1887

Abstract

Epstein–Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier , 77, 2807–2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFPLMP2 recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2 virus to immortalize primary human B cells when compared to that of wild-type virus.

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1999-08-01
2020-01-24
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References

  1. Brielmeier, M., Mautner, J., Laux, G. & Hammerschmidt, W. ( 1996; ). The latent membrane protein 2 gene of Epstein–Barr virus is important for efficient B cell immortalization. Journal of General Virology 77, 2807-2818.[CrossRef]
    [Google Scholar]
  2. Burkhardt, A. L., Bolen, J. B., Kieff, E. & Longnecker, R. ( 1992; ). An Epstein–Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases. Journal of Virology 66, 5161-5167.
    [Google Scholar]
  3. Caldwell, R. G., Wilson, J. B., Anderson, S. J. & Longnecker, R. ( 1998; ). Epstein–Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9, 405-411.[CrossRef]
    [Google Scholar]
  4. Chalfie, M. ( 1995; ). Green fluorescent protein. Photochemistry and Photobiology 62, 651-656.[CrossRef]
    [Google Scholar]
  5. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. ( 1994; ). Green fluorescent protein as a marker for gene expression. Science 263, 802-805.[CrossRef]
    [Google Scholar]
  6. Chang, R. S. & Lung, M. L. ( 1994; ). A modified procedure for the propagation of wild-type Epstein–Barr virus in cultures of marmoset blood cells. Journal of Virological Methods 46, 167-178.[CrossRef]
    [Google Scholar]
  7. Chen, F., Zou, J. Z., di Renzo, L., Winberg, G., Hu, L. F., Klein, E., Klein, G. & Ernberg, I. ( 1995; ). A subpopulation of normal B cells latently infected with Epstein–Barr virus resembles Burkitt lymphona cells in expressing EBNA-1 but not EBNA-2 or LMP1. Journal of Virology 69, 3752-3758.
    [Google Scholar]
  8. Cormack, B. P., Valdivia, R. H. & Falkow, S. ( 1996; ). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-38.[CrossRef]
    [Google Scholar]
  9. Fruehling, S. & Longnecker, R. ( 1997; ). The immunoreceptor tyrosine-based activation motif of Epstein–Barr virus LMP2A is essential for blocking BCR-mediated signal transduction. Virology 235, 241-251.[CrossRef]
    [Google Scholar]
  10. Fruehling, S., Lee, S. K., Herrold, R., Frech, B., Laux, G., Kremmer, E., Grasser, F. A. & Longnecker, R. ( 1996; ). Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction. Journal of Virology 70, 6216-6226.
    [Google Scholar]
  11. Fruehling, S., Swart, R., Dolwick, K. M., Kremmer, E. & Longnecker, R. ( 1998; ). Tyrosine 112 of latent membrane protein 2A is essential for protein tyrosine kinase loading and regulation of Epstein–Barr virus latency. Journal of Virology 72, 7796-7806.
    [Google Scholar]
  12. Heim, R. & Tsien, R. Y. ( 1996; ). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology 6, 178-182.[CrossRef]
    [Google Scholar]
  13. Heim, R., Cubitt, A. B. & Tsien, R. Y. ( 1995; ). Improved green fluorescence (letter). Nature 373, 663-664.
    [Google Scholar]
  14. Herold, B. C., WuDunn, D., Soltys, N. & Spear, P. G. ( 1991; ). Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. Journal of Virology 65, 1090-1098.
    [Google Scholar]
  15. Hummeler, K., Henle, G. & Henle, W. ( 1966; ). Fine structure of a virus in cultured lymphoblasts from Burkitt lymphoma. Journal of Bacteriology 91, 1366-1368.
    [Google Scholar]
  16. Kaye, K. M., Izumi, K. M. & Kieff, E. ( 1993; ). Epstein–Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proceedings of the National Academy of Sciences, USA 90, 9150-9154.[CrossRef]
    [Google Scholar]
  17. Kempkes, B., Pich, D., Zeidler, R. & Hammerschmidt, W. ( 1995a; ). Immortalization of human primary B lymphocytes in vitro with DNA. Proceedings of the National Academy of Sciences, USA 92, 5875-5879.[CrossRef]
    [Google Scholar]
  18. Kempkes, B., Pich, D., Zeidler, R., Sugden, B. & Hammerschmidt, W. ( 1995b; ). Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein–Barr virus DNA. Journal of Virology 69, 231-238.
    [Google Scholar]
  19. Kieff, E. ( 1996; ). Epstein–Barr virus and its replication. In Fundamental Virology, pp. 1109-1162. Edited by B. Fields, D. Knipe & P. Howley. New York: Raven Press.
  20. Kim, O. J. & Yates, J. L. ( 1993; ). Mutants of Epstein–Barr virus with a selective marker disrupting the TP gene transform B cells and replicate normally in culture. Journal of Virology 67, 7634-7640.
    [Google Scholar]
  21. 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, USA 71, 3283-3286.[CrossRef]
    [Google Scholar]
  22. Klein, G., Clements, G., Zeuthen, J. & Westman, A. ( 1976; ). Somatic cell hybrids between human lymphoma lines. II. Spontaneous and induced patterns of the Epstein–Barr virus (EBV) cycle. International Journal of Cancer 17, 715-724.[CrossRef]
    [Google Scholar]
  23. Laux, G., Perricaudet, M. & Farrell, P. J. ( 1988; ). A spliced Epstein–Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome. EMBO Journal 7, 769-774.
    [Google Scholar]
  24. Laux, G., Economou, A. & Farrell, P. J. ( 1989; ). The terminal protein gene 2 of Epstein–Barr virus is transcribed from a bidirectional latent promoter region. Journal of General Virology 70, 3079-3084.[CrossRef]
    [Google Scholar]
  25. Longnecker, R. ( 1998; ). Molecular biology of Epstein–Barr virus. In Human Tumor Viruses, pp. 133-172. Edited by D. McCance. Washington DC: ASM Press.
  26. Longnecker, R. & Miller, C. ( 1996; ). Regulation of Epstein–Barr virus latency by latent membrane protein 2. Trends in Microbiology 4, 38-42.
    [Google Scholar]
  27. Longnecker, R., Druker, B., Roberts, T. M. & Kieff, E. ( 1991; ). An Epstein–Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase. Journal of Virology 65, 3681-3692.
    [Google Scholar]
  28. Longnecker, R., Miller, C. L., Miao, X.-Q., Marchini, A. & Kieff, E. ( 1992; ). The only domain which distinguishes Epstein–Barr virus latent membrane 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro, and LMP2A is therefore nonessential. Journal of Virology 66, 6461-6469.
    [Google Scholar]
  29. Longnecker, R., Miller, C. L., Miao, X.-Q., Tomkinson, B. & Kieff, E. ( 1993a; ). The last seven transmembrane and carboxy terminal cytoplasmic domains of Epstein–Barr virus latent membrane protein 2 (LMP2) are dispensable for lymphocyte infection and growth transformation in vitro. Journal of Virology 67, 2006-2013.
    [Google Scholar]
  30. Longnecker, R., Miller, C. L., Tomkinson, B., Miao, X. Q. & Kieff, E. ( 1993b; ). Deletion of DNA encoding the first five transmembrane domains of Epstein–Barr virus latent membrane proteins 2A and 2B. Journal of Virology 67, 5068-5074.
    [Google Scholar]
  31. Marchini, A., Tomