Characterization of a 75-kDa epstein-barr virus capsid protein using a new monoclonal antibody H250

© INSTITUTPASTEUR/ELSEVIER Paris 1989

Res. Virol.

1989, 140, 531-543

CHARACTERIZATION OF A 75-kDa EPSTEIN-BARR VIRUS CAPSID PROTEIN USING A NEW MONOCLONAL ANTIBODY H250 A. Sanchez-Pinel, J. Bernad, H. Rives, J. Icart and J. Didier Laboratoire de Bactdriologie-Virologie, Facultd de Mddecine Toulouse-Rangueil, 31400 Toulouse

SUMMARY A monoclonal antibody (mAb) designated H250, directed against an Epstein-Barr virus (EBV) capsid antigen, was obtained following immunization of BALB/c mice with naked particles from the producer cell line B95.8. This antigen was present in the producer lines B95.8, P3HR1, M81, RI and CA, and absent from the non-producer lines B JAB, Raji and 1022. H250 did not inhibit the transformation of cord blood lymphocytes by the B95.8 virus, nor did it inhibit EA induction on Raji cells by the P3HR1 virus. In addition, H250 showed no fluorescence on living B95.8 cells. This indicates that H250 does not recognize a membrane antigen. By indirect immunofluorescence, no fluorescence was observed on induced Raji cells or on PAA-treated B95.8 cells. Thus, H250 recognized a late antigen of the EBV virus replication cycle. Agglutination of naked virus by H250 showed it was directed against a capsid antigen. Positive fluorescence was observed on cells treated with tunicamycin, indicating that H250 recognized a protein. The molecular weight of this protein was obtained by Western blot and was approximately 75 kDa. The blocking tests carried out with H250 seemed to indicate that this Ab appeared late in patient sera during primary infection. KEV-WOROS:EBV, Capsid, Protein antigen, mAb H250; Characterization, Pathogenesis.

Submitted January 26, 1989, accepted October 23, 1989.

532

A. SANCHEZ-PINEL

ET AL.

INTRODUCTION

The structural proteins of the Epstein-Barr virus (EBV) were established in 1976 by Dolyniuk from the B95.8 cell line (Dolyniuk et al., 1976a). Thirtythree proteins were identified, with molecular weights ranging from 28 to 290 kDa. However, the complexity of the viral antigens and the polyspecificity of the human sera used to detect them prevented complete study of these proteins. The production of monoclonal antibodies (mAb) directed against EBV has made it possible to characterize and purify some viral antigens. From 1980, mAb directed against the major glycoprotein of the viral envelope, gp 350/250, were obt~iined (Hoffman et al., 1980; Thorley-Lawson and Geilinger, 1980). Later, other mAb appeared, directed against the antigens of the viral envelope (MA or membrane antigen) (Mueller-Lantzsch et aL, 1981 ; Strnad et al., 1982; Boudouma et al., 1986; Balachandran et al., 1986), the viral capsid (VCA) (Kishishita et al., 1984; Takada et aL, 1983 ; Wroman et al., 1985), restricted components (EAR) or diffuse components (EAD) of the early antigen (Epstein, 1984; Luka et al., 1986; Sairenji et al., 1987), or against EBNA (Hearing et al., 1985). These mAb have been useful for the purification of the corresponding proteins by affinity chromatography and for the development of ELISA assays to detect the presence of specific Ab directed against EBV in human sera (Luka et al., 1984; Uen et al., 1988). With these mAb, genomic mapping of some viral antigens has also been carried out: gp 350/250 was mapped to the B a m H I L fragment (Hummel et al., 1984), gp 85 to the B a m H I X fragment (Heineman et al., 1988), and the EAD complex to the B a m H I M fragment (Pearson et al., 1983). In this article, we present the first mAb, designated H250, developed to 75-kDa protein. This mAb identifies the protein as a viral capsid antigen (VCA).

a

BL CS DAB

= Burkitt l y m p h o m a . = culture supernatant. = 3,3'-diaminobenzidine (tetrahydro chloride dihydrate). EA = early antigen. EAD = d i f f u s e c o m p o n e n t s o f the E A complex. EAR = restricted c o m p o n e n t s of the E A complex. E B N A = EB nuclear antigen. EBV = Epstein-Barr virus. gp = glycoprotein. IEM = i m m u n e electron microscopy. IIF = indirect immunofluorescence. IM = infectious mononucleosis.

