Differential expression of Epstein-Barr virus membrane antigens defined with monoclonal antibodies

148,114-120

VIROLOGY

Differential TAKASHI

(1986)

Expression of Epstein-Barr Virus Membrane Defined with Monoclonal Antibodies

YOKOCHI,*+’

*Department of bficrobio~, the Regiml

Primate

EDWARD

A. CLARK,f

YOSHINOBU

AND

Fukui Medical School, Fukui 910-11, Japan, Research Center, University of Washingtcm, Received

April

SO, 1985; accepted

September

Antigens KIMURA*

and department Seattle, Woshingta

of Genetics

and

98195

18, 1985

Four different mouse hybrid cell lines producing IgG2b monoclonal antibodies against Epstein-Barr virus (EBV) membrane antigens (MA) were analyzed. The antigens defined by these antibodies, MA-2, MA-4, MA-5, and MA-7, were expressed only on EBV-producing cells. The antigens were induced on PSHR-1 cells by treatment with tumor-promoting agent (TPA) plus n-butyrate, and this induction was inhibited by treatment with phosphonoacetic acid (PAA) or acyclovir. Most MA monoclonal antibodies neutralized the infectivity of EBV in vitro in the presence of complement. The monoclonal antibody MA-4 precipitated two polypeptides with mol wt of 340K and 240K, while other monoclonal antibodies MA-2, MA-5, and MA-7, did only 340K peptide. The frequency of positive cells in MA-induced cells varied for each monoclonal antibody tested. It was also found that MA-4 (anti-340K and 240K) antibody reacted on both chemically induced cells and EBVsuperinfected cells, but others did only on chemically induced cells. It was suggested that MA had a different pattern of expression between chemically induced cells and EBVsuperinfected cells. 0 1986 Academic Press. Inc.

1980), and others recognized the strainspecific determinants (Mueller-Lantzsch et aL, 1981; Qualtiere et al, 1982). However, the detailed expression of the MA complex is still poorly understood, In the present study, we isolated a series of monoclonal antibodies which are specific for EBV MA determinants and found that MA was expressed in a distinct pattern on the chemically induced and EBV-superinfected cells.

INTRODUCTION

Infection of cells with Epstein-Barr virus (EBV) leads to the expression of a series of antigens during the course of virus growth. They are EBV nuclear antigen (EBNA), early antigen (EA), viral capsid antigen (VCA), and membrane antigen (MA) (Ernberg and Klein, 1979). Recently, monoclonal antibodies have been developed for a better characterization of the MA complex (Hoffman et aL, 1980; ThorleyLawson and Geilinger, 1980; MuellerLantzsch et aL, 1981; Qualtiere et al., 1982; Strnad et al, 1982). These studies have revealed that there are two high molecular weight polypeptides in the MA complex, which seem to share common antigenic determinants (Thorley-Lawson and Geilinger, 1980; Mueller-Lantzsch et aL, 1981; Strnad et al, 1982). Some of the monoclonal antibodies against these molecules neutralized the infectivity of EBV (Hoffman et aL, 1980; Thorley-Lawson and Geilinger, ’ To whom

0042-6822/86 Copyright All rights

reprint

requests

should

$3.00

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Production of mono&ma1 antibodies. C57BL/6J mice were inoculated intraperitoneally with 1 ml of supernatant of the EBV-producing B95-8 cell line three times at weekly intervals. Ten days after the last injection, they were injected intravenously with 0.5 ml of concentrated B95-8 virus (1.5 X lo7 transforming units/ml) obtained from Life Science Inc. (St. Petersburg, Fla.). Three days later spleen cells were fused with the nonsecreting variant cell line P3/NS-l/l-Ag4-1 (NS-1) by the method described in detail elsewhere

be addressed.

