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J Arch Virol (1992) 127:379-386

© Springer-Verlag 1992 Printed in Austria

Identification of a protein encoded in the EB-viral open reading frame B M R F 2 Brief R e p o r t Susanne Modrowl, Brigitte Hiiflacher2,*, and H. Wolf 1 1Max yon Pettenkofer-Institut, Munich 2Gesellschaft ffir Strahlen- und Umweltforschung,Neuherberg, Federal Republic of Germany Accepted March 16, 1992 Summary. Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in the open reading frame BMRF 2 we could identify a protein-complex of 53/ 55 kDa in chemically induced B 95-8, P3HR1 and Raji cell lines. This protein could be shown to be membrane-associated, as predicted by previous computer analysis of the secondary structure and hydrophilicity pattern, and may be a member of EBV-induced membrane proteins in lytically infected cells.

After primary infection Epstein-Barr virus (EBV), a member of the human herpes virus family, replicates in certain epithelial cells of the oropharynx, where limited amounts of infectious virus are produced continuously [3, 16, 21, 23]. These particles may infect peripheral B-lymphocytes circulating in the oropharynx, which acquire the capacity of unlimited growth and may contribute to the maintenance of the pool of EBV-positive B-lymphocytes in the host [15]. In these permanently growing cells the virus remains essentially latent and only a small number of viral gene products (EBNAs, EBERs, LYDMA) are synthesized. EBV-positive B-cell lines may be established from seropositive persons, from Burkitt lymphoma biopsies and by in vitro infection of cord blood lymphocytes. In some cell lines (e.g., B 95-8, P3HR1), a low percentage of cells constantly enter the productive cycle resulting in the production of early and late viral proteins and of infectious virus; host cells are destroyed as a conse* Present address: Mikrogen GmbH, Munich, Federal Republic of Germany.

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quence of the lytic cycle; it is not known whether this cell lysis is based on virusspecific components. The number of cells entering the lytic cycle may be increased by addition of various agents, especially by TPA (phorbol-12-myristate13-acetate) [26] and butyric acid [12]. Raji-cells, a non-producer cell line can also be stimulated by the chemical agents, but only early viral proteins are synthesized. This class of viral gene products includes one protein, which is able to induce fusion of immobilized cells [2]. Three viral regions of the viral genome have recently been shown to be highly important for the induction and the fate of the lytic cycle: firstly, the Barn HI R/Z region encoded gene products, which are synthesized very early in the productive infection and can induce the lytic cycle [6, 8, 21] and secondly, the Bam HI M region, which is known to encode two nuclear antigens, BMLF 1 and BMRF 1. BMLF 1 has trans-activator properties on a variety of eukaryotic and viral promoters [11] and belongs to the first proteins synthesized in the lytic cycle [22], the BMRF 1-gene product, a DNA-binding nuclear antigen, has been shown to be a main component of the class of EA-proteins [-5, 19]. Recently, a third EBV trans-activator protein has been described in the open reading frame BI'LF 4 [13]. From DNA sequencing data [1] and c-DNA cloning [18] of the BamHI M region a third open reading frame, BMRF 2, is known to be localized between BMRF 1 and BMLF 1, which is transcribed as a bicistronic message together with BMRF 1 early in virus replication. A second message encoding BMRF 2 alone was identified in addition; which one serves in the synthesis of BMRF 2 is not known. The predicted protein is highly hydrophobic; no protein or function has been described for BMRF 2 till now, using immunoprecipitation experiments with human EBV-positive sera and hybrid-selected translation [20]. We used an amino acid sequence analysis program written for a Microvax (Digital Equipment), which combines values for local hydrophilicity, flexibility, surface probability and antigenicity with predicted secondary structure parameters according to Chou and Fasman [4] or Gamier et al. [7] for analysis of the BMRF 2-encoded polypeptide. All values were averaged over 7 amino acids. By an additional algorithm, prediction of amphipathic a-helical or 13-pleated regions were included; for those predictions the alternate periodic occurrence of hydrophobic and hydrophilic residues (3.6 for a-helical regions and 2.0 for 13-sheets) were combined with the potentials for the respective secondary structures derived from the predictions described above. The combination of all parameters allowed the identification of only one highly hydrophilic, 13-turn rich region in the center of the protein from residue 180-215, which is supposedly the only antigenic sequential epitope located in a surface accessible area. At the amino terminal region, a stretch of hydrophobic amino acids is followed by a potential cleavage site at residue 21, which represents a signal peptide responsible for the transport of the amino terminal region through the membrane of the endoplasmic reticulum [9]. The sequences between amino acids 70-160 and 220-355 contained 2 (I, II) and 3 (III, IV, V)

