1887

Abstract

To develop a transformation system with a conditional Epstein—Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-77-2-227
1996-02-01
2024-12-12
Loading full text...

Full text loading...

/deliver/fulltext/jgv/77/2/JV0770020227.html?itemId=/content/journal/jgv/10.1099/0022-1317-77-2-227&mimeType=html&fmt=ahah

References

  1. Abbot S. D., Rowe M., Cadwallader K., Ricksten A., Gordon J., Wang F., Rymo L., Rickinson A. B. 1990; Epstein–Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein. Journal of Virology 64:2126–2134
    [Google Scholar]
  2. Ali S., Metzger J., Bornert J. M., Chambon P. 1994; Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor. EMBO Journal 12:1153ߝ1160
    [Google Scholar]
  3. Allday M. J., Crawford D. H., Griffin B. E. 1989; Epstein-Barr virus latent gene expression during the initiation of B-cell immortalization. Journal of General Virology 10:1755–1764
    [Google Scholar]
  4. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Seguin C., Tufnell P. S., Barell B. G. 1984; DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature 310:207–211
    [Google Scholar]
  5. Bornkamm G. W., Hudewentz J., Freese U. K., Zimber U. 1982; Deletion of the nontransforming Epstein-Barr virus strain P3HR-1 causes fusion of the large internal repeat to the DST region. Journal of Virology 43:952–968
    [Google Scholar]
  6. Calender A., Billaud M., Aubry J. P., Banchereau J., Vuillaume M., Lenoir G. M. 1987; Epstein–Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells. Proceedings of the National Academy of Sciences, USA 84:8060–8064
    [Google Scholar]
  7. Cohen J. I., Wang F., Mannick J., Kieff E. 1989; Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proceedings of the National Academy of Sciences, USA 86:9558–9562
    [Google Scholar]
  8. Cordier M., Calender A., Billaud M., Zimber U., Rousselet G., Pavlish O., Banchereau J., Tursz T., Bornkamm G. W., Lenoir G. M. 1990; Stable transfection of Epstein–Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23. Journal of Virology 64:1002–1013
    [Google Scholar]
  9. Cotter T. G., Lennon S. V., Glynn J. M., Green D. R. 1992; Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Research 52:997–1005
    [Google Scholar]
  10. Crawford D. H., Epstein M. A., Bornkamm G. W., Achong B. G., Finerty S., Thompson J. L. 1979; Biological and biochemical observations on isolates of EB virus from the malignant epithelial cells of two nasopharyngeal carcinomas. International Journal of Cancer 24:294–302
    [Google Scholar]
  11. Cuomo L., Ramquist T., Trivedi P., Wang F., Klein G., Masucci M. G. 1992; Expression of the Epstein-Barr virus (EBV)-encoded membrane protein LMP1 impairs the in vitro growth, clonability and tumorigenicity of an EBV-negative Burkitt lymphoma line. International Journal of Cancer 51:949–955
    [Google Scholar]
  12. Eilers M., Picard D., Yamamoto K. R., Bishop J. M. 1989; Chimaeras of myc oncoprotein and steroid receptors cause hormonedependent transformation of cells. Nature 340:66–68
    [Google Scholar]
  13. Eissner G., Kohlhuber F., Grell M., Ueffing M., Scheurich P., Hieke A., Multhoff G., Bornkamm G. W., Holler E. 1995; Critical involvement of transmembrane TNF-α in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin. Blood 86:4175–4184
    [Google Scholar]
  14. Fahraeus R., Jansson A., Ricksten A., Sjoblom A., Rymo L. 1990; Epstein-Barr virus-encoded nuclear antigen 2 activates the viral latent membrane protein promoter by modulating the activity of a negative regulatory element. Proceedings of the National Academy of Sciences, USA 87:7390–7394
    [Google Scholar]
