Introduction
The Epstein-Barr virus (EBV) is endemic in human populations and is normally carried as a persistent, asymptomatic infection in the immunocompetent host. However, when primary infection is delayed until adolescence or beyond, EBV can cause the lymphoproliferative disorder infectious mononucleosis (IM). EBV is strongly associated with the development of B cell lymphomas in immunocompromised hosts, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma (reviewed in Miller, 1990 and Rickinson, 1994) . More recently, EBV has been implicated in the development of other malignancies including some types of Hodgkin's disease, T cell lymphomas and carcinomas (Shibata et al., 1991; Rowlands et al., 1993; Pallesen et al., 1993) , suggesting that EBV can infect several different cell types in vivo. In vitro, EBV replication has been studied almost exclusively in lymphocytes and their derived cell lines due to difficulties in establishing suitable * Author for correspondence. Fax 646.837.3107. e-mail rachelwalker@example.net epithelial cell cultures. Studies designed to begin characterization of EBV replication in epithelial cells in vivo have been carried out using oral hairy leukaplakia (OHL) biopsies (Lau et al., 1993) . No major differences in EBV late gene expression in OHL cells and B95-8 cells have so far been determined, but it remains possible that some EBV-encoded genes are cell-type specific.
Introduction
All herpesviruses have a membrane-derived outer envelope containing several virus-encoded glycoproteins which have been shown to play important roles in the interaction between the free virion and its target cell, and in particular they are involved in the initial binding of the virion to the cell surface and the subsequent fusion of viral and cell membranes. Some viral glycoproteins have been studied in detail because of their ability to induce protective immune responses, making them attractive candidates for inclusion in vaccine development.
Introduction
By comparison with EBV, the a-herpesvirus herpes simplex type 1 (HSV-1) has been relatively well studied and to date, it has been shown to encode 11 antigenically distinct glycoproteins, of which five (gB, gD, gH, gK and gL) are essential for virus replication in cultured cells (reviewed in Roizman & Sears, 1993) . EBV contains a predominant envelope glycoprotein, gp340/220, which is 0001-3020 © 1995 SGM encoded by the BLLF1 gene and has a short region of homology with HSV-1 gC (Beisel et al., 1985; Herrold et al., 1991) . Both gp340 and gp220 contain a long serineand threonine-rich external domain, a single transmembrane domain and a short carboxy-terminal cytoplasmic domain. The relative molecular mass of nascent gp340 is approximately 135 kDa with the remaining mass represented by a large amount of both N-and Olinked sugars. Gp340/220 is responsible for the initial binding of the virus to the B lymphocyte cell-surface molecule CD21 (Fingeroth et al., 1984; Nemerow et al., 1987; Tanner et al., 1987 Tanner et al., , 1988 . Homologues of HSV-1 gH, gL and gB are found in all sub-groups of herpesviruses including EBV (Cranage et al., 1988; Yaswen et al., 1993; Emini et al., 1987) . Gp85, encoded by the BXLF2 gene (Heineman et al., 1988; Oba et al., 1988) and gp25, encoded by the BKRF2 gene are considered to be the gH and gL homologues respectively, and appear to form at least part of a complex which is necessary for fusion of EBV with the cell membrane (Miller & Hutt-Fletcher, 1988; Haddad & Hutt-Fletcher, 1989; Yaswen et al., 1993) . Further glycoprotein molecules may also be required for the fusion process (Shieh et al., 1992; Shieh & Spear, 1994; Yaswen et al., 1993) . The EBV gene product of BALF4, gp110, is the predicted homologue of HSV-1 gB (Pellet et al., 1985; Emini et al., 1987 ), but appears to have a different distribution in that it is restricted to the nuclear membranes and endoplasmic reticulum of EBV-infected cells (Gong et al., 1987; Gong & Kieff, 1990) . HSV-1 gB is an abundant glycoprotein found on the surface of the virion and is important for virus entry into the cell and cell-to-cell spread of the virus (Sarmiento et al., 1979; Cai et al., 1988; Navarro et al., 1992) , whereas the function of gpll0 is unknown. Two further glycoproteins, also with unknown functions, have previously been identified in EBV-infected cells. These are gp55/78, a heavily glycosylated protein with an apparent molecular mass of only 28 kDa in the absence of carbohydrate, encoded by the BILF20RF (Mackett et al., 1990) , and gp43, which was identified by a monoclonal antibody raised against purified EBV particles and has yet to be assigned to a particular ORF (Sanchez-Pinel et al., 1991) . The BZLF20RF of EBV codes for a 42 kDa glycoprotein which associates with the EBV gH-gL complex (L. Hutt-Fletcher, personal communication).
