1887

Abstract

Co-operation between the Epstein–Barr virus (EBV)-coded leader protein EBNA-LP and the nuclear antigen EBNA2 appears to be critical for efficient virus-induced B cell transformation. Here we report the genetic analysis of EBNA-LP function using two transient co-transfection assays of co-operativity, activation of latent membrane protein 1 (LMP1) expression from a resident EBV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regions (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within all three CRs in the EBNA-LP W1W2 repeat domain completely abrogated function, through inhibition of nuclear localization in the cases of CR1 and CR2 but not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had relatively little effect, yet loss of the whole Y2 sequence blocked activity. Alanine substitution of serine residues within potential phosphorylation sites identified two mutants of particular interest. Substitution of three such residues (S) within W1W2 not only abrogated EBNA-LP activity but was associated with a complete loss of EBNA2 detectability in co-transfected cells, implying possible destabilization of the co-expressed EBNA2 protein. More importantly the individual substitution of S completely blocked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We infer critical roles for the CR3 domain and for the S residue in EBNA-LP’s co-operative function.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-12-3067
2001-12-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/12/0823067a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-12-3067&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. Alfieri, C., Birkenbach, M. & Kieff, E. ( 1991; ). Early events in Epstein–Barr virus infection of human B lymphocytes. Virology 181, 595-608.[CrossRef]
    [Google Scholar]
  3. Bell, A. I., Skinner, J., Kirby, H. & Rickinson, A. ( 1998; ). Characterization of regulatory sequences at the Epstein–Barr virus BamHI W promoter. Virology 252, 149-161.[CrossRef]
    [Google Scholar]
  4. 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.[CrossRef]
    [Google Scholar]
  5. 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]
  6. Dufva, M., Olsson, M. & Rymo, L. ( 2001; ). Epstein–Barr virus nuclear antigen 5 interacts with HAX-1, a possible component of the B cell receptor signalling pathway. Journal of General Virology 82, 1581-1587.
    [Google Scholar]
  7. Fourie, A. M., Sambrook, J. F. & Gething, M. J. ( 1994; ). Common and divergent peptide binding specificities of hsp70 molecular chaperones. Journal of Biological Chemistry 269, 30470-30478.
    [Google Scholar]
  8. 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.[CrossRef]
    [Google Scholar]
  9. Hammerschmidt, W. & Sugden, B. ( 1989; ). Genetic analysis of immortalizing functions of Epstein–Barr virus in human B lymphocytes. Nature 340, 393-397.[CrossRef]
    [Google Scholar]
  10. Han, I., Harada, S., Weaver, D., Xue, Y., Lane, W., Orstavik, S., Skalhegg, B. & Kieff, E. ( 2001; ). EBNA-LP associates with cellular proteins including DNA-PK and HA95. Journal of Virology 75, 2475-2481.[CrossRef]
    [Google Scholar]
  11. Harada, S. & Kieff, E. ( 1997; ). Epstein–Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. Journal of Virology 71, 6611-6618.
    [Google Scholar]
  12. Henkel, T., Ling, P. D., Hayward, S. D. & Peterson, M. G. ( 1994; ). Mediation of Epstein–Barr virus EBNA2 transactivation by recombination signal-binding protein J kappa. Science 265, 92-95.[CrossRef]
    [Google Scholar]
  13. Holmes, J. K. & Solomon, M. J. ( 1996; ). A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2. Journal of Biological Chemistry 271, 25240-25246.[CrossRef]
    [Google Scholar]
  14. Jayachandra, S., Low, K. G., Thlick, A. E., Yu, J., Ling, P. D., Chang, Y. & Moore, P. S. ( 1999; ). Three unrelated viral transforming proteins (vIRF, EBNA2, and E1A) induce the MYC oncogene through the interferon-responsive PRF element by using different transcription coadaptors. Proceedings of the National Academy of Sciences, USA 96, 11566-11571.[CrossRef]