kDa MA mAb MW NPC p PAA PAGE PVDF s.c. SDS TPA VCA vp

= = = = = = = = = = = = = =

ki l oda l t on. m e m b r a n e antigen. m o n o c l o n a l antibody. mol e c ul a r weight. n a s o p h a r y n g e a l carcinoma. protein. p h o s p h o n o a c e t i c acid. p o l y a c r y l a m i d e gel electrophoresis. polyvinylidene difluoride. subcutaneous(ly). s odi um dodecyl sulphate. 12-0-tetradecanoyl phorbol-13-acetate. viral capsid antigen. viral protein.

A 75-kDa E P S T E I N - B A R R C A P S I D P R O T E I N

533

MATERIALS AND METHODS Cell lines. All cell lines were described by M. Boudouma et al., 1986. Six EBV-producer cell lines were used : 2 marmoset lymphoblastoid cell lines (LCL) B95.8, M81 originating from lymphocytes of patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC) respectively, 3 human spontaneous LCL Ly72 derived from B lymphocytes present in the tumour of a patient with NPC, and RI and CA, obtained after culture of peripheral lymphocytes from a patient with IM and a patient who had received a kidney transplant, respectively; and 1 human lymphomatous cell line P3HR1, a subclone of the Jijoye line which originated from a Burkitt's lymphoma biopsy. Three cell lines were used as negative controls for the selection of hybridomaproducing Ab directed against EBV : these included 1 human EBV-genome-positive non-producer Raji cell line derived from BL, and 2 EBV-genome-negative cell lines • B JAB, a human cell line derived from BL and 1022, a herpes ateles-producer marmoset cell line. Virus production. The B95.8 producer cell line was induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) (Zur Hausen et al., 1978) for 3 days at 20 ng/ml for a cell density of 5 × 105 cell/ml. The culture supernatant contained the enveloped and transforming virus. This liquid, filtered on 0.45 Izm, was used for the test of transformation inhibition of cord blood lymphocytes by the mAb. VCA production of these cells was monitored by indirect immunofluorescence (IIF). Naked virus was produced from nuclei of 109 cells. Cells were washed twice in PBS, then diluted in hypotonic buffer (50 mM NaCI, 20 mM Tris HCI pH 7.4, 4 mM MgC12, 1 mM Na3 EDTA) for 15 min at 4°C. They were then disrupted in a "Dounce" homogenizer. The nuclei were brought to a concentration of 10S/ml and purified by passage in "Ficoll 400" (Pharmacia) (1/3 Ficoll 400 for 2/3 suspension containing the nuclei) for 15 min at 1,800 rpm. The pellet was recovered with 1 ml PBS and the virus obtained by two successive freezings and thawings. Two clarifications were then carried out: the first at 3,000 rpm for 5 min and the second at 10,000 rpm for 10 min at 4°C. The presence of the virus was monitored by electron microscopy. This virus suspension was used for mouse immunization, agglutination tests and immunoblotting. Phosphonoacetic acid treatment. Phosphonoacetic acid (PAA) reversibly inhibits the production of late proteins by interfering with the viral DNA polymerase which allows replication of viral DNA (Nyormoi et al., 1976). The use of PAA thus indicated whether the Ag recognized by the mAb could be assigned to the early or late phase of the viral replication cycle. To this end, the mAb was tested by IF on B95.8 cells activated with T P A and simultaneously treated with 200 ~tg/ml PAA for 4 days. Two human sera, one with anti-VCA antibodies (titre 320), the other with anti-VCA and anti-EA antibodies (titres 640 and 320, respectively) and a mAb corresponding to the major late glycoprotein of the envelope, gp 350/250 (Biorad), were used as controls for the reaction. The absence of production of late proteins was monitered by IIF by comparison with cells treated with TPA and PAA, and control cells treated with T P A alone.

534

A. SANCHEZ-PINEL

ET AL.