Introduction
The EBV ORF BDLF3 is also predicted to encode a glycoprotein on the basis that it contains a putative amino-terminal signal sequence, a carboxy-terminal transmembrane domain and nine potential N-linked glycosylation sites. The ORF contains 705 base pairs coding for 234 amino acids of which one-third are either serine or threonine suggesting that, like gp340, the protein may also be modified by O-linked glycosylation and/or phosphorylation. As yet there is no known sequential or positional homology between the BDLF3 sequence and any other herpesvirus ORF.
Introduction
This paper describes the identification and characterization of the BDLF3 gene product as it exists in EBVinfected B lymphoblastoid cells. In order to determine whether EBV-immune human sera contain antibodies to a BDLF3 protein product, recombinant forms of BDLF3-encoded protein were expressed as a fusion protein with glutathione S-transferase in E. coli cells, and as a full-length product in insect cells infected with a BDLF3 recombinant baculovirus. These products were then used in Western blots to demonstrate that over onethird of sera tested reacted with at least one of the recombinant BDLF3 proteins. We have also shown that polyclonal rabbit sera raised against a 20 amino acid peptide representing the carboxy-terminal sequence of BDLF3 reacts with a product in lysates of the EBVinfected lymphoblastoid cell line B95-8, both by Western blotting and by indirect immunofluorescence on acetonefixed cells. The relative molecular mass of this putative BDLF3 protein is between 100 and 150 kDa.
Methods
Cells. The Spodopterafrugiperda insect cell lines Sf9 and Sf21 were propagated as monolayer cultures at 27 °C in Graces Insect ceiI medium (Gibco) supplemented with 10 % fetal calf serum, 100 IU/ml penicillin and 100 ~tg/ml streptomycin, and passaged twice a week. The EBV-mfected B cell lines B95-8, P3HR-1, AG876 and Jijoye, and the EBV-negative B cell line EB4 were maintained at 37 °C in RPMI 1640 (Glbco) supplemented with 10% fetal calf serum, 100 IU penicillin, 100 lag/ml streptomycin and 2 mM-glutamine and passaged once or twice per week.
Source of BDLF3 sequence and cloning into pBlueseript H KS(-).
The complete BDLF3 sequence is contained within the EcoRI E fragment of the B95-8 strain of EBV. A small sample of pBR322 containing EcoRI E was kindly provided by Dr P. J. Farrell (Ludwig Institute for Cancer Research, London, UK). Digestion of this plasmid with StuI removes a 997 bp fragment and contains the complete BDLF3 ORF beginning 19bp upstream of the start codon. Linkers containing a XbaI restriction site [Stratagene #1081 d(pGCTCTAGAGC) ] were ligated to this fragment which was then inserted into the Xbal site of pBluescript II KS(-) (Stratagene) to make pBS-BDLF3. Standard molecular cloning techniques were used throughout as described in Sambrook et al. (1989) .
Source of BDLF3 sequence and cloning into pBlueseript H KS(-).
In vitro translation of BDLF3. pBS-BDLF3 described above contains the BDLF3 sequence downstream of the bacteriophage T7 RNA polymerase promoter and was used with the TNT T7 coupled reticulocyte lysate system (Promega) for in vitro [aSS] methioninelabelled translation of BDLF both with and without canine pancreatic microsomal membranes. The manufacturer's protocols were followed and after incubation of the reaction mixtures for 90 min at 30 °C; the synthesized proteins were separated by SDS~PAGE. The gels were fixed in 10% methanol-10% acetic acid, washed, soaked in 0.8 Msalicylate for fluorography, dried and exposed directly to X-ray film (Hyperfilm-MP, Amersham) for 4-5 h at -70 °C.
Source of BDLF3 sequence and cloning into pBlueseript H KS(-).
Immunoprecipitation of in vitro translated proteins. Rabbit and human sera were used to immunoprecipitate the in vitro translated BDLF3 products as described in Heineman et al. (1988) . Samples (5 gl) of the reaction mixtures were diluted to 0.5 ml in PBS containing 0.5 % Triton X-100, 1% BSA and 1 mM-PMSF. Samples were incubated on ice for 30 min before addition of 5 td serum and overnight incubation at 4 °C. Next, 50 lal of a 1 : 1 slurry of Protein A~Sepharose CL-4B (Sigma) in PBS was added to each sample for 2 h followed by washing the beads five times in PBS containing 0"5% Triton X-100 and 1 rnM-PMSF, and once in 10 mM-Tris-HC1 pH 8.0, 1 mM-EDTA. SDS-PAGE sample buffer (25 gl) was added before boiling the samples for 3 rain and separation of the released proteins by SDS-PAGE.