    [Google Scholar]
  15. Kaiser, C., Laux, G., Eick, D., Jochner, N., Bornkamm, G. W. & Kempkes, B. ( 1999; ). The proto-oncogene c-myc is a direct target gene of Epstein–Barr virus nuclear antigen 2. Journal of Virology 73, 4481-4484.
    [Google Scholar]
  16. Kawaguchi, Y., Nakajima, K., Igarashi, M., Morita, T., Tanaka, M., Suzuki, M., Yokoyama, A., Matsuda, G., Kato, K., Kanamori, M. & Hirai, K. ( 2000; ). Interaction of Epstein–Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP. Journal of Virology 74, 10104-10111.[CrossRef]
    [Google Scholar]
  17. Kieff, E. ( 1996; ). Epstein–Barr virus and its replication. In Fields Virology , pp. 2343-2396. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:Lippincott–Raven.
  18. Kitay, M. K. & Rowe, D. T. ( 1996a; ). Cell cycle stage-specific phosphorylation of the Epstein–Barr virus immortalization protein EBNA-LP. Journal of Virology 70, 7885-7893.
    [Google Scholar]
  19. Kitay, M. K. & Rowe, D. T. ( 1996b; ). Protein–protein interactions between Epstein–Barr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins. Virology 220, 91-99.[CrossRef]
    [Google Scholar]
  20. 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]
  21. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. ( 1987; ). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154, 367-382.
    [Google Scholar]
  22. Laux, G., Adam, B., Strobl, L. J. & Moreaugachelin, F. ( 1994a; ). The Spi-1/Pu.1 and Spi-B Ets family transcription factors and the recombination signal binding-protein RBP-J-Kappa interact with an Epstein–Barr-virus nuclear antigen-2 responsive cis-element. EMBO Journal 13, 5624-5632.
    [Google Scholar]
  23. Laux, G., Dugrillon, F., Eckert, C., Adam, B., Zimber-Strobl, U. & Bornkamm, G. W. ( 1994b; ). Identification and characterization of an Epstein–Barr-virus nuclear antigen 2-responsive cis-element in the bidirectional promoter region of latent membrane-protein and terminal protein-2 genes. Journal of Virology 68, 6947-6958.
    [Google Scholar]
  24. Lee, M. A., Diamond, M. E. & Yates, J. L. ( 1999; ). Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein–Barr virus. Journal of Virology 73, 2974-2982.
    [Google Scholar]
  25. Mannick, J. B., Cohen, J. I., Birkenbach, M., Marchini, A. & Kieff, E. ( 1991; ). The Epstein–Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. Journal of Virology 65, 6826-6837.
    [Google Scholar]
  26. Mannick, J. B., Tong, X., Hemnes, A. & Kieff, E. ( 1995; ). The Epstein–Barr virus nuclear antigen leader protein associates with hsp72/hsc73. Journal of Virology 69, 8169-8172.
    [Google Scholar]
  27. Moghaddam, A., Koch, J., Annis, B. & Wang, F. ( 1998; ). Infection of human B lymphocytes with lymphocryptoviruses related to Epstein–Barr virus. Journal of Virology 72, 3205-3212.
    [Google Scholar]
  28. Nitsche, F., Bell, A. I. & Rickinson, A. ( 1997; ). Epstein–Barr virus leader protein enhances EBNA-2-mediated transactivation of latent membrane protein 1 expression: a role for the W1W2 repeat domain. Journal of Virology 71, 6619-6628.
    [Google Scholar]
  29. Peng, R., Gordadze, A. V., Fuentes-Panana, E. M., Wang, F., Zong, J., Hayward, G. S., Tan, J. & Ling, P. D. ( 2000a; ). Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses. Journal of Virology 74, 379-389.[CrossRef]
    [Google Scholar]
  30. Peng, R., Tan, J. & Ling, P. D. ( 2000b; ). Conserved regions in the Epstein–Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2. Journal of Virology 74, 9953-9963.[CrossRef]
    [Google Scholar]
  31. Rabin, H., Strnad, B. C., Neubauer, R. H., Brown, A. M., Hopkins, R. F. & Mazur, R. A. ( 1980; ). Comparisons of nuclear antigens of Epstein–Barr virus (EBV) and EBV-like simian viruses. Journal of General Virology 48, 265-272.[CrossRef]
    [Google Scholar]