Treatment with tunicamycin. Tunicamycin inhibits fixation of the oligosaccharide fragment to the asparagine residue of the glycoprotein (Heifetz et al., 1979). After treatment of the B95.8 cells with tunicamycin, the viral glycoproteins lose their N-glycosylated radical and the enveloped virus loses its transforming power (Hutt-Fletcher et al., 1986). B95.8 cells were washed, adjusted to 2 × 106 cell/ml and tunicamycin was added at 5 ~tg/ml of culture medium. The cells were incubated for 3 h at 37°C in the presence of 5 % CO2. Twenty ng/ml of TPA were then added. Three days later, the B95.8 ceils were tested by IIF with mAb H250 and the reference mAb which recognized the gp 350/250 mAb. A transformation test was carried out with the culture supernatants of these cells. The negativation of IF with the 350/250 mAb and the disappearance of the transforming power of the enveloped virus showed the action of tunicamycin.

Production of mAb H250. Several BALB/c mice were given a subcutaneous (s.c.) injection of 150 ~tl of nonsonicated naked virus + 150 g.l of complete Freund's adjuvant, followed by 4 s.c. injections of 250 ~tl of sonicated naked virus at 2-week intervals and, on the day before fusion, an i.v. injection of 100 ~tl of non-sonicated naked virus. Fusion was carried out with Sp2/0-Agl4 cells (Shulman et al., 1978). Positive hybridomas were selected and cloned according to the techniques described by Boudouma et al., 1986. In our case, however, after fusion, the cells were distributed in 96-well microtitre plates containing 5 x 104 celIs/per well. Massive production of Ab in the form of ascitic fluid was obtained after injection of hybrid cells in mice treated with 300 g.1 of pristane for 6 days. The mAb subclass was determined from concentrated culture supernatant by radial immunodiffusion with defined Ab against various mouse immunoglobulin isotypes. H250, an IgM, was purified from ascites liquid according to the method described by Garcia-Gonzalez et aL (1988). Briefly, the ascitic fluid was twice dialysed gainst water and then resuspended in 0.1 M Tris HCI, 1 M NaCI pH 8.

Immunofluorescence assays. The mAb was characterized by immunofluorescence assays (Boudouma et al., 1986). Three tests were carried out on fixed cells: for EBNA on Raji cells, for EA on induced Raji cells and for VCA and MA on B95.8 cells and on all producer cell lines. One test was carried out on live B95.8 ceils, for MA.

Naked virus agglutination and immune electron microscopy (IEM). A total of 100 ~zlof culture supernatant (CS) of the hybridoma was added to 100 ~tl of the suspension of naked virus and left for 1 h at 37°C. Two negative controls were carried out, one with RPMI replacing the CS and the other with mAb H140 corresponding to a 56-kDa membrane antigen (Boudouma et aL, 1986). Agglutination and non-agglutination of the naked virus by the mAb was observed by electron microscopy.

A 75-kDa E P S T E I N - B A R R

CAPSID PROTEIN

535

Inhibition of the biological activity of the virus. Inhibition o f transformation by the B95.8 virus. The B95.8 virus is able to transform cord blood B lymphocytes. It has previously been shown that certain mAb inhibit this transformation by preventing the virus from attaching to its receptor (Wells et al., 1982) or by inhibiting the fusion of EBV envelopes with cord blood.B-lymphocyte membranes (Miller and Hutt-Fletcher, 1988). The present test was carried out as previously described (Boudouma et al., 1986) and with clarified diluted ascitic fluid. Inhibition o f EA induction by the P3HR1 virus. P3HRl-superinfected Raji cells express EA. This property of the P3HR1 virus was inhibited by Ab directed against viral envelope Ag. We carried out this test with mAb H250 using clarified diluted ascitic fluid. The techniques used were those described by Boudouma et al., 1986.

Blocking tests. The presence of a human Ab similar to mAb in patient sera was shown by IIF in a blocking test of mAb fluorescence by human Ab. A total of 73 serums were tested: 22 primary infections, 31 active post-primary infections, 14 latent infections and 6 EBV-negative sera. These EBV serological profiles and the technique we used have been previously described by Boudouma et al. (1986).