Production of a glutathione S-transferase (GST)-BDLF3 fusion protein.
PCR was used to amplify the region of BDLF3 found between the putative signal sequence and transmernbrane domains. Primers were designed to incorporate BamHI and EcoRI sites for unidirectional ligation into pGEX-3X (Pharmacia). The oligonucleotide sequences used (5'-3') were TGTGGATCCCATCTAGTGGCAGTTCG and AGAGAATTCCCAGAGAGGAAGACCGT (restriction enzyme sites underlined). The presence of an in-frame BDLF3 sequence in pGEX-BDLF3 was confirmed by sequencing across the GST/BDLF3 junction using the GEX-3X forward primer GCATGGCCTTTGC-AGGG (5'-3'). Purification of GST-BDLF3 fusion protein and GST alone was carried out from 1 litre cultures of E. colt cells transformed with either pGEX-BDLF3 or pGEX-3X and induced with 0.i mM-IPTG for 5 h (Smith & Johnson, 1988) . The cells were lysed using a probe sonicator, and after pelleting cell debris, the supernatants were passed over pre-packed glutathione 4B columns (Pharmacia), and bound proteins were eluted using 5 mM-glutathione in Tris-HC1 (pH 8.0). The fractions were analysed by SDS-PAGE and stored at -70 °C until required.
Production of a glutathione S-transferase (GST)-BDLF3 fusion protein.
Western blotting. Proteins were separated on SDS-polyacrylamide gels using the Bio-Rad Mini-Protean II apparatus and transferred to nitrocellulose paper for 3 h at 100 V. The blots were stained in Ponceau S (Sigma) to check transfer and protein loading before destaining and blocking in 5 % milk buffer (MB) and overnight incubation at 4 °C with the primary antibody diluted in MB. The blots were washed and incubated in the appropriate alkaline-phosphatase conjugated anti-IgG in MB for 1 h at room temperature, and developed in Fast Blue/ctnaphthyl phosphate substrate (Sigma).
Preparation of polyclonal antisera in rabbits.
Purified GST-BDLF3 fusion protein or GST alone (90 gg) prepared as an emulsion in complete Freund's adjuvant was injected subcutaneously at four different sites in New Zealand White rabbits. Three subsequent doses were given in incomplete Freund's adjuvant. The animals were bled 10 days after each injection and final bleed-out sera were taken 12 weeks after the first injection. Sera were tested for reactivity to GST-BDLF3 fusion protein, GST alone and protein expressed from a BDLF3 recombinant baculovirus (described below) by Western blotting. Serum 8382 is reactive against the GST-BDLF3 fusion protein and serum 8029 is reactive against GST alone.
Preparation of polyclonal antisera in rabbits.
A peptide representing amino acids 215 to 234 from the carboxyterminus of the BDLF30RF was synthesized by Dr G. Bloomberg, Dept. of Biochemistry, University of Bristol, UK. A cysteine residue was attached to the amino-terminal end of the peptide to enable it to be coupled to carrier proteins resulting in a final sequence of -CDEALLTNAVTRDPSLYKGLV. The peptide (10 rag) was coupled to either keyhole limpet haemocyanin (KLH) or to bovine serum albumin (BSA) using a kit supplied by Immune Systems, UK and the linker sulphosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). The coupled peptide (0.8 nag) was used to immunize rabbits as described above followed by three subsequent doses of 0"4 mg/rabbit, before final bleed-out sera were taken. Sera 099 and 865 were raised against KLH-peptide and sera 310 and 535 were raised against BSA-peptide. Sera were screened against the peptide alone and both coupled peptides using a standard ELISA and tested for reactivity against BDLF3 recombinant baculovirus-expressed protein in Western blots.
Construction of a BDLF3 recombinant baculovirus.
The XbaI fragment containing BDLF3 from pBS-BDLF3 was used to construct a recombinant baculovirus transfer vector derived from pVL1393. pVL-BDLF3 contains the complete sequence for BDLF3 under the control of the polyhedrin gene promoter. Homologous recombination between pVL-BDLF3 and Pharmingen Baculogold linearized baculovirus DNA (AMS Biotechnology) took place after co-transfection of the insect cell line Spodopterafrugiperda Sf21, and virus stocks derived from single clones were obtained using the methods described in Summers & Smith (1987) . The recombinants were screened for the presence of BDLF3 DNA by Southern blotting using a radiolabelled BDLF3-specific probe, and one recombinant, AcBDLF3, was used in further studies.