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stretches of hydrophobic amino acids in a-helical or [3-pleated sheet configuration. Only one of them showed the characteristics of an optimal transmembrahe region (III), consisting of 23 hydrophobic residues with a-helical structures. The other four hydrophobic regions (I, II, IV, V) showed the properties of amphipathic transmembrane regions (Fig. 1), For the identification and further characterization of the protein in EBVinfected cells we synthesized an oligopeptide derived from the most antigenic region localized in the middle of the protein from residue 186-197 (NHz-T-SG-L-E-R-R-R-S-I-F-C-COOH) using a 430A peptide synthesizer (Applied Biosystems, Weiterstadt, Federal Republic of Germany), as described [14]. For the production of antisera, the amino acids glycine, glycine and lysine were covalently linked to the amino terminus of the peptide and both amino groups of the terminal lysine were esterified with palmitic acid by following the procedure of solid phase synthesis [10]. Rabbits were inoculated several times with 500 gg of palmitoylated peptide, antipeptide antibodies were determined in ELISA-tests using 200 ng of purified peptide per well as described [14]; the sera showed titers of 1 : 20,000.

Fig. 1. Linear graph of the BMRF 2-encoded protein. Values for hydrophilicity, surfaceprobability, flexibility,antigenicityand secondary structures are shown [25], Amphipathic a-helical regions are indicated by oscillating lines in combination with a-helical structure predictions. Potential transmembrane spanning regions are indicated as shaded areas

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The rabbit antipeptide sera were used to identify the corresponding native EBV-protein in virus positive lymphoblastoid cell lines. For this approach the Burkitt-lymphoma derived cell line Raji was used, induction of viral protein synthesis leeds to the synthesis of viral early proteins but not to late polypeptides or infectious virus. This is in contrast to cell line P3HR1, which is infected by a EBV-strain growing preferentially in the tytic infection cycle. TPA/butyric acid induction results in the production of early proteins and of late structural polypeptides in combination with infectious virus particles. The marmosetderived B-cell line B 95-8 is infected by an EBV-isolate originally derived from a patient with infectious mononucleosis. The virus grows preferentially in the latent cycle, small amounts of early and late viral proteins may be induced by chemical induction. The cell fines were grown in RPMI 1640 and induced with TPA/butyric acid, as described [20]. 1 x 10 6 cells of the respective preparations were pelleted, washed with phosphate buffered saline (PBS) and suspended in 100~tl lysis buffer (2% SDS, 5% mercaptoethanol, 20mM Tris-HC1, pH 7.4, 10% sucrose, bromphenolblue), sonicated and heated for 5 rain at 100 °C. 50 gl of the mixture per slot were applied to SDS-polyacrylamide gels, proteins were separated overnight and transferred to nitrocellulose sheets by electroblotting [-21]. The sheets were incubated for 1 h in blocking solution (154mM NaC1, 10mM Tris-HC1, pH 7.5, 0.5% Tween, 5% dry milk) followed by a 2h incubation with the rabbit antipeptide serum and preimmune serum as control in dilution 1:50 in 10mM Tris-HC1, pH7.5, 154raM NaC1, 0.5% Tween. The sheets were washed thoroughly, detection of the immunocomplexes was done using swine anti-rabbit IgG (peroxidase-labelled, DAKO, Hamburg, Federal Republic of Germany), staining was done with diaminobenzidine as described [21]. In TPA/butyric acid induced Raji-, B 95-8- and P3HRl-cells two rather diffuse protein bands of 53 and 55 kDa could be identified after incubation with antipeptide serum (Fig. 2 A, slots I-VII). The fact that these protein bands could not be identified in EBV-positive uninduced cells which produce only the latent viral gene products allowed to classify the BMRF 2 encoded proteins to be a member of the early EBV gene products. In Raji cells TPA/butyric acid treatment leads to the synthesis of early viral gene products but not to infectious virus (Fig. 2 A, slots IV and V). The BMRF 2 gene product could be identified, synthesis was, however, weaker than in P3HR1 cells, where EBV exists preferentially in the lyric propagation cycle after TPA/butyric acid induction (Fig. 2 A, slot II). The p 53/55 protein was very weakly produced in B 95-8 cells, also chemical induction did not lead to a significantly higher synthesis rate (Fig. 2 A, slots VI and VII). In B 95-8 cells EBV exists preferentially in the latent cycle, only a few cells may be induced to lyric virus propagation. By comparison of the synthesis rate of p 53/55 in the various cell lines we classified the BMRF 2 protein to be a member of the EA-class of EBV polypeptides. This proteincomplex was not present in EBV-negative BJA-B cells, preimmune serum obtained from rabbits prior to peptide inoculation showed no reaction. In order