Western blot. Naked virus suspension (10 ~tl) diluted with 10 ~tl of Laemmli's SDS buffer and boiled for 3 min were analysed by SDS-PAGE on a 5-to-15 % polyacrylamide gradient (Laemmli, 1970). Transfer to the polyvinylidene difluoride (PVDF) membrane was carried out overnight. The membrane was then saturated in a solution of PBS containing 5 % skim milk, washed in PBS containing 0.2~% Tween-20 and incubated with purified mAb H250 for 12 h at 4°C or 1 h at 37°C. The membrane was washed 4 times for 5 min in PBS + 0.2 % Tween-20, then put in contact with peroxidase-labelled anti-mouse IgM (Immunotech), again washed 4 times and revealed with a solution containing 0.5 mg/ml DAB Tris HC1 50 mM, pH 7.5 and 10 ml H202 at 10 vol/ml.

RESULTS

Characterization of mAb by immunofluorescence. During screening, m A b H 2 5 0 s h o w e d positive cytoplasmic fluorescence on B95.8 cells whether activated by T P A or n o t (fig. 1) and negative fluorescence on the n o n - p r o d u c e r Raji cell line, the induced Raji cell line and EBV-negative B J A and 1022 cell lines. A n t i b o d y titre was 40 for culture supernatant and 5120 for clarified ascitic fluid. This A b also p r o d u c e d fluorescence with all p r o d u c e r cell lines tested whatever their origin. These results thus indicate that m A b H 2 5 0 recognized a viral antigen present in cells p r o d u c i n g

536

A. SANCHEZ-PINEL

ET AL.

a virus derived from IM, BL, NPC and from a renal transplant recipient. No fluorescence was seen in IIF carried out on live cells. This indicated that H250 was not directed against an MA.

Blocking tests. Results of blocking of H250 antibody fluorescence by human sera are shown in table I. Most primary infections did not block the fluorescence of mAb H250; there were 5 positives out of 22. All post-primary sera with active or latent profiles blocked mAb H250. Six negative sera were tested and none of them blocked the mAb H250.

Localization of the antigen corresponding to mAb H250 in the viral cycle. When B95.8 cells were activated with TPA, the percentages of positive cells were 15 to 18 % and 18 to 20 % with reference human sera possessing anti-VCA antibodies and a n t i - V C A / E A antibodies, respectively, and 15 % with mAb H250. When B95.8 cells were activated with T P A and treated with PAA, 3 to 5 % of ceils showed fluorescence of an EA type with the reference human serum possessing anti-VCA/EA antibodies. Reference h u m a n sera possessing anti-VCA antibodies and mAb H250 showed no fluorescence ( < 1 % cells), indicating that mAb H250 was directed against a late Ag of the virus replication cycle of EBV.

FIG. 1. --Immunofluorescencestaining ofB95.8 cells with mAb H250. Magnificationx 3,000. Note small cytoplasmicinclusion staining.

A

75-kDa EPSTEIN-BARR

CAPSID

537

PROTEIN

Protein or glycoprotein nature of Ag recognized by m A b H250. Cells o f the B95.8 line were t r e a t e d with 5 g.g/ml t u n i c a m y c i n for 3 days a n d s i m u l t a n e o u s l y activated with T P A . A c o n t r o l slide w i t h o u t t u n i c a m y c i n was also tested. The reference mAb directed against membrane gp 350/250 s h o w e d n o f l u o r e s c e n c e o n cells t r e a t e d w i t h t u n i c a m y c i n , w h e r e a s it w a s p o s i t i v e o n u n t r e a t e d cells. T h u s , t u n i c a m y c i n d i d i n d e e d i n h i b i t N - g l y c o sylation of the viral gp. MAb H250 showed the same fluorescence and the s a m e p e r c e n t a g e o f p o s i t i v e cells o n t u n i c a m y c i n - t r e a t e d a n d u n t r e a t e d cells. This indicated that H250 was directed against a viral antigen without an N-glycosylated group, which could favour the hypothesis of the protein nature of this antigen.

TABLE I. - - Serological profiles and blocking tests.