Construction of a BDLF3 recombinant baculovirus.
Expression of recombinant BDLF3 protein in insect cells. Sf9 cells were infected at an m.o.i, of between 1 and 10 with BDLF3 recombinant baculovirus (AcBDLF3) or a recombinant baculovirus containing the fl-galactosidase gene (AcBg), or were mock-infected. After 72 h cells were harvested, washed in phosphate-buffered saline (PBS) and lysed in buffer containing 1% Triton X-100, 150 mM-NaC1, 50 mM-Tris-HC1 (pH 8.0) to release cytoplasmic and membrane-bound proteins. After centrifugation, the remaining pellets were lysed in the same buffer with 1% SDS to give a nuclear lysate. Protease inhibitors were routinely included in the lysis buffers used for preparing cell lysates and were added to a final concentration of 16 ~tg/ml benzarnidine. HC1, 10 gg/ml each of phenanthroline, aprotinin, leupeptin and pepstatin A, and I mM-PMSF. Aliquots derived from approximately 4 x 104 cells per lane were separated by SDS-PAGE and screened by rabbit and human sera in Western blots.
Construction of a BDLF3 recombinant baculovirus.
Indirect immunofluorescence. Individual layers of Sf9 cells were either infected with recombinant bactflovirus at an m.o.i, of 10 or were mockinfected. Three days post-infection the cells were harvested, washed and resuspended in PBS at a density of 2 × 106/ml. B cell lines were either left untreated (uninduced) or exposed to 20 ng/ml 12-tetradecanoyl phorbol-13-acetate (TPA) for 2-3 days to induce the production of EBV lytic cycle antigens (induced). The cells were harvested and resuspended as above. Suspensions were spotted onto multispot microscope slides (Hendley), air-dried and fixed for 10 min in 80% acetone. Rabbit anti-BDLF3 peptide sera were diluted 1 : 250 in PBS containing either 10 % normal goat serum (NGS) or 5 % fat-free milk powder, and incubated with the fixed cells for 1 h at 37 °C in a humidified box. The slides were washed two times in PBS and exposed to goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody (Sigma) at a dilution of 1 : 100 in blocking solution for 1 h at 37 °C. Cells were washed three times in PBS before mounting in either Citifluor (Agar) or Vectashield (Vector), observing under a fluorescence microscope and photography.
Construction of a BDLF3 recombinant baculovirus.
For two-colour immunofluorescence, fixed, TPA-induced B95-8 cells were incubated with a mixture of rabbit anti-BDLF3 peptide serum 099 and mouse monoclonal antibody F34-5H7 directed against gp340 (Qualtiere et al., 1987) , followed by a mixture of anti-rabbit FITCconjugated IgG1 and anti-mouse Texas Red-conjugated IgG2A (Southern Biotechnology Associates). Slides were viewed under a fluorescence microscope allowing the same field of cells to be observed and photographed in the presence of selective band filters.
Construction of a BDLF3 recombinant baculovirus.
Preparation of B cell lysates. Cells were pelleted at 4 °C and washed in PBS before resuspending in lysis buffer (1% Triton X-100, 140 mMNaC1, 10 mM-Tris-HC1 pH 7-5) and protease iuhibitors for 45 min on ice. Cell debris and nuclei were removed by centrifuging at 5000 r.p.m. for 15 min followed by 13000 r.p.m, for 20 rain in a Sigma 202MK microcentrifuge. Lysate derived from approximately 106 cells per lane was used in Western blots. B95-8-infected cell membranes and B95-8 virus preparations were made as described previously (Morgan et al., 1983; Epstein et al., 1986) . Membranes derived from approximately 10 ~ cells per lane were used on Western blots.
Immunoprecipitation from B cell lysates. Normal rabbit serum (NRS)
(20 ~tl/ml lysate) was added and samples incubated on ice for 1 h. After addition of 140 ~tl of 1 : 1 slurry of Protein A Sepharose in PBS, the samples were incubated for a further 1 h at 4 °C, centrifuged briefly, and the supernatants divided into 200 It aliquots. The samples were incubated overnight with 5-10 B1 test serum. Protein A-Sepharose in PBS (70 ~tl) was added for 1-2 h before washing the beads and elution of proteins for SDS-PAGE as described above.