114

EXPRESSION

OF

EBV

(Nowinski & ak, 1979). Microcultures reactive with TPA plus n-butyrate-induced P3HR-1 cells were cloned twice by a limiting dilution method with a thymocyte feeder layer, and the hybrid clone cells were inoculated into (C57BL/6J X BALB/ c) Fl mice to obtain monoclonal antibodyrich ascites fluid. Antibody isotype was determined by the slide immunodiffusion. All monoclonal antibodies had the immunofluorescence titers in excess of more than 1:3000. Immunojlwrescence (IF) assay. IF-positive cells were stained by the indirect IF method using a 1:lOO dilution of the ascites fluids (Yokochi et al, 1984), and assayed for surface IF using Leitz Diavert fluorescence microscope. Suspensions of IF-positive cells were also analyzed with the aid of a fluorescence-activated cell sorter (Cell sorter CS-20, Showa Denko K. K., Japan) as previously described (Loken and Herzenberg, 1975). Induct&. and inhibition of MA expression. Cells were cultured in RPMI-15 containing 100 rig/ml 12-O-tetradecanylphorbol-13 acetate (TPA) and 2 mM n-butyrate (Sigma, St. Louis, MO.) for induction of MA (Kawanishi et a& 1981b). In superinfection experiments Raji cells at a concentration of 5 X 105/ml were suspended in l-weekold spent supernatant of the P3HR-1 cllB cell line at 37” for 2 hr, and then diluted to 1 X lo5 cells/ml in RPMI-15. After various lengths of incubation MA immunofluorescence was tested. Phosphonoacetic acid (PAA) and acyclovir were used at a concentration of 200 pg/ml in RPMI-15. Neutralization tests. An aliquot of 0.4 ml of P3HR-1 cllB supernatant was mixed with 0.3 ml of monoclonal antibody. The mixture was held at 4” for 30 min and then added to 0.1 ml of an 1:3 dilution of rabbit complement, (Low-Tox-H, Cedarlane, Ontario). After incubation at 37” for 30 min each mixture was tested for the ability to induce EBV EA in Daudi cells by the indirect IF method using human serum from an EA seropositive nasopharyngeal carcinoma (NPC:) patient (Henle et aL, 1970). Labeling of proteins, immunoprecipitat&m, and gel electrophcn-esis. P3HR-1 cllB

MEMBRANE

ANTIGENS

115

cells were cultured in RPMI-15 containing TPA and n-butyrate for induction of MA. After a 48-hr incubation, cell surface proteins were labeled by the ‘%I-lactoperoxidase method (Vitteta et al, 1971). Cell lysates were prepared using 0.5% NP-40 detergent in 0.15 M NaCl, 0.05 M Tris, and 5 mM EDTA, at pH 8.0 (Kessler, 1975) and the lysate was then adjusted to a concentration of 0.1% sodium dodecyl sulfate (SDS). The lysate was immunoprecipitated with antibody and protein A-Sepharose CL-4B(Pharmacia Fine Chemical, Uppsala, Sweden). Radiolabeled proteins were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of SDS in 7.5% slab gels under reducing conditions (Laemmli, 1970). Radiolabeled bands in the gels were identified by autoradiography. Molecular weight standards obtained from Bio-Rad were used: myosin (200,000), human y-globulin (150,000), phosphorylase B (97,400), bovine serum albumin (69,000), and ovalbumin (46,000). Double IF staining. TPA plus n-butyratetreated (MA-induced) P3HR-1 cllB cells were incubated with MA-5 or MA-7 antibody, followed by treatment with rhodamine-labeled rabbit anti-mouse IgG antibody (Tago Inc.), and then incubated with FITC-labeled MA-4 antibody. The numbers of FITC-positive cells, rhodamine-positive cells, and both FITC- and rhodamine-positive cells were calculated. RESULTS

Characterkation of MA monockmal antibodies. The determinants recognized by the MA-2,4,5, and 7 monoclonal antibodies were expressed on only EBV-producing cell lines, such as B95-8 and P3HR-1 cllB, but not on EBV-nonproducing cell lines such as Raji, Daudi, and Jijoye nor on a EBVnegative cell line Ramos. Treatment of virus-producing cell lines with TPA plus nbutyrate enhanced the expression of the antigens defined by MA monoclonal antibodies, while the same treatment failed to induce MA in nonproducing cell lines (Table 1). The induction of EBV MA was blocked by the simultaneous treatment

116

YOKOCHI,

CLARK, TABLE

REACTIVITY

OF MA

MONOCLONAL

ANTIBODIES

AND

KIMURA

1 ON MA-INDUCED % IF-positive

Human Treatment B95-8 PAA (3) acyclovir

(days)

(EA-

(7)

PSHR-1 . TPA TPA + TPA +

+ n-butyrate + n-butyrate PAA (3) + n-butyrate acyclovir (3)

(3)

Raji TPA

+ n-butyrate

(3)

a Five

OR MA-INHIBITED cells” Monoclonal

serum

CELLS

antibody

MA-2

MA-4

MA-5

MA-7

12.5 3.0 0.5

9.7 0.4 0

25.0 5.1 0.5

5.5 0.2 0

10.1 0.6 0

1.8 45.2

0.9 9.0

3.0 40.2

0.6 5.9

0.5 11.7

4.4

1.6

4.9

0.9

1.5

4.9

2.1

7.0

1.5

1.9

0 0

0 0

0 0

0 0

0 0

MA+

VCA+)

cllB

hundred

cells counted

per test.

with PAA or acyclovir. It should be noted that the percentage of positive cells varied for each antibody tested. MA-2, 4, and 7 monoclonal antibodies neutralized the infectivity of P3HR-1 virus in the presence of complement (more than 80% reduction), whereas the MA-5 antibody was far less effective (28% reduction) at a concentration of the same IF staining titer. Antigens detected by MA monoclonal antibodies were analyzed by immunoprecipitation and ‘7.5% PAGE (Fig. 1). These monoclonal antibodies consistently precipitated polypeptides with the following approximate molecular weights: MA-2, 5, and ‘7, 340K; MA-4, both 340K and 240K. Irrelevant antibody (Ig G2b) did not precipitate any peptides. Kinetics of MA expressim Because each MA determinant was expressed in a characteristic fashion, it was of interest to examine the kinetics of the expression of each determinant. Two kinds of MA induction experiments were done: (1) treatment of P3HR-1 cllB cells with TPA plus n-butyrate, and (2) superinfection of Raji cells with

PQHR-1 virus. In TPA plus n-butyratetreated P3HR-1 cllB cells the expression of MA-4 antigen increased progressively

4 340K 4 240K

12

3

4

5

FIG. 1. Autoradiograph of the SDWpolyacrylamide gel electrophoresis of lrr’I-labeled proteins precipitated from TPA plus n-butyrate-induced P3HR-1 cllB ceils with monoclonal antibodies. Lane: 1, MA-2; 2, MA-4; 3, MA-8 4, irrelevant control antibody (IgG2b); 5, MA-7.

EXPRESSION

60

OF EBV MEMBRANE

117

ANTIGENS

A

0

1

FIG. 2. Kinetics of MA expression in TPA plus n-butyrate-induced superinfected Raji cells (B). MA-2 (W-m); MA-4 (O-O); MA-5 (O-O);

through Day 4, whereas MA-2,5, and 7 antigens peaked on Day 3 and then waned (Fig. 2A). MA-4 had the highest frequency of IF-positive cells among MA monoclonal antibodies tested and MA-2, 5, and 7 had similar frequency each other. On the other hand, the expression of the antigens in superinfected Raji cells increased after Day 1, peaked at Day 2 and 3, and thereafter waned (Fig. 2B). The percentage of each IF-positive cells at Day 3 were 7.6% (MA4), 2.4% (MA-2), 0.4% (MA-‘7), and 0.2% (MA-5), respectively. Characteristically, unlike in chemically induced P3HR-1 cllB cells, MA-5 and 7 antigens were hardly detected on superinfected Raji cells. This phenomenon was quantitatively confirmed by the flow cytometry (Fig. 3A and B). The reactivity of MA-7 antibody was almost the same as MA-5 (data not shown). Double IF staining. For an investigation of the expression of each MA determinant in a single cell, double IF staining was employed (Table 2). MA-4 positive cells were not necessarily MA-5 positive, while all MA-5 positive cells expressed MA-4 anti-

2

3

4

Time (days)

Ttme (days)

P3HR-1 cllB cells (A) and MA-7 (A-A).

gen. The relationship between MA-4 positive cells and MA-7 positive cells was essentially similar to that observed above. DISCUSSION

