic detergents released proteins p35, p17, p14, and p12 from the virion. Of these proteins,...
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