IF VCA

IF VCA

titre

Blocking

22 P I : 40 160 20 40 160 640 80 40 160 160 320 10 320 40 40 160 160 40 40 40 320 40

+ + + + + -

titre 31AI: 23various diseases 640 640 320 160 640 160 640 1980 2560 2560 640 1280 320 320 320 160 160 160 160 1280 160 320 320

IF VCA Blocking

+ + + + + + + + + + + + + + + + + + + + + + +

titre 6 AIDS 640 80 160 160 160 160 2 BL 160 640

Blocking

+ + + + + + + +

14 LI: 40 160 40 40 40 20 80 160 20 10 40 20 20 40

+ + + + + +

+ + + + + + + +

6 EBV-: < 10 Serologicalprofiles: PI = primary infection; AI = active infection; LI = latent infection; BL = Burkitt's lymphoma; AIDS and EBV. -corresponds to human sera without blocking activity for H250. + corresponds to human sera with blocking activity for H250. VCA IF titres demonstrate that blocking activity does not depend on VCA titres.

538

A. SANCHEZ-PINEL E T AL.

Biological properties of H250. The biological tests carried out were negative. H250 did not inhibit transformation of cord blood lymphocytes by the B95.8 virus whether used pure or diluted, nor did it inhibit EA induction by P3HR1 whatever the dilution.

Agglutination test of naked virus by H250 and IEM. Images of agglutination of naked particles were observed by electron microscopy. Nine agglutinated particles are shown in figure 2. Rare clumps of particles observed in controls did not present characteristics of agglutination by the Ag-Ab reaction (fig. 3) and no more than three particles were ever spontaneously agglutinated. On the other hand, using mAb H250, we were able to count 9 to 50 agglutinated particles. These images show that H250 recognized an antigen present on the capsid of the EBV.

2 FIG. 2. - - Agglutination of naked viral particles by mAb 1-1250. Magnification x 120,000; bar corresponds to 0.1 ~tM.

FIG. 3. - - Negative control of naked viral particle agglutination. Magnification x 120,000; bar corresponds to 0.1 g.M. Intervals between viral particles are shorter than in immune agglutination.

A 75-kDa E P S T E I N - B A R R C A P S I D P R O T E I N

539

Determination of the molecular weight of the Ag corresponding to mAb H250 by immunoblotting. The molecular weight o f the protein recognized by H250 was obtained from the same suspension o f n a k e d virus as t h a t which enabled us to carry out agglutination tests. A control preparation was m a d e using the cells o f the nonp r o d u c e r Raji line treated according to the same p r o t o c o l as B95.8 cells. A 75-kDa b a n d appeared only with the suspension o f n a k e d virus obtained f r o m B95.8 cells (fig. 4).

t~. {-

94--

7567-

(

r£

:

43--

MW(kd)

].

2

FIG. 4. -- Determination by immunoblotting with mAb H250 of molecular weight of corresponding antigen. A band was found et 75 kDa when B95.8 naked viral particles were used as source of antigen (lane 1). No band was found when Raji cells were used as source of antigen (lane 2).

540

A. SANCHEZ-PINEL

ET AL.