Immunoprecipitation from B cell lysates. Normal rabbit serum (NRS)
Enzymatic deglycoo,lation. The methods used are described in Klupp et al. (1992 Klupp et al. ( , 1994 and all enzymes used were purchased from Boehringer Mannheim B95-8 virion preparations were centrifuged at full speed in a microcentrifuge for 30 rain and the resulting supernatants were discarded. For removal of terminal sialic acid residues, samples of virlons were incubated with 2 mU Vibrio cholerae neuraminidase in 50 mM-sodium acetate (pH 5.2)-4 mM-CaCI~ for 1 h at 37 °C. For subsequent removal of O-linked carbohydrates, de-sialated samples of virions were pelleted for 30 min and resuspended in 20 mM-Trisphosphate (pH 7.4), and incubated for 17 h at 37 °C with 1 mU BSAfree O-glycosidase. For removal of N-linked carbohydrates, samples of virions were resuspended in 10 BI 1% SDS for 3 min at 95 °C and then 90 ~tl 50 mM-potassmm phosphate-0.6 % CHAPS-25 mM-EDTA was added to give a final concentration of 0.1% SDS. N-Glycosidase F (2 U) was added before incubating the tube for 18 h at 37 °C. For each enzyme deglycosylation a sample of virlons was included which had been resuspended in buffer alone and incubated in otherwise identical conditions. SDS PAGE sample buffer was added to each tube and the samples were boiled for 5 min before separating the proteins on SDS-7.5 % polyacrylamlde gels and Western blotting.
Results

Recognition of recombinant BDLF3 protein by human EB V-immune sera
Recombinant BDLF3 protein was expressed initially in E. coli cells as a GST-BDLF3 fusion protein with a molecular mass of 48 kDa by SDS-PAGE. The fusion protein and GST alone were partially purified and used on Western blots for screening human sera for the presence of antibody directed against BDLF3 protein. A total of 62 available human sera were tested and those which reacted with the fusion protein but not GST alone were considered to be positive. Sera from five EBVnegative donors all failed to react to the fusion protein.
Recognition of recombinant BDLF3 protein by human EB V-immune sera
The test sera had been obtained from patients with EBVrelated disease as well as from apparently normal EBV sero-positive donors. The results are summarized in Table 1 , column (a) and examples of Western blots are shown in Fig. 1 . Over one-third of the human sera tested reacted with GST-BDLF3 protein on Western blots demonstrating that a BDLF3 product is expressed in vivo.
Recognition of recombinant BDLF3 protein by human EB V-immune sera
However, as the fusion protein represents only 68 % of the BDLF30RF and is not glycosylated in E. coli cells, it was decided to express a full-length recombinant BDLF3 protein using the baculovirus system. The plasmid pVL-BDLF3 was constructed as described in Methods and used to make the recombinant baculovirus AcBDLF3 which expresses BDLF3 protein under the control of the late polyhedrin promoter. The insect cell lines used, Sf21 and Sf9, are capable of glycosylating proteins and whilst this may be of a different pattern to that found in mammalian cells, recombinant proteins expressed in this system may attain a near native conformation.
Recognition of recombinant BDLF3 protein by human EB V-immune sera
The GST-BDLF3 fusion protein was used to raise polyclonal antiserum in rabbits (serum 8382) in order to screen for the presence of recombinant BDLF3 protein products in AcBDLF3-infected insect cell lysates by Western blot. Serum 8382 detected a series of specific heterogeneous products from AcBDLF3-infected cell lysates of apparent molecular masses 28-32 kDa and 50-60 kDa which were not present in AcBg-infected cell lysates or in mock-infected insect cell lysates (data not shown). Over one-half of the 42 human sera screened to recombinant baculovirus BDLF3 protein reacted with the lower molecular mass species on Western blots with a few of the sera also recognizing the higher molecular mass, glycosylated species (Table 1, column b; Fig. 1 ). Sera from EBV-negative donors failed to react to any of the recombinant baculovirus BDLF3 products. 
