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Volume 75, Issue 11

DNA-binding Studies of the Epstein-Barr Virus Nuclear Antigen 2 (EBNA-2): Evidence for Complex Formation by Latent Membrane Protein Gene Promoter-binding Proteins in EBNA-2-positive Cell Lines Free

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA- 2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Transactivation of the TP1 promoter and of the promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates transactivation. To determine whether EBNA-2 also trans- activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides −135 to − 176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein- protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.

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  • Accepted:
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© The Journal of General Virology 1994
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1994-11-01
2023-11-28
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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. Aman P., Rowe M., Kai C., Finke J., Rymo L., Klein E., Klein G. 1990; Effect of the EBNA-2 gene on the surface antigen phenotype of transfected EBV-negative B-lymphoma lines. International Journal of Cancer 45:77–82
     [Google Scholar]
  3. Anagnostopoulos I., Herbst H., Niedobitek G., Stein H. 1989; Demonstration of monoclonal EBV genomes in Hodgkin’s disease and Ki-1-positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization. Blood 74:810–816
     [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., Barrell B. G. 1984; DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature; London: 310207–211
     [Google Scholar]
  5. Baeuerle P. A., Baltimore D. 1988; Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell 53:211–217
     [Google Scholar]
  6. Billaud M., Busson P., Huang D., Mueller-Lantzch N., Rousselet G., Pavlish O., Wakasugi H., Seigneurin J. M., Tursz T., Lenoir G. M. 1989; Epstein-Barr virus (EBV)- containing nasopharyngeal carcinoma cells express the B-cell activation antigen blast2/CD23 and low levels of the EBV receptor CR2. Journal of Virology 63:4121–4128
     [Google Scholar]
  7. Bornkamm G., 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 DSL region. Journal of Virology 43:952–968
     [Google Scholar]
  8. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. 1988; Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. Journal of Cell Biology 106:761–771
     [Google Scholar]
  9. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  10. Calender A., Billaud M., Aubry J., Banchereau J., Vuillaume M., Lenoir G. 1987; 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 U.S.A: 848060–8064
     [Google Scholar]
  11. Calender A., Cordier M., Billaud M., Lenoir G. M. 1990; Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein-Barr virus (EBV). International Journal of Cancer 46:658–663
     [Google Scholar]
  12. 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 U.S.A: 869558–9562
     [Google Scholar]
  13. Cordier M., Calender A., Billaud M., Zimber U., Rousselet G., Pavlish O., Banchereau J., Tursz T., Bornkamm G., 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]
  14. Cordier-Bussat M., Calender A., Vuillaume M., Bornkamm G. W., Lenoir G. M. 1993; Expression of the Epstein-Barr virus (EBV) latent membrane protein is tightly regulated, independently of EB nuclear antigen 2 and of EBV integration or copy number. Virus research 27:55–69
     [Google Scholar]
  15. Crawford D. H., Epstein M. A., Bornkamm G. W., Achong B. G., Finerty S., Thompson J. 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]
  16. Dambaugh T., Hennessy K., Chamnankit L., Kieff E. 1984; U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2. Proceedings of the National Academy of Sciences U.S.A: 817632–7636
     [Google Scholar]
  17. Dignam J. D., Lebovitz R. M., Roeder R. G. 1983; Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11:1475–1489
     [Google Scholar]
  18. Fahraeus R., Fu H. L., Ernberg I., Finke J., Rowe M., Klein G., Falk K., Nilsson E., Yadav M., Busson P., Tursz T., Kallin B. 1988; Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. International Journal of Cancer 42:329–338
     [Google Scholar]
  19. Fahraeus R., Jansson A., Rickstein A., Sjöblom 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 U.S.A: 877390–7394
     [Google Scholar]
  20. Fahraeus R., Jansson A., Sjöblom A., Nilsson T., Klein G., Rymo L. 1993; Cell phenotype-dependent control of Epstein-Barr virus latent membrane protein 1 gene regulatory sequences. Virology 195:71–80
     [Google Scholar]
  21. Ghosh D., Kieff E. 1990; Cis-acting regulatory elements near the Epstein-Barr virus latent infection membrane protein transcriptional start site. Journal of Virology 64:1855–1858
     [Google Scholar]
  22. Grässer 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]
  23. Hammerschmidt W., Sugden B. 1989; Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes. Nature; London: 340393–397
     [Google Scholar]
  24. Herbst H., Dallenbach F., Hummel M., Niedobitek G., Pillari S., Muller-Lantzsch N., Stein H. 1991; Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proceedings of the National Academy of Sciences U.S.A: 884766–4770
     [Google Scholar]