  32. Rikkonen, M., Peranen, J. & Kaariainen, L. ( 1992; ). Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology 189, 462-473.[CrossRef]
    [Google Scholar]
  33. Robertson, E. S., Grossman, S. R., Johannsen, E., Miller, C., Lin, J., Tomkinson, B. & Kieff, E. ( 1995; ). Epstein–Barr-virus nuclear-protein 3C modulates transcription through interaction with the sequence-specific DNA-binding protein J-Kappa. Journal of Virology 69, 3108-3116.
    [Google Scholar]
  34. Sample, J., Hummel, M., Braun, D., Birkenbach, M. & Kieff, E. ( 1986; ). Nucleotide sequences of mRNAs encoding Epstein–Barr virus nuclear proteins: a probable transcriptional initiation site. Proceedings of the National Academy of Sciences, USA 83, 5096-5100.[CrossRef]
    [Google Scholar]
  35. 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]
  36. Sinclair, A. J., Palmero, I., Peters, G. & Farrell, P. J. ( 1994; ). EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein–Barr virus. EMBO Journal 13, 3321-3328.
    [Google Scholar]
  37. Sung, N. S., Kenney, S., Gutsch, D. E. & 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. Szekely, L., Jiang, W. Q., Pokrovskaja, K., Wiman, K. G., Klein, G. & Ringertz, N. ( 1995; ). Reversible nucleolar translocation of Epstein–Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion. Journal of General Virology 76, 2423-2432.[CrossRef]
    [Google Scholar]
  39. Tomkinson, B., Robertson, E. & Kieff, E. ( 1993; ). Epstein–Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. Journal of Virology 67, 2014-2025.
    [Google Scholar]
  40. Tong, X., Drapkin, R., Yalamanchili, R., Mosialos, G. & Kieff, E. ( 1995a; ). The Epstein–Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE. Molecular and Cellular Biology 15, 4735-4744.
    [Google Scholar]
  41. Tong, X., Drapkin, R., Reinberg, D. & Kieff, E. ( 1995b; ). The 62-Kda and 80-Kda subunits of transcription factor IIH mediate the interaction with Epstein–Barr virus nuclear protein-2. Proceedings of the National Academy of Sciences, USA 92, 3259-3263.[CrossRef]
    [Google Scholar]
  42. Tong, X., Wang, F., Thut, C. J. & Kieff, E. ( 1995c; ). The Epstein–Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein. Journal of Virology 69, 585-588.
    [Google Scholar]
  43. 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]
  44. Wang, L., Grossman, S. R. & Kieff, E. ( 2000; ). Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter. Proceedings of the National Academy of Sciences, USA 97, 430-435.[CrossRef]
    [Google Scholar]
  45. Yokoyama, A., Tanaka, M., Matsuda, G., Kato, K., Kanamori, M., Kawasaki, H., Hirano, H., Kitabayashi, I., Ohki, M., Hirai, K. & Kawaguchi, Y. ( 2001; ). Identification of major phosphorylation sites of Epstein–Barr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA2 is regulated by phosphorylation. Journal of Virology 75, 5119-5128.[CrossRef]
    [Google Scholar]
  46. Young, L. S., Dawson, C. W. & Eliopoulos, A. G. ( 2000; ). The expression and function of Epstein–Barr virus encoded latent genes. Journal of Clinical Pathology: Molecular Pathology 53, 238-247.[CrossRef]
    [Google Scholar]
  47. Zhao, B., Marshall, D. R. & Sample, C. E. ( 1996; ). A conserved domain of the Epstein–Barr virus nuclear antigens 3A and 3C binds to a discrete domain of J kappa. Journal of Virology 70, 4228-4236.
    [Google Scholar]
  48. 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]
  49. Zimber-Strobl, U., Strobl, L. J., Meitinger, C., Hinrichs, R., Sakai, T., Furukawa, T., Honjo, T. & Bornkamm, G. W. ( 1994; ). Epstein–Barr virus nuclear antigen 2 exerts its transactivating function through interaction with recombination signal binding-protein RBP-J-Kappa, the homolog of Drosophila Suppressor of Hairless. EMBO Journal 13, 4973-4982.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-12-3067
Loading
/content/journal/jgv/10.1099/0022-1317-82-12-3067
Loading

Data & Media loading...

Most Cited This Month

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