DISCUSSION The use of mAb for the study of EBV proteins has made it possible to purify and characterize certain viral antigens of infected or EBV-transformed ceils (Mann et al., 1985). However, few publications have dealt with the antigenic structure of the virion itself. The EBV capsid appears to be composed of seven proteins (Diller and Kallin, 1988) ; two of these, gp 125 and p 160, have been characterized using mAb. In our study, after immunization of BALB/c mice with naked particles, we obtained a hybridoma producing an anti-EBV IgM H250, probably directed against a capsid antigen. The biological tests carried out using mAb H250 indicate that the Ag it recognizes is not an envelope antigen. In fact, whatever the dilution used, mAb H250 did not inhibit the transformation of cord blood lymphocytes by B95.8 EBV and did not inhibit EA induction in Raji cells by PBHRI EBV. Also, no fluorescence was observed on live B95.8 cells. Thus, mAb H250 does not recognize a protein of the MA complex. Moreover, no fluorescence was observed on induced Raji cells, and the Ag recognized by mAb H250 was PAA-sensitive. It thus belongs to the late phase of the EBV virus replication cycle. The molecular weight of the Ag recognized by mAb H250 obtained by immunoblotting from naked particles was 75 kDa. These results, taken overall, and agglutination of naked particles by mAb H250 as seen in electron microscopy, make it possible to affirm that mAb H250 recognizes a 75-kDa VCA. This Ag may be similar to vp 15-16 (MW 77-78) described by Dolynluk et al. (1976b), who purified the enveloped virus and the naked virus from the B95.8 cell line and mapped the structural proteins of this virus. A 75-kDa protein was also described by Kawanishi et al. (1981) by immunoprecipitation with human sera from P3HRI ceils treated with N-butyrate. According to Kawanishi, the 75-kDa protein corresponds to vp 15-16. Dolyniuk found no glycoprotein among the VCA. This appears to be in agreement with our results; indeed, the Ag recognized by mAb H250 was not sensitive to tunicamycin, which could attest to the protein nature of this Ag. However, this test carried out by IIF was not entirely conclusive, as mAb H250 may have recognized the protein fraction in the glycoprotein. Confirmation is thus necessary: we intend to purify the naked virus from tunicamycin-treated cells in order to perform immunoblotting and determine the MW of this protein in the presence of tunicamycin. If we again obtain a 75-kDa band, we will be able to confirm that the Ag recognized by mAb H250, is not N-glycosylated. Positive fluorescence obtained with mAb H250 on all producer cell lines tested, and blocking of fluorescence of mAb H250 by the majority of human sera tested containing anti-VCA, provide further evidence that this mAb does indeed recognize an EBV antigen. Non-blocking sera originated only from cases of primary infections. This result is in favour of the late appearance

A 75-kDa E P S T E I N - B A R R C A P S I D P R O T E I N

541

of anti-p75 Ab during primary infection. Thus, immunologically, p75 seems to behave like the VCA-160 protein. In fact, Pearson (1985) observed a difference in the timing of the appearance of anti-VCA Ab during IM, and protein 160 (Luka et al., 1984) was immunologically recognized later than gp 125 during primary infection. Results of blocking tests must be checked by immunoblotting and ELISA assays with purified p75. Purification tests by affinity chromatography are in progress. In conclusion, using a mAb designated H250, we characterized a new EBV capsid Ag of 75 kDa which was different from the two EBV capsid Ag already described (p160 and gp 125). p75 seems to correspond to vp 15-16 present in the electrophoregram of EBV proteins carried out by Dolyniuk, and to p75 described by Kawanishi.

RI~$UMI~ CARACTI~RISATION D'UNE PROTI~INE DE LA CAPSIDE DU VIRUS EPSTEIN-BARR ( E B V ) DE 75 k D a A L'AIDE D'UN NOUVEL ANT1CORPS MONOCLONAL H 2 5 0

Nous avons obtenu apr6s immunisation de souris BALB/c avec des particules virales nues provenant de la lign6e productrice B95.8 un anticorps monoclonal (AcM) H250 dirig6 contre un antigone de la capside de I'EBV (virus Epstein-Barr). Cet Ag est pr6sent dans les lign6es productrices B95.8, P3HR1, Ly72, M81, RI et CA, et il est absent dans les lign6es non productrices BJA, RAJI, 1022. L'AcM H250 n'inhibe pas la transformation des lymphocytes du sang de cordon par le virus B95.8 et n'inhibe pas l'induction de I'EA (antigOne pr6coce) sur cellules Raji par le virus P3HR1. De plus, rAcM H250 ne pr6sente aucune fluorescence sur cellules B95.8 vivantes. Cela indique que I'AcM H250 ne reconna~t pas un antigone de membrane. En immunofluorescence indirecte, aucune fluorescence n'est observ6e sur eellules Raji induites, ni sur cellules B95.8 tralt6es par l'acide phosphoac6tique; donc I'AcM H250 reconna~t un antigone tardif du cycle viral de I'EBV. L'agglutination du virus nu par I'AcM-H250 d6montre que celui-ci est dirig6 contre un antig6ne de la capside. Une fluorescence positive a 6t6 observ6e sur cellules tralt6es par la tunicamycine, indiquant que I'AcM H250 reconna~t une prot6ine. Le poids mol6culaire de cette prot6ine a 6t6 obtenu par Western-blot, et il est approximativement de 75 kDa. Les tests de blocage effectu6s avec I'AcM H250 semblent d6montrer que cet anticorps appara~t tardivement dans le s6rum de malades au tours de la primoinfection. MOTS-CLI~S: EBV, Capside, AntigOne prot6ique, AcM H250; Caract&isation, Pathogen~se.