  15. Falk M. H., Hultner L., Milner A., Gregory C. D., Bornkamm G. W. 1993; Irradiated fibroblasts protect Burkitt lymphoma cells from apoptosis by a mechanism independent of bcl-2. International Journal of Cancer 55:485–491
    [Google Scholar]
  16. Grasser F. A., Haiss P., Gottel S., Mueller-Lantzsch N. 1991; Biochemical characterization of Epstein-Barr virus nuclear antigen 2A. Journal of Virology 65:3779–3788
    [Google Scholar]
  17. Grossman S. R., Johannsen E., Tong X., Yalamanchili R., Kieff E. 1994; The Epstein–Barr virus nuclear antigen 2 transactivator is directed to response elements by the J Kappa recombination signal binding protein. Proceedings of the National Academy of Sciences, USA 91:7568–7572
    [Google Scholar]
  18. Hammerschmidt W., Sugden B. 1988; Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein–Barr virus. Cell 55:427–433
    [Google Scholar]
  19. Hammerschmidt W., Sugden B. 1989; Genetic analysis of immortalizing functions of Epstein–Barr virus in human B lymphocytes. Nature 340:393–397
    [Google Scholar]
  20. Henkel T., Ling P. D., Hayward S. D., Peterson M. G. 1994; Mediation of Epstein–Barr virus EBNA2 transactivation by recombination signal-binding protein Jκ . Science 265:92–95
    [Google Scholar]
  21. Kempkes B., Pich D., Zeidler R., Sugden B., Hammerschmidt W. 1995a; Immortalization of human B lymphocytes by a plasmid containing 71 kbp of Epstein–Barr virus DNA. Journal of Virology 69:231–238
    [Google Scholar]
  22. Kempkes B., Spitkovsky D., Jansen-Durr P., Ellwart J. W., Kremmer E., Delecluse H. J., Rottenberger C., Bornkamm G. W., Hammerschmidt W. 1995; B-cell proliferation and induction of early G1-regulating proteins by Epstein–Barr virus mutants conditional for EBNA2. EMBO Journal 14:88–96
    [Google Scholar]
  23. Klein G., Lindahl T., Jondal M., Lbibold W., Menezes J., Nilsson K., Sundstrom C. 1974; Continuous lymphoid cell lines with characteristics of B cells (bone-marrow-derived), lacking the Epstein-Barr virus genome and derived from three human lymphomas. Proceedings of the National Academy of Sciences, USA 71:3283–3286
    [Google Scholar]
  24. Knutson J. C. 1990; The level of c-fgr RNA is increased by EBNA-2, an Epstein-Barr virus gene required for B-cell immortalization. Journal of Virology 64:2530–2536
    [Google Scholar]
  25. Kremmer E., Kranz B. R., Hille A., Klein K., Eulitz M., Hoffmann-Fezer G., Feiden W., Herrmann K., Delecluse G. J., Delsol G., Bornkamm G. W., Mueller-Lantzsch N., Grasser F. A. 1995; Rat monoclonal antibodies differentiating between the Epstein–Barr virus nuclear antigen 2A (EBNA2A) and 2B (EBNA2B). Virology 208:336–342
    [Google Scholar]
  26. Kumar V., Green S., Staub A., Chambon P. 1986; Localization of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO Journal 5:2231–2236
    [Google Scholar]
  27. Laux G., Dugrillon C., Eckert B., Adam B., Zimber-Strobl U., Bornkamm G. W. 1994; Identification and characterization of an Epstein-Barr virus nuclear antigen 2-responsive element in the bidirectional promoter region of latent membrane protein and terminal protein 2 genes. Journal of Virology 68:6947–6958
    [Google Scholar]
  28. Lenoir G. M., Vuillaume M., Bonnardel C. 1985; The use of lymphomatous and lymphoblastoid cell lines in the study of Burkitt’s lymphoma. JARC Science Publications309–318
    [Google Scholar]
  29. Ling P. D., Rawlins D. R., Hayward S. D. 1993; The Epstein-Barr virus immortalizing protein EBNA-2 is targeted to DNA by a cellular enhancer–binding protein. Proceedings of the National Academy of Sciences, USA 90:9237–9241
    [Google Scholar]
  30. Matsunami N., Hamaguchi Y., Yamamoto Y., Kuze K., Kangawa K., Matsuo H., Kawaichi M., Honjo T. 1989; A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif. Nature 342:934–937