In vitro translation of BDLF3
pBS-BDLF3, which contains the BDLF3 sequence downstream of the T7 RNA polymerase promoter was used for the in vitro translation of BDLF3 protein. Two non-glycosylated products of apparent molecular mass 29 and 30 kDa by SDS-PAGE were obtained. After translation on canine pancreas microsomal membranes, an additional labelled species appeared as a 58 kDa band by SDS-PAGE, indicating that core glycosylation had occurred. No products were obtained from the parental pBluescript vector alone (Fig. 2a) . The proteins translated in the presence of microsomal membranes were HC is a human serum from a known EBV seronegative donor. Proteins were separated on SDS 12 % polyacrylamide gels, transferred to nitrocellulose and blocked in milk buffer before overnight incubation with human serum diluted in milk buffer. The blots were subsequently incubated with alkaline phosphatase-conjugated anti-human IgG and developed using Fast Blue/a-naphthyl phosphate substrate. The positions of molecular mass markers are indicated alongside. Lanes 1, GST-BDLF3 fusion (molecular mass 48 kDa); lanes 2, GST alone (molecular mass 27 kDa); lanes 3, AcBDLF3-infected insect cell cytoplasmic lysate; lanes 4, AcBDLF3-infected insect cell nuclear lysate; lanes 5, AcBg-infected total cell lysate; lanes 6, mock-infected insect cell lysate. incubated with human EBV-seropositive sera, with human EBV-seronegative sera, with rabbit anti-GST-BDLF3 fusion protein serum and with rabbit anti-GST serum. The human EBV-immune sera immunoprecipitated both non-glycosylated and glycosylated BDLF3 products, whereas rabbit anti-fusion protein serum only immunoprecipitated the non-glycosylated species. No products were immunoprecipitated by human EBVseronegative sera or by rabbit anti-GST serum (Fig. 2 b) . These data provide further evidence that human EBVimmune sera contain antibodies directed against BDLF3-encoded protein, and suggest that it is a glycoprotein.
Production of an anti-BDLF3 peptide serum
The antibodies raised against the G S T -B D L F 3 fusion protein do not recognize the glycosylated BDLF3 product when translated in vitro, and do not immunoprecipitate specific protein from EBV-infected lymphoblastoid cell lines. It was therefore decided to raise antibodies in rabbits against the carboxy-terminal peptide representing amino acids 215-234 of BDLF3. This region of the sequence is predicted to be the cytoplasmic domain of the protein and to be non-glycosylated. Peptides representing polypeptide chain termini have often been shown to induce antibodies which subsequently recognize the native protein (Friede et al., 1994) . The BDLF3 peptide was synthesized, coupled to either K L H or BSA and used to immunize four rabbits. Sera collected after the third boost were used for further analysis of the BDLF3 protein. All the anti-BDLF3 peptide sera demonstrated reactivity to the BDLF3 peptide by ELISA, and to recombinant baculovirus BDLF3 by Western blot (Fig. 3 a) . Furthermore, unlike the anti-fusion protein serum, anti-BDLF3 peptide sera were able to immunoprecipitate the glycosylated species after in vitro translation of the BDLF3 sequence on canine pancreas microsomal membranes. Pre-immune sera were negative in this respect (Fig. 3 b) .
Indirect immunofluorescence using anti-BDLF3 peptide sera
Because the anti-BDLF3 peptide sera is directed against the carboxy-terminus of the BDLF3 protein, and this is predicted to be the intracellular domain, indirect immunofluorescence was carried out on acetone-fixed cell preparations. The lymphoblastoid cell lines B95-8, P3HR-1, AG876 and Jijoye contain a few cells which show positive immunofluorescence after incubation with anti-BDLF3 peptide serum followed by an FITC 
(c-d)
17PA-induced, acetone-fixed B95-8 cells stained by two-colour immunofluorescence (c) for BDLF3 expression using anti-BDLF3 peptide serum 099 and FITC-conjugated IgG1, and (d) for gp340 expression using mouse MAb F345H7 and Texas Red-conjugated IgG2A. Cells that are positive for BDLF3
(c-d)
protein are also positive for gp340. Scale bars: 15 gm (a, b); 10 gm (e, d). (Fig. 4) . B95-8 cells were used in a twoc o l o u r i m m u n o f l u o r e s c e n c e assay to d e t e r m i n e that after T P A i n d u c t i o n , those cells which express the B D L F 3 p r o t e i n also express the late a n t i g e n gp340 (Fig. 4) .
7" 3"

L. A. Nolan and A. J. Morgan

Detection of BDLF3 protein in B95-8 cells by Western blot
The W e s t e r n blots were p e r f o r m e d by separating the proteins in S D S -9 % p o l y a c r y l a m i d e gels u n d e r r e d u c i n g conditions, t r a n s f e r r i n g to nitrocellulose a n d p r o b i n g with r a b b i t a n t i -B D L F 3 peptide sera at a 1:1000 dilution. B cell lysates were p r e p a r e d from b o t h uninduced and TPA-induced B95-8 cells and from the EBV-negative cell line EB4. Partially purified B95-8 virion and B95-8-infected cell membrane preparations were also lysed and included on the blots. The results obtained using the anti-carboxy-terminal peptide antiserum 099 are shown in Fig. 5(a) . A diffuse band or bands ranging in size from approximately 100-150 kDa was detected in all preparations derived from the B95-8 cell line; this band was not present with the EB4 cell line or detected by pre-immune sera. The large apparent molecular mass of this band suggests that the BDLF3 product is heavily glycosylated in vivo. Anti-BDLF3 peptide sera also immunoprecipitate this same band from both uninduced and induced B95-8 cells, with less intense staining in the lane containing uninduced cell lysate (Fig. 5b) . This result is consistent with that obtained by indirect immunofluorescence on fixed B95-8 cells. We were unable to detect a BDLF3 product from P3HR-1 and AG876 cell lysates by Western blotting which may indicate that BDLF3 protein is expressed at lower levels in these cell lines.
