VIRUS GENES 6:3,273-280, 1992 © Kluwer Academic Publishers, Manufactured in The Netherlands

Neutralizing Monoclonal Antibodies Against and 13 Subunits of the Ustilago maydis Virus Encoded Toxin I. GINZBERG, I S. ROSENBLUM, i Y. KOLTIN, I AND N.I. SMORODINSKY 2

1Department of Microbiology-Biotechnotogy 2The Hybridoma Unit, G.S. Wise Faculty of Life Sciences, Tel-Aviv Universi~, Ramat-Aviv, Israel Received July 31, 1991 Accepted October 9, 1991 Requests for reprints should be addressed to Y. Koltin, Department of Microbiology-Biotechnology, G.S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Israel 69978.

Key words: monoclonal antibodies, Ustilago maydis, dsRNA virus, fungal toxins

Abstract The toxins secreted by Ustilago rnaydis are encoded by dsRNA viruses. The KP6 toxin encoded by subtype P6 consists of two polypeptides oLand 13, which are not covalently bound. Neutralizing monoclonal antibodies (MoAbs) were raised against each subunit. Some of the anti-13 MoAbs identify different epitopes in the antigen. The MoAbs were used to affinity purify c~ and 13 polypeptides from culture media and to detect the precursor of the mature toxin.

Introduction

Double-stranded RNA viruses have been detected in more than 100 fungal species (1). In Saccharomyces cerevisiae and in Ustitago maydis the viruses encode a toxin that affects sensitive cells of the same species and related species (2-4). The U. maydis viruses include three subtypes, known as P1, P4, and P6, and each encodes a specific toxin designated KP1, KP4, and KP6, respectively. The best characterized toxin is KP6, and it consists of two noncovalently linked subunits, a (8.6 kD) and 13 (9.1 kD), which are encoded from a single transcript as a preprotoxin that is processed into the secretable mature subunits (5,6). For improved purification purposes, to gain insight into the processing of the toxin, and to study the interaction of the toxin with the target, monoclonal antibodies (MoAbs) for each of the subunits were derived and characterized.

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Methods

Strains All of the U. maydis strains used have been described elsewhere (5). These were KP6 wild type (75-1), KP6 lacking the et subunit (75-INK3), KP6 lacking the t3 subunit (75-INK13), an isogenic strain that does not contain the information for the toxin (75-1UI), and a sensitive strain (strain 18).

Partial purification of KP6 toxin Toxin was purified from 75-1 culture supernatant using an ion-exchange column, CM Sephadex C-25 (Pharmacia), followed by size separation on Sephadex G-50 (Pharmacia), as previously described (5).

lsotopic labeling of KP6 Labeled toxin was obtained by growing 75-1 cells for 50 min at 27°C in defined medium (7) with labeled sulphur (40 txCi/ml, Amersham) as the sole source of sulphur. The supernatant of the culture was collected.

Gel electrophoresis Proteins were characterized by 18% NaDodSO 4 polyacrylamide gel electrophoresis (PAGE), as previously described (8).

Preparation of MoAbs Partially purified toxin was linked to K L H (keyhold limpet hemocyanin) using glutaraldehyde to increase its immunogenicity. Immunization was performed in BALB\c mice using 50-100 p~g of the conjugate. This antigen was applied subcutaneously every 3 weeks for a period of 2 months. Prior to recovery of the spleen for the hybridization, the mice were immunized once with 100 lxg of the unconjugated toxin. Hybridization of the spleen cells with NS 0 myeloma cells, subsequent cultures, and cloning were performed as described (9).

Immunological procedures Radioimmunoassay (RIA), immunoprecipitation, and Western blots were performed as described (10).

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Affinity purification by MoAbs The antibodies were precipitated from ascities fluids by ammonium sulphate (36% of saturation) and were dialyzed against phosphate saline buffer. Each antibody (12 mg) was coupled to activated Sepharose 4B beads (Pharmacia) (2 ml) by CNBr (11). Columns (1.5 ml) were washed with TNE buffer (10 mM Tris-HC1 pH 7.5, 150 mM NaCI, I mM EDTA).

Labeling of MoAb and inhibition of binding assay Hybridoma cells producing MoAbs were grown in Dulbeco's Modified Eagles Medium (DMEM, Biological Industries, Israel) without methionine (37°C, 7% COz). After 1 hr 150 ixCi/ml of 75Se-methionine (Amersham) was added to the culture for 5 hr. Culture filtrate was collected and was designated 75Se-MoAb. Microtiter plates were coated with 20 ixg/ml of toxin and incubated with known amounts of cold MoAbs, as determined by RIA for IgG levels (12), for 5 hr at room temperature. After washing the microtiter plates, 60,000 cpm/well of 75Se-MoAb was added and the plates were incubated at 4°C overnight. The plates were washed and the amount of bound 75Se-MoAb was detected in a gamma counter.