ACKNOWLEDGEMENTS

We are grateful to the Clonatec Society for generously providing us with their IgM purification technique. We thank L. Lapchine for illustrations used in this manuscript. We also thank P. Jullia and A. Rouquayrol for their excellent technical assistance.

A. SANCHEZ-PINEL

542

ET AL.

REFERENCES

BALACHANDRAN,N., PITTARI, J. & HUTT-FLETCHER, L.M. (1986), Detection by monoclonal antibodies of an early membrane protein induced by EpsteinBarr virus. J. Virol., 60, 369-375. BOUDOUMA, M., BERNAD, J., PINEL, A., ICART, J., RIVES, H., ABBAL, M., DEWlLDE,A. & DIDIER,J. (1986), Characterization of an Epstein-Barr virus membrane antigen (56/52 Kd) by a monoclonal antibody with neutralizing properties. Ann. Virol. (Inst. Pasteur), 137E, 369-379. DILLNER, J. & KALLIN,B. (1988), The Epstein-Barr virus proteins. Advanc. Cancer Res., 50, 95-158. DOLYNIUK, M., PRITCHETr, R. & KIEFF, E. (1976a), Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus. J. Virol., 17, 935-949. DOLYNIUK,M., WOLFF,E. & KIEFF, E. (1976b), Proteins of Epstein-Barr virus. - II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine and polysaccharide containing components of enveloped virus. J. Virol., 18, 289-297. EPSTEIN, A.L. (1984), Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells. J. Virol., 50, 372-379. GARCIA-GONZALEZ,M., BETTINGER,S., OTT, S., OLIVIER, P., KADOUCHE,J. & POULEa--rv, P. (1988), Purification of murine IgG3 and IgM monoclonal antibodies by euglobulin precipitation. J. Immunol. Methods, 111, 17-23. HEARING, J.C., LEWIS,A. & LEVlNE,A.J. (1985), Structure of the Epstein-Barr virus nuclear antigen as probed with monoclonai antibody. Virology, 142, 215-220. HEIFETZ, A., KEENAN, R.W. & ELBEIN, A.D. (1979), Mechanism of action of tunicamycin on the UDP-GIcNac: dolichylphosphate GlcNac-l-phosphate transferase. Biochemistry, 18, 2186-2192. HEINEMAN, T., GONG, M., SAMPLE, J. & KIEFF, E. (1988), Identification of the Epstein-Barr virus gp85 gene. J. Virol., 62, 1101-1107. HOFFMAN, G.J., LAZABOWlTZ,S.G. & HAYWARD,S.D. (1980), Monoclonal antibody against a 250,000 dalton glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen. Proc. nat. Acad. Sci. (Wash.), 77, 2979-2983. HUMMEL,M., THORLEY-LAWSON,D. & KIEFF, E. (1984), An Epstein-Barr virus DNA fragment encodes messages for the two major envelope glycoproteins (gp 350/300 and gp 220/200). J. Virol., 49, 413-417. HUTT-FLETCHER,L.M., BALACHANDRAN,N. & LE BLANC,P.A. (1986), Modification of Epstein-Barr virus replication by tunicamycin. J. Virol., 57, 117-123. KAWANISHI,M., SUGAWARA,K. & ITO, Y. (1981), Epstein-Barr virus-induced polypeptides: a comparative study with superinfected Raji, IUdR-treated, and N-butyrate-treated P3HR1 cells. Virology, 109, 72-81. KISHISHITA,M., LOKA,J., VROMAN,B., PODUSLO,J.F. & PEARSON,G.R. (1984), Production of monoclonal antibody to a late intracellular Epstein-Barr virusinduced antigen. Virology, 133, 363-375. LAEMMLI,O.K. (1970), Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-685. LUKA, J., CHASE, R.C. & PEARSON, G.R. (1984), A sensitive enzyme-linked immunosorbent assay (ELISA) against the major EBV-associated antigens. - I. Correlation between ELISA and immunofluorescencetiters using purified antigens. J. ImmunoL Methods, 67, 145-156. LUKA, J., MILLER, G., JORNVALL,H. & PEARSON,G.R. (1986), Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein. J. Virol., 58, 748-756. -