The MA complex has been reported to consist of two major glycoproteins of gp 350K and gp 220K (Thorley-Lawson and Edson, 1979), which presumably correspond to the 340K and 240K polypeptides identified in the present study. These two molecules seem to be antigenically related, since polyclonal and monoclonal antibodies can precipitate them at the same time (Thorley-Lawson and Geilinger, 1980). However, MA-2,5, and 7 antibodies reacted only with the 340K peptide. This may mean that there is the specific determinant expressed only on the 340K molecule, not shared with the 240K, and may deny the possibility that two major components exist as a complex form. Mueller-Lantzsch et al. (1981) have reported the monoclonal anti-p340 strain-specific antibody which is reactive with QIMR-WIL EBV strain but

118

YOKOCHI, A

B

CLARK,

MA-4

MA-4

FIG. 3. Flow cytometry analysis of MA expression cells (A) and superinfected Raji cells (B). The figure (z axis) and the cell number (g axis).

not with B95-8 and P3HR-1 EBV virus. It is not unlikely that our anti&OK antibody may inversely recognize the strain-specific determinant expressed on B95-8 and P3HR-1 virus but not on QIMR-WIL strain. Further, it should be noted that there is a significant number of MA-4 (340/240K) positive but MA-5 or ‘7 (340K) negative cells in the double IF staining (Table 2). This indicates that there exists the population in which only the 240K polypeptide is expressed. Another population expressing only the 340K polypeptide possibly presents. The previous finding (Thorley-Lawson and Geilinger, 1980; Thorley-Lawson, 1984) that the B95-8 cell line expresses very low level of gp 220 (which is a major com-

AND

KIMURA MA-5

MA-5

in TPA plus n-butyrate-induced is expressed with the intensity

PQHR-1 cllB of fluorescense

ponent in P3HR-1 cell line) while the gp 350 is expressed in excess could be explained by the difference of the dominant population in two cell lines. Of particular interest is the finding that there was differential expression of MA defined by the monoclonal antibodies between chemically induced P3HR-1 cllB producer cells and EBV-superinfected Raji cells. For example, MA-2, 5, and 7 (anti340K) reacted with more than half of MA-4 (anti-340K1240K) positive cells induced by TPA plus n-butyrate, while in superinfected cells these monoclonal antibodies reacted at a much lower frequency with MA-4 positive cells (Figs. 2 and 3). Probably the 340K molecule defined by MA-

EXPRESSION TABLE DOUBLE

OF EBV MEMBRANE

ANTIGENS

2

119

REFERENCES

IMMUNOFLUORESCENCEMA STAINING

I., and KLEIN, G. (1979). EB virus-induced antigens. In “The Epstein-Barr Virus” (M. A. Epstein and B. D. Anchong, eds.), pp. 39-60. SpringerVerlag, Berlin. HENLE, W., HENLE, G., ZAJAC, B., PEARSON, G., WAUBKE, R., and SCRIBA, M. (1970). Differential reactivity of human sera with early antigens induced by Epstein-Barr viruses. Science (Washing@ D. C.) 169,188-190. HOFFMAN, G. J., LAZAROWITZ, S. G., and HAYWARD, S. D. (1980). Monoclonal antibody against a 250,000clalton glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neuralizing antigen. ERNBERG,

% Positive cells” Monoclonal antibody

MA-4+

MA-4-

1

MA-5+ MA-5-

74.1 25.5

0 -

2

MA-7+ MA-7-

71.9 24.3

3.0 -

Expt. No.

DPercentage IF-positive cells based on counting 400 MA-positive cells.

Proc.

2,5, and 7 is well induced on P3HR-1 cllB by chemical treatment but less in superinfected Raji cells. Qualtiere et al. (1982) has reported the similar finding that 2F5.6 monoclonal antibody did not react with TPA-activated P3HR-1 cells in an IF assay nor did it precipitate the Mr 220/250 glycoprotein from this cell line, but that the 2F5.6 antibody did precipitate the Mr 220/ 250 glycoprotein from superinfected Raji cells. Kawanishi et al. (1981a) also reported three kinds of polypeptides with mol wt of 150K, 140K, and 120K which were expressed only in chemically induced producer cells but not in superinfected Raji cells. Recently Hummel et al. (1934) have suggested that the 350K and 220K polypeptides are encoded by 3.4- and 2%kb mRNAs, respectively and these mRNAs overlap considerably in their sequences suggesting that they are derived from the same gene by different splicing process. Factors, such as chemical treatment and superinfection may affect the splicing of different size of RNAs transcribed from the same gene.