Results and Discussion

The hybridoma supernatants were screened for specific antibodies for KP6 by RIA using culture filtrate of the killer strain (75-1). A filtrate from an isogenic strain (75-1U1) that does not contain the dsRNA segment that encodes the toxin was used as a control. Labeled 1251goat anti-mouse IgG was used to detect the specific antibodies for toxin. Only 3 out of 4000 hybridomas gave a positive signal displaying binding to the filtrate from the killer strain and not to the control. These colonies were cloned, and the subclones were designated 24F, 24G, and 27H. Another 24 colonies produced antibodies that bound to filtrates from the control and from the killer strain. Two colonies (designated 23B and 26H) were cloned and used as control antibodies for nonspecific binding. The specificity of the MoAbs was tested by immunoprecipitation of 35S labeled KP6 toxin. The precipitates were analyzed by 18% NaDodSO4-PAGE. The results indicated that only the 13 subunit was immunoprecipitated from the toxin that contains both a and 13, as shown in Fig. 1. MoAbs 27H and 24G appeared to precipitate 13very efficiently, whereas 24F was less effective for immunoprecipitation, although it is clearly specific for 13. Similar results were obtained using Western blots. As no antibodies against oL were recovered in the initial effort, an additional hybridization was performed using the partially purified a subunit conjugated to

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Fig. 1. Immunoprecipitation of 13by MoAbs. 35S-labeled proteins from culture filtrate of strain 75-1

were reacted with the MoAbs. For immunoprecipitation goat anti-mouse Fab and protein A Sepharose (Pharmacia) were added. The precipitates were analyzed in 18% NaDodSO4-PAGE. Lanes 1-7: MoAbs 24F (1), 24G (2), 27H (3), 23B (4), 26H (5), serum from an immunized mouse (6), and serum from a nonimmunized mouse (7). Lane 8: Culture filtrate of strain 75-1 as a marker for a and 13.

K L H as the immunogen. Subunit ~t was obtained f r o m the supernatant o f an isogenic strain (75-INK13) that carries a virus mutant that e x p r e s s e s only the cx subunit (5,13). T h e entire immunization procedure, cloning, subcloning, and the screening for M o A b s were repeated. As cx subunit displayed p o o r a d h e r e n c e to the wells of the microtiter plates, the conjugate KLH-et was used to detect anti-o~ antibodies from the h y b r i d o m a supernatants. O f 1203 colonies tested, one colony, designated 26st, e x p r e s s e d antibodies specific for ix, as can be seen in Fig. 2. All the monoclonal antibodies w e r e identified by immunodiffusion as IgG 1 using goat anti-mouse IgG1, IgG2, IgA, and IgM. To determine w h e t h e r the M o A b s neutralize the activity of the toxin, two-fold dilutions o f the antibodies w e r e incubated with the toxin for 2 hr at r o o m t e m p e r a ture. Inhibition o f killing activity was tested by spotting 10-vd samples f r o m the mixtures onto a lawn of sensitive cells (strain 18) (Fig. 3). The results indicate that all four antibodies neutralize the activity and that the decrease in neutralization is proportional to the dilution of the antibodies. The three anti-13 M o A b s - - 2 4 F , 24G, and 2 7 H - - a r e apparently distinct and recognize different epitopes of t3. Their specificity for c o m m o n epitopes was tested b y inhibition of binding of labeled 24F to 13 by cold 24F, 24G, and 27H. Microtiter plates were coated with KP6 and were incubated with k n o w n a m o u n t s of cold 24F, 24G, and 27H. After washing, 75Se-24F was added and the plates

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Fig. 2. Western blot of proteins from a culture filtrate of strain 75-1 revealed with anti-a MoAb. Lanes: (1) MoAb 26st; (2) serum from a nonimmunized mouse; (3) serum from an immunized mouse; 4: proteins from culture filtrate of 75-t stained with Ponceau S (Sigma),

Fig. 3. Neutralization of the toxin by MoAbs 24G and 26st. A total of 10 o,l of two-fold dilutions of ascites fluids in phosphate buffer were incubated with 10 p,l of culture filtrate of 75-1 for 2 hr at room temperature. The mixtures (10 ~1) were spotted onto a lawn o f a sensitive strain 18 and the plates were incubated at 28°C for 24 hr, Center o f plates: filtrate from 75-1 not incubated with antibodies, Plates A-B: Sample 1, filtrate o f 75-1 incubated with serum of a nonimmunized mouse; samples 2-13, filtrate of 75-1 incubated with two-fold dilutions of anti-13 MoAb 24G, Plate C: Samples 1-8, filtrate of 75-1 incubated with two-fold dilutions of anti-c~ MoAb 26st.