-

A 75-kDa E P S T E I N - B A R R

CAPSID PROTEIN

543

MANN, K.P., STAUNTON,D. & THORLEY-LAWSON,D.A. (1985), Epstein-Barr virusencoded protein found in plasma membranes of transformed cells. J. ViroL, 55, 710-720. MILLER, N. (~¢ HUTT-FLETCHER,L.M. (1988), A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus. J. Virol., 62, 2366-2372. MUELLER-LANTZSCH, N., GEORO-FRIES, B., HERBST, H., ZUR HAUSEN, H. & BRAUN, D.G. (1981), Epstein-Barr virus strain and group-specific antigenic determinants detected by monoclonal antibodies, lnt. J. Cancer, 28, 321-327. NYORMOI, O., THORLEY-LAWSON,D.A., ELKINGTON,J. & STROMINGER,J.L. (1976), Differential effect of phosphonoacetic acid on the expression of EpsteinBarr viral antigens and virus production. Proc. nat. Acad. Sci. (Wash.), 73, 1745-1748. PEARSON, G.R., VROMAN, B., CHASE, B., SCULLEY,T., HUMMEL) M. & KIEFF) E. (I 983), Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies. J. ViroL, 47) 193-201. PEARSON,G.R. (1985), Advances in the identification of EBV-specific proteins : an overview, in "Epstein°Barr virus and associated diseases" (P.H. Levine, D.V. Ablashi, G.R. Pearson, S.D. Kottaridis) (pp. 411-425). Martinus Nijhoff Publ., Boston. SAIKENJI) T., NGUYEN, Q.V.) WODA, B. & HUMPHREYS, R.E. (1987), Immune response to intermediate filament-associated Epstein-Barr virusqnduced early antigen. J. ImmunoL, 138, 2645-2652. SHULMAN) M.) WILDE) C.D. & KOHLER, G. (1978), A better cell line for making hybridomas secreting specific antibodies. Nature (Lond.), 276, 269-270. STRNAD, B.C., SCHUSTER, T.) KLEIN, R.) HOPKINS, R.F. III, WITMER, T., NEUBAUER,R.H. & RABIN)H. (1982), Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen. J. ViroL, 41, 258-264. TAKADA, K., FUJIWARA, S., YANO, S. & OSATO, W. (1983), Monoclonal antibody specific for capsid antigen of Epstein-Barr virus. Med. Microbiol. Immunol., 171, 225-231. THORLEY-LAWSON,D.A. & GEILINGER,K. (1980), Monoclonal antibodies against the major glycoprotein (gp 350/220) of Epstein-Barr virus neutralize infectivity. Proc. nat. Acad. Sci. (Wash.), 77, 5307-5311. UEN, W.C., LUKA, J. & PEARSON,G.R. (1988), Development of an enzyme-linked immunosorbent assay (ELISA) for detecting IgA antibodies to the EpsteinBarr virus. Int. J. Cancer, 41, 479-482. VROMAN,B., LUKA, J., RODRIGUEZ,M. & PEARSON,G.R. (1985), Characterization of a major protein with a molecular weight of 160.000 associated with the viral capsid of Epstein-Barr virus. J. Virol., 53, 107-113. WELLS,A.) KOtDE,N. 8(; KLEIN, G. (1982), Two large virion envelope glycoproteins mediate Epstein-Barr virus binding to receptor-positive cells. J. Virol., 41, 286-297. ZUR HAUSEN, H., O'NEILL, F.J. & FREESE, U.K. (1978), Persisting oncogenic herpesvirus induced by the tumour promoter TPA. Nature (Lond.), 272, 373-375.