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Fig. 2. Western blot experiments detecting BMRF 2-specific proteins by rabbit antipeptide serum. The significant proteins expressed by BMRF 2 open reading frame of 53/55 kDa m'e indicated by arrowheads. A Total cell lysate: I BJA-B cells; H and III P3HRl-cells; I V and V Raji-cells; VI and VII B 95-8 cells; in I, II, IV, and VI cells were treated with TPA/butyric acid. B Nuclear and membrane protein preparation: I BJA-B cells, nuclear extract; H BJA-B cells membrane preparation; III Raji cells, nuclear extract; I V Raji cells, membrane preparation. All cell lines were induced by TPA/butyric acid. C Endo ~N-acetylgalactosaminidase treatment; I BJA-B cells, untreated; H BJA-B cells treated with a-N-acetylgatactosaminidase; HI Raji cells, untreated; IV Raji cells, treated with a-N-acetylgalactosaminidase. All cells were induced by TPA/butyric acid

to s h o w the specificity o f the reaction, the rabbit s e r u m was p r e i n c u b a t e d with the free peptide before used in W e s t e r n blotting; this t r e a t m e n t r e d u c e d the intensity o f the 53/55 k D a p r o t e i n b a n d drastically. W h e n a p o o l o f EBV-positive sera was used for the identification o f viral proteins, this p r o t e i n b a n d was n o t

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detected. In a similar way previous experiments using hybrid-selected translation and immunoprecipitation did not lead to the identification of a protein corresponding to open frame BMFR 2 [20]. It is not yet clear if this finding is due to very low amounts of immunoglobulins directed against that specific protein in EBV-positive human sera or if it is not possible to correlate a defined band with this polypeptide, since several viral proteins in induced cells show molecular weights in the range of 45-55 kDa. Since the 53/55 kDa band is rather diffuse, parts of the protein may exist with posttranslational modifications; the amino acid sequence, however, shows no potential sites for the addition of N-glycosylation-groups. In order to test the possibility of O-glycosylation events, a lysate of induced Raji cells was incubated with endo-a-N-acetylgalactosaminidase (Boehringer Mannheim, Federal Republic of Germany). This treatment resulted in a reduction of the 55 kDa protein and intensification of the 53 kDa band (Fig. 2 C, slots III and IV). When cellular membranes of induced Raji cells were stripped of the nuclei, the nuclear fraction showed only a low content of the 55 kDa protein probably due to contamination with cellular membrane particles which cannot be totally removed by mechanical disruption treatment. This may however be due to the assembly process of herpesviruses, which takes place at the site of the inner nuclear membrane. Most of the polypeptide was part of the cell membrane preparation (Fig. 2 B, slots II and IV); the 53 kDa protein was present in the nuclear and membrane fraction and may represent the unmodified form of the BMRF 2-protein. In conclusion, using a rabbit serum against a synthetic peptide derived from an antigenic region, we identified a protein complex of 53/55 kDa in chemically induced EBV-positive B-cell lines (Fig. 2). The presence of this protein in the non-producer cell line Raji, where no structural or late virus proteins are synthesized after TPA induction, suggests that the identified polypeptide may be part of the early viral protein complex. Computer analysis proposed membrane association for the BMRF2-encoded protein. The 55 kDa protein copurifies with the membrane compartment of induced cells and may be modified by Oglycosylation groups. Other modifications like phosphorylation or sulfylation, however, cannot be excluded and may contribute to the rather diffuse band of 53/55 kDa.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft(Wo 272-2). The authors thank A. Willer (GSF) for immunisation of rabbits and J. Mfirz for typing the manuscript.