Deglycosylation of B95-8 BDLF3 protein
The apparent molecular mass of the native BDLF3 protein detected in B95-8 cells ranges from 100-150 kDa by SDS-PAGE, whereas the molecular mass of the unglycosylated polypeptide is only 29-31 kDa after in vitro translation. The BDLF3 sequence contains nine potential N-linked glycosylation sites (Asn-X-Ser/Thr) and 36 % of the total amino acid content is made up of serine and threonine residues, indicating that the polypeptide chain may also contain O-linked carbohydrate. We attempted to enzymatically remove terminal sialic acid residues, O-linked sugars and N-linked sugars from a preparation of B95-8 virus, and to analyse the resulting proteins by SDS-PAGE and Western blotting using anti-BDLF3 peptide serum diluted 1:1000 (Fig. 6) .
Deglycosylation of B95-8 BDLF3 protein
Removal of the sialic acid residues by neuraminidase resulted in an increase in the apparent molecular mass (lane 3) to a range of 140-175 kDa, due to the decrease in the overall negative charge of the glycoprotein. Sequential digestion with neuraminidase and Oglycosidase (lane 5) resulted in a small decrease in molecular mass to a range of 130-170 kDa compared with the values after neuraminidase treatment alone. Treatment with N-glycosidase F resulted in a decrease in the apparent molecular mass of the BDLF3 protein to a range of 82-119 kDa (lane 7). These data indicate that the BDLF3 protein from B95-8 virus is glycosylated.
Discussion
Analysis of the B95-8 virus genome predicted that the B D L F 3 0 R F would code for an integral membrane protein from an unspliced message. We have demonstrated the presence of a novel EBV glycoprotein in lysates of B95-8-infected cells by using anti-BDLF3 peptide serum. The major species of this glycoprotein ran in SDS-gels as a diffuse band with a molecular mass of 100-150 kDa. Two approaches were used to express recombinant BDLF3 products and the first was to express the sequence found between the putative signal and membrane anchor domains of BDLF3 as a GST fusion protein in E. coli cultures. Protein extracts from pGST-BDLF3 transformed cells were analysed by Western blot for specific recognition of the BDLF3 portion of the fusion protein using a panel of 62 human EBV-immune sera. Approximately one-third of these were found to contain antibodies directed against this BDLF3 product with the highest proportion of positive sera found in those taken from patients with undifferentiated nasopharyngeal carcinoma (NPC) and the lowest proportion in those taken from patients with acute infectious mononucleosis (IM). All known EBV-seronegative donors tested were negative in this respect. Bacterially expressed fusion proteins used in Western blot assays only present linear epitopes and are therefore of limited use in serological surveys implying that the actual numbers of EBV-immune sera reactive against native BDLF3 protein could be much higher. The true serological picture might be better assessed using BDLF3 protein presented in a more realistic conformational state.
Discussion
Therefore, the second approach taken towards the production of recombinant BDLF3 protein was to use the baculovirus expression system, which has been used previously to produce large quantities of recombinant protein including herpesvirus glycoproteins which, in some cases, were antigenically, immunologically and functionally similar to the respective native proteins (Luckow & Summers, 1988; Bishop, 1992; Ghiasi, 1994) . As no specific antibodies for detection of BDLF3 protein were available to us, polyclonal antisera were raised in rabbits against the GST-BDLF3 fusion protein and used to screen for the presence of BDLF3 protein in recombinant baculovirus using Western blots because the level of expression of recombinant BDLF3 protein was insufficient to allow identification by Coomassie blue staining. The reason for this is unknown but may be a function of BDLF3 itself through its effect upon other functions of the host insect cell. It has so far proved impossible to predict how well a particular protein will be synthesized using the baculovirus system (Bishop, 1992; King & Possee, 1992) but post-translational modification of a product can be rate-limiting. In addition, the recombinant BDLF3 protein is being expressed during the terminal phase of the insect cell lifecycle and consequently the processing of newly synthesized proteins is likely to become less and less efficient. Human EBV-immune sera but not EBVseronegative sera recognized recombinant baculovirus BDLF3 products by Western blot and were able to immunoprecipitate both unglycosylated and glycosylated BDLF3 products after translation in vitro. As the BDLF3 sequence contains nine potential N-linked glycosylation sites and a high percentage of threonine and serine residues, it might have been expected that the native protein would be heavily glycosylated via both N-and Olinkages, and that serum raised against the polypeptide backbone would not recognize the native protein.