Nat1

This work was supported by grants from the Ministry of Education, Science and Culture of Japan and by American Cancer Society IM-260 (E.A.C.). We thank Dr. Aharam Ablashi for providing cell lines and Dr. Gertrude Elison for the gift of acyclovir. We are grateful to Yoshiko Inoue and Junko Yamamoto for the technical assistance.

Sci. USA 77,2979-2983.

406-409.

KESSLER, S. W. (1975). Rapid isolation of antigens from cells with a staphyloccocal protein A-antibody absorbent: Parameters of the interaction of antibodyantigen complexes with protein A. J. Immund 115, 1617-1624. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LOKEN, M. R., and HEF&NBERG, L. A. (1975). Analysis of cell populations with a fluorescence-activated cell sorter. Ann N. I: Ad Sci 254.163-171. MUELLER-LANIZSCH, N., GEORG-FRIES,B., HERBST, H., ZUR HAUSEN, H., and BRAUN, D. G. (1981). EpsteinBarr virus strain- and group-specific antigenic determinants detected by monoclonal antibodies. Int. J. Cancer 28,321-327. NOWINSKY,

ACKNOWLEDGMENTS

Acaa!

HUMMEL, M., THORLEY-LAWSON, D., and KIEFF, E. (1984). An Epstein-Barr virus DNA fragment encodes messages for the two major envelop glycoproteins (gp3501300 and gp229/200). J. fi& 49,413417. KAWANISHI, M., SUGAWARA, K., and ITO, Y. (1981a). Epstein-Barr virus-induced polypepticles: A comparative study with superinfected Raji, IUdRtreated, and n-butyrate-treated P3HR-1 cells. virdogy 169,72-81. KAWANISHI, M., SUGAWARA, K., and ITO, Y. (1981b). Epstein-Barr virus-induced early polypeptides in Raji and NC37 cells activated by diterpene ester TPA in combination with n-butyrate. I&w 115,

R. C., LOSTROM,

M. E., TAM, M. R., STONE,

M. R., and BURNETTE, W. N. (1979). The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses. VirdoQy 93.111-126. QUALTIERE, L. F., CHASE, R., VROMAN, B., and PEARSON, G. R. (1982). Identification of Epstein-Barr virus strain differences with monoclonal antibody to a membrane glycoprotein. Prcc NatL Ad Sci USA 79.616-620.

120

YOKOCHI,

CLARK,

STRNAD, B. C., SCHUSTER, T., KLEIN, R., HOPKINS, R. F., III, WHITMER, T., NEUBAUER, R. H., and RABIN, H. (1982). Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen. J. viral. 41,258-264. THORLEY-LAWSON, D. A. (1984). EBV associated membrane antigens on virions, producer cells and transformed lymphocytes. In “The Role of Viruses in Human Cancer” (G. Giraldo and E. Beth, eds.), Vol. II, pp. 153-168. Elsevier, New York. THORLEY-LAWSON, D. A., and EDSON, C. M. (1979). Polypeptides of the Epstein-Barr virus membrane antigen complex. J. viral. 32,458-467. THORLEY-LAWSON, D. A., and GEILINGER, K. (1980).

AND KIMURA Monoclonal antibodies against the major glycoprotein (gp350/220) of Epstein-Barr virus neutralize infectivity. Proc Nat1 Awd. Sci USA 77,5307-5311. VITETTA, E. S., BAUR, S., and UHR, J. W. (1971). Cell surface immunoglobulin. II. Isolation and characterization of immunoglohulin from spleen lymphocytes. J. Exp. Med. 132,242-264. YOKOCHI, T., BRICE, M., RABINOVITCH, P. S., PAPAYANNOPOLJLOU,T., and STAMATOYANNOPOULOS, G. (1984). Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast. Blood, 63,13761384.