    [Google Scholar]
  31. Meitinger C., Strobe L. J., Marschall G., Bornkamm G. W., Zimber-Strobl U. 1994; Crucial sequences within the Epstein-Barr virus TP1 promoter for EBNA2-mediated transactivation and interaction of EBNA2 with its responsive element. Journal of Virology 68:7497–7506
    [Google Scholar]
  32. Mosmann T. 1983; Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65:55–63
    [Google Scholar]
  33. Picard D., Salser S. J., Yamamoto K. R. 1988; A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell 54:1073–1080
    [Google Scholar]
  34. Polack A., Hartl G., Zimber U., Freese U. K., Laux G., Takaki K., Hohn B., Gissmann L., Bornkamm G. W. 1984; A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein–Barr virus isolates. Gene 27:279–288
    [Google Scholar]
  35. Rabson M., Gradoville L., Heston L., Miller G. 1982; Nonimmortalizing P3 J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye. Journal of Virology 44:834–844
    [Google Scholar]
  36. Rooney C., Howe J. G., Speck S. H., Miller G. 1989; Influence of Burkitt’s lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus. Journal of Virology 63:1531–1539
    [Google Scholar]
  37. Sung N. S., Kenney S., Gutsch D., Pagano J. S. 1991; EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus. Journal of Virology 65:2164–2169
    [Google Scholar]
  38. Torsteinsdottir S., Andersson M. L., Avila-Carino J., Ehlin-Henriksson B., Masucci M. G., Klein G., Klein E. 1989; Reversion of tumorigenicity and decreased agarose clonability after EBV conversion of an IgH/myc translocation-carrying BL line. International Journal of Cancer 43:273–278
    [Google Scholar]
  39. Waltzer L., Logeat F., Brou C., Israel A., Sergeant A., Manet E. 1994; The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein–Barr virus EBNA2 protein to its DNA responsive elements. EMBO Journal 13:5633–5638
    [Google Scholar]
  40. Wang F., Gregory C. D., Rowe M., Rickinson A. B., Wang D., Birkenbach M., Kikutani H., Kishimoto T., Kieff E. 1987; Epstein–Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23. Proceedings of the National Academy of Sciences, USA 84:3452–3456
    [Google Scholar]
  41. Woisetschlaeger M., Jin X. W., Yandava C. N., Furmanski L. A., Strominger J. L., Speck S. H. 1991; Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. Proceedings of the National Academy of Sciences, USA 88:3942–3946
    [Google Scholar]
  42. Yates J., Warren N., Reisman D., Sugden B. 1984; A cis-acting element from the Epstein–Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proceedings of the National Academy of Sciences, USA 81:3806–3810
    [Google Scholar]
  43. Zimber-Strobl U., Suentzenich K. O., Laux G., Eick D., Cordier M., Calender A., Billaud M., Lenoir G. M., Bornkamm G. W. 1991; Epstein–Barr virus nuclear antigen 2 activates transcription of the terminal protein gene. Journal of Virology 65:415–423
    [Google Scholar]
  44. Zimber-Strobl U., Kremmer E., Grasser F., Marschall G., Laux G., Bornkamm G. W. 1993; The Epstein–Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. EMBO Journal 12:167–175
    [Google Scholar]
  45. Zimber-Strobl U., Strobl L. J., Meitinger C., Hinrichs R., Sakai T., Furukawa T., Honjo T., Bornkamm G. W. 1994; The Epstein–Barr virus nuclear antigen 2 exerts its function through interaction with recombination signal binding protein RBP-J Kappa, the homologue of Drosophila suppressor of hairless . EMBO Journal 13:4973–4982
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-77-2-227
Loading
/content/journal/jgv/10.1099/0022-1317-77-2-227
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error