Discussion
Another EBV glycoprotein, gp340/220, is extensively glycosylated but few if any neutralizing antibodies are directed against linear peptide epitopes in the amino acid backbone and therefore must be directed against discontinuous protein determinants or carbohydrate (Morgan et aL, 1984; Beisel et al., 1985; Pither et al., 1992a, b) . However, EBV-immune sera do contain some types of antibody directed against a few linear epitopes within gp340, as shown by the ability of these human sera to detect bacterially expressed fusion proteins containing small regions of the gp340 backbone (Pither et al., 1992a) .
Discussion
As the level of baculovirus-expressed BDLF3 proteins was too low to enable purification for use as an antigen, it was decided to raise an antiserum in rabbits against the carboxy-terminal peptide of the BDLF3 sequence. The 20 amino acid peptide represents the putative cytoplasmic domain of the BDLF3 protein and as such has no predicted glycosylation sites within it. We have shown that this anti-peptide serum (i) recognizes BDLF3 products in Western blots following expression by recombinant baculovirus; (ii) immunoprecipitates both glycosylated and unglycosylated BDLF3 products after in vitro translation in the presence of dog pancreas microsomes; (iii) demonstrates positive immunofluorescence on acetone-fixed AcBDLF3-infected insect cells, on cells from the EBV-infected B cell lines B95-8, P3HR-1, AG876 and Jijoye and on cells from six tamarin B cell lines transformed with B95-8 virus; (iv) reacts in a two-colour indirect immunofluorescence assay with a product expressed on the same TPA-induced B95-8 cells that are expressing the lytic phase antigen gp340; (v) recognizes a 100-150kDa product from B95-8 cell lysates on Western blots and immunoprecipitates this product from B95-8 cell lysates; and (vi) recognizes a 100-150 kDa product from partially purified preparations of B95-8 virus and B95-8 cell membranes by Western blot. We have not formally demonstrated that BDLF3 is expressed as a late lytic protein by determining whether its expression is affected by treatment of the B95-8 cells with phosphonoacetic acid (PAA). However, Hummel & Kieff (1982) found that out of 50 RNAs expressed by EBV late in productive infection, only mRNAs whose abundance was sensitive to viral DNA synthesis inhibitors mapped to the BamHI D fragment of the EBV genome.
Discussion
After enzymatic deglycosylation with neuraminidase, O-glycosidase or N-glycosidase F, the 100-150kDa product showed altered electrophoretic mobility by SDS-PAGE, suggesting that the BDLF3 product is a glycoprotein. However, the extent and type of this glycosylation has not been fully defined. Heavy glycosylation seems likely because of the large differences in apparent molecular mass in SDS-PAGE between the native molecule and products expressed in bacteria and in in vitro translation. It still remains possible that the product found in the B95-8 cells has a decreased mobility by SDS-PAGE because it represents the product of a spliced gene, although no splice donor or acceptor sites have been identified in the BDLF30RF. The unusually wide bandwidth of the natural product in SDS-PAGE is also probably a reflection of heavy glycosylation which itself may be variable. The high frequency of serine and threonine in the BDLF3 polypeptide chain would be consistent with there being considerable O-linked carbohydrate.
Discussion
There is no indication of the role that BDLF3 protein plays in the life cycle of the virus. We have not yet found either sequential or positional homology between the BDLF3 sequence and any other herpesvirus ORF. The apparent heavy glycosylation may serve to minimize the humoral immunogenicity of the molecule, restrict proteolytic attack or somehow facilitate the attachment or localization of the virus at mucosal surfaces through carbohydrate interactions. There is a need for specific antibodies which are reactive with extraceUular epitopes on the BDLF3 protein. These would enable us, as a first step, to determine where in the infected B cell BDLF3 protein is expressed, and whether such antibodies are virus-neutralizing. If the immune response to BDLF3 protein is found to be significant, then inclusion in future development of EBV vaccines may be justified, irrespective of whether the actual function of the glycoprotein has been determined.

{'paper_id': '37450313', 'title': 'The Epstein--Barr virus open reading frame BDLF3 codes for a 100-150 kDa glycoprotein', 's2_url': 'https://api.semanticscholar.org/CorpusID:37450313', 'source': 'S2ORC'}