  25. Hinuma Y., Konn M., Yamaguchi J., Wudarski D. J., Blakeslee J. R., Grace T. J.Jr 1967; Immunofluorescence and herpes- type virus particles in the P3HR-1 Burkitt lymphoma cell line. Journal of Virology 1:1045–1051
     [Google Scholar]
  26. Jeang K. T., Hayward S. D. 1983; Organization of the Epstein-Barr virus DNA molecule. III. Location of the P3HR-1 deletion junction and characterization of the Notl repeat units that form part of the template for an abundant 12-O-tetradecanoyl- phorbol 13-acetate-induced mRNA transcript. Journal of Virology 48:135–148
     [Google Scholar]
  27. Jones M. D., Foster L., Sheedy T., Griffin B. E. 1984; The EB virus genome in Daudi Burkitt’s lymphoma cells has a deletion similar to that observed in a non-transforming strain (P3HR-1) of the virus. EMBO Journal 3:813–821
     [Google Scholar]
  28. Kanda K., Decker T., Aman P., Wahlström M., Von Gabain A., Kallin B. 1992; The EBNA2-related resistance towards alpha interferon (IFN-a) in Burkitt’s lymphoma cells effects induction of IFN-induced genes but not the activation of transcription factor ISGF-3. Molecular and Cellular Biology 12:4930–4936
     [Google Scholar]
  29. Kieff E., Liebowitz D. 1990; Epstein-Barr virus and its replication. In Virology, 2nd edn. pp 1889–1920 Fields B. N., Knipe D. M. Edited by New York: Raven Press;
     [Google Scholar]
  30. King W., Thomas-Powell A., Raub-Traub N., Hawke M. C., Kieff E. 1980; Epstein-Barr virus RNA: viral RNA in a restringently infected, growth transformed cell. Journal of Virology 36:506–518
     [Google Scholar]
  31. Klein G., Lindahl T., Jondal M., Leibold 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 U.S.A: 713283–3286
     [Google Scholar]
  32. 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]
  33. Ling P. D., Rawlins D. R., Hayward S. D. 1993; The Epstein-Barr virus immortalizing protein EBNA2 is targeted to DNA by a cellular enhancer-binding protein. Proceedings of the National Academy of Sciences U.S.A: 909237–9241
     [Google Scholar]
  34. Middleton T., Gahn T. A., Martin J. M., Sugden B. 1991; Immortalizing genes of Epstein-Barr virus. Advances in Virus Research 40:19–55
     [Google Scholar]
  35. Miller G. 1990; Epstein-Barr virus: biology, pathogenesis and medical aspects. In Virology, 2nd edn. pp 1921–1958 Fields B. N., Knipe D. M. Edited by New York: Raven Press;
     [Google Scholar]
  36. Murray R. J., Wang D., Young L. S., Wang F., Rowe M., Kieff E., Rickinson A. B. 1988; Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP. Journal of Virology 62:3747–3755
     [Google Scholar]
  37. Polack A., Hartl G., Zimber U., Freese K. -U., Laux G., Takadi K. , Gissmann L., Bornkamm G. W. 1984; A complete set of overlapping cosmid clones of M-ABA virus derived from a nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates. Gene 27:279–288
     [Google Scholar]
  38. Rickinson A. B., Young L. S., Rowe M. 1987; Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells. Journal of Virology 61:1310–1317
     [Google Scholar]
  39. Sample J., Young L., Martin B., Chatman T., Kieff E., Rickinson A. 1990; Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes. Journal of Virology 64:4084–4092
     [Google Scholar]
  40. Schickedanz J., Scheidtmann K. H., Walter G. 1986; Kinetics of nuclear transport and oligomerization of simian virus 40 large T antigen. Virology 148:47–57
     [Google Scholar]
  41. 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]
  42. Tsang S. -F., Wang F., Izumi K. M., Kieff E. 1991; Delineation of the cis-acting element mediating EBNA-2 transactivation of latent infection membrane protein. Journal of Virology 65:6765–6771
     [Google Scholar]
  43. Wang D., Liebowitz D., Kieff E. 1985; An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43:831–840
     [Google Scholar]
  44. 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 U.S.A: 843452–3456
     [Google Scholar]
  45. Wang F., Tsang S. -F., Kurilla M. G., Cohen J. I., Kieff E. 1990; Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. Journal of Virology 64:3407–3416
     [Google Scholar]
  46. Wang F., Kikutani H., Tsang S. -F., Kishimoto T., Kieff E. 1991; Epstein-Barr virus nuclear protein 2 transactivates a cis- acting CD23 DNA element. Journal of Virology 65:4101–4106
     [Google Scholar]
  47. Weiss L. M., Mohaved L. A., Warnke R. A., Sklar J. 1989; Detection of Epstein-Barr genomes in Reed-Sternberg cells of Hodgkin’s disease. New England Journal of Medicine 320:502–506
     [Google Scholar]
  48. Zimber U., Adldinger FI. K., Lenoir G. M., Vuillaume M., Knebel-Doeberitz M. V., Laux G., Desgranges C., Wittmann P., Freese U. K., Schneider U., Bornkamm G. 1986; Geographical prevalence of two types of Epstein-Barr virus. Virology 154:56–66
     [Google Scholar]
  49. Zimber-Strobl U., Suent,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]
  50. Zimber-Strobl U., Kremmer E., Grässer 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]
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DNA-binding Studies of the Epstein-Barr Virus Nuclear Antigen 2 (EBNA-2): Evidence for Complex Formation by Latent Membrane Protein Gene Promoter-binding Proteins in EBNA-2-positive Cell Lines
J. Gen. Virol. 75, 3067 (1994); https://doi.org/10.1099/0022-1317-75-11-3067
/content/journal/jgv/10.1099/0022-1317-75-11-3067
/content/journal/jgv/10.1099/0022-1317-75-11-3067
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