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3. Becker J, Leser U, Marschall M, Langford A, Jilg W, Gelderblom H, Reichart P, Wolf H (1991) Expression of proteins encoded by Epstein-Barr virus trans-activator genes depends on the differentiation of epithelial cells in oral hairy leukoplakia. Proc Natl Acad Sci USA 88:8332-8336 4. Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45-148 5. Cho MS, Milman G, Hayward SD (1985) A second Epstein-Barr virus early antigen gene in the Barn HI fragment M encodes a 48-50 kilodalton nuclear protein. J Virol 56:860-866 6. Countryman J, Miller G (1985) Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA. Proc Natl Acad Sci USA 82:8084-8089 7. Garnier J, Osguthorpe O J, Robson B (1978) Analysis of the accuracy and implication of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:97-120 Hardwick JM, Liebermann PM, Hayward SD (1988) A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. J Virol 62:2274-2284 9. v Heijine G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683-4690 10. Hopp TP (1984) Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty-acid carrier. Mol Immunol 21:13-16 11. Liebermann PM, O'Hare P, Hayward GS, Hayward DS (1986) Promiscuous transactivation of gene expression by an Epstein-Barr virus encoded early nuclear protein. J Virol 60:140-148 12. Luka J, Kallin B, Klein G (1979) Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by N-butyrate. Virology 94:228-231 13. Marschall M, Schwarzmann F, Leser U, Oker B, Alliger P, Mairhofer H, Wolf H (1991) The BI'LF4 trans-activator of Epstein-Barr virus is modulated by type and differentiation of the host cell. Virology 181: 172-179 14. Modrow S, Wolf H (1986) Characterization of two related Epstein-Barr-virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro transformed cell lines. Proc Natl Acad Sci USA 83:5703-5707 15. Modrow S, Jilg W, Meerwarth I, Mairhofer H, Haus M, Wolf H (1987) Differential expression of EB-viral and cellular surface markers on Burkitt lymphoma and lymphoblastoid cell lines. In: Levine PH, Ablashi DV, Nonoyama M, Pearson GR, Glaser R (eds) Epstein-Barr virus and human disease. Humana Press, Clifton, NJ, pp407411 16. Morgan DG, Miller G, Niedermann JC, Smith HW, Dowaliby JM (1979) Site of Epstein-Barr virus replication in the oropharynx. Lancet i: 1154-1157 17. Oguro MO, Shimizu N, Ono Y, Takada K (1987) Both the rightward and leftward open reading frame within the Barn HI M DNA fragment of the Epstein-Barr virus act as trans-activators of gene expression. J Virol 61:3310-3313 18. Pfitzner AJ, Strominger JL, Speck SH (1987) Characterization of a cDNA clone corresponding to a transcript from the Epstein-Barr virus Barn HI M fragment: evidence for overlapping mRNAs. J Virol 61:2943-2956 19. Sample J, Tanaka A, Lanez G, Nonoyama M (1984) Identification of Epstein-Barr virus genes expressed during the early phase of virus replication and during lymphocyte immortalization. Virology 139:1-10 20. Seibl R, Wolf H (1985) Mapping of Epstein-Barr virus proteins on the genome by translation of hybrid-selected RNA from induced P3HR1 cells and induced Raji cells. Virology 141:1 13 .

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2t. Seibl R, Motz M, Wolf H (1986) Strain-specific transcription and translation of the Barn HI Z area of Epstein-Barr virus. J Virol 60:902-909 22. Seibl R, Leser U, Marschatl M, Modrow S, Fuchs K, Wolf H (1987) Identification and characterization of regulatory proteins of EBV. In: Levine PH, Ablashi DV, Nonoyama M, Pearson GR, Glaser R (eds) Epstein-Barr virus and human disease. Humana Press, Clifton, NJ, pp 211-216 23. Sixby JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS (1984) Epstein-Barr virus replication in oropharyngeal epithelial cells. N Engl J Med 310:1225-1230 24. Wolf H, Haus M, Wilmes E (1984) Persistence of Epstein-Barr virus in the parotid gland. J Virol 51:795-798 25. Wolf H, Modrow S, Motz M, Jameson B, Hermann G, F6rtsch B (1988) An integrated family of amino acid sequence analysis programs. Cabios 4:18%191 26. zur Hausen H, O'Neil F J, Freese UK (1978) Persisting oncogenic herpesvirus induced by the tumor promotor TPA. Nature 272:373-375 Authors' address: Susanne Modrow, Institut ffir Medizinische Mikrobiologie und Hygiene, Universit/it Regensburg, Franz-Josef-Straug-Allee 11, D-W-8400 Regensburg, Federal Republic of Germany. Received October 14, 1991

Identification of a protein encoded in the EB-viral open reading frame BMRF2.

Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in th...
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