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Fig. 4. Specificity of the anti-13 MoAbs. Microtiter plates coated with 20 p.g/ml of toxin were incubated with known amounts of unlabeled MoAbs 24F, 24G, and 27H for 5 hr at room temperature. The plates were washed and incubated with 60,000 cpm/weU of 7SSe-24F overnight at 4°C. The plates were washed and counted. The percent inhibition of binding of labeled 7SSe-24F as a function of the concentration of the unlabeled MoAbs is shown.

were incubated at 4°C overnight to enable its binding to its specific determinants on KP6 that remained free after the binding of the cold MoAbs. The plates were washed, and the amount of bound 75Se-24F was detected in a gamma counter. The counts were calculated as the percentage of inhibition of binding of 75Se-24F by the cold MoAbs as a function of their concentration. The results shown in Fig. 4 suggest that the MoAbs identify different antigenic determinants in 13. The binding of 75Se-24F was effectively inhibited by unlabeled 24F (80% inhibition), was partially inhibited by 24G (40%), and was not inhibited at all by 27H. Thus it appears that 24F and 27H interact with different epitopes, and it is likely that partial overlap exists between 24F and 24G. Three of the MoAbs were tested for use in affinity purification of the subunits and [3. The MoAbs tested were 26st for oLand 24F, and 24G for 13. Then 30 ml of culture filtrate from the killer strain (75-1) were passed through each column. The flowthrough was collected and tested for residual activity by spotting I0 ~xl on a lawn of the sensitive strain. No toxic activity was noticed in the flowthrough of all three columns. Examination of the flowthrough in 18% NaDodSO4-PAGE confirmed that ot bound to the column of 26st and [3 was bound to both columns of 24F and 24G (Fig. 5). Biologically active o~ was eluted with 0.1 N acetic acid. After dialysis of the eluate against water, the eluted subunit was tested for its killing activity of sensitive cells by addition of the complementary subunit. The eluted ct was found to be active. Elution of 13encountered greater difficulties and was unsuccessful with 0.1 N acetic acid; 0.1 acetic acid/0.15 M NaCI, or with 1 N acetic acid/8 M urea.

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Fig. 5. Affinity purification with MoAbs 24F, 24G, and 26st. The flowthrough and the eluates were dialyzed, concentrated, and analyzed in 18% NaDodSO4-PAGE. a: 1. Culture filtrate of strain 75-1NK3 expressing only 13.2. Culture filtrate of strain 75-1NK13 expressing only c~. 3. Culture filtrate of the killer strain 75-1.4. Prestained low molecular weight markers. 5. Flowthrough from column of 24F. 6. Eluate from column 24F eluted 5 M MgCI2. 7. Flowthrough from column of 24G. 8. Eluate from column 24G eluted with 5 M McCI2. b: 1. Flowthrough from column of 26st. 2. Eluate from column 26st eluted with 0.1 N acetic acid. Subunits a and 13are indicated. The pretoxin 29 kD is marked with an arrow in Fig. 5a.

However, the use of 5 M MgCI2 was found to be effective. The elution was performed in a stepwise manner with t .5 and 3 M MgCI 2 to elute initially nonspecific bound proteins. The 13 subunit was eluted with 5 M MgCI2. The eluate was dialyzed, concentrated, and tested for its biological activity on a lawn of sensitive cells using et for complementation. The eluted 13 was found to be biologically active. The eluted ot and 13 were analyzed on 18% N a D o d S O 4 - P A G E (Fig. 5). The eluted material appears as distinct bands in the gel at the expected position. Through the use of this series o f monoclonal antibodies efforts have been initiated to detect the precursor of the mature toxin. The use of such antibodies along with a genetic approach is essential for studies on the expression and processing of the toxin and to study the secretory pathway of these molecules. Pulse-chase experiments to determine the rate of synthesis and secretion of the mature toxin have shown that the synthesis, processing, and secretion occur within less than 10 rain. In Western blots and immunoprecipitation, not even traces of the precursors can be detected. By affinity purification, however, one additional polypeptide of ca. 29 kD, similar in size to the predicted preprotoxin based on in vitro translation and sequence data (6), has been already recovered (Fig. 5A, lane 6) and is currently under investigation. Furthermore, this series of M o A b s are currently in use to study the expression of the viral information in foreign cells and to study the interaction of the subunits with the target cells.

Acknowledgment This study was supported by a grant from the U.S.-Israel Binational Science Foundation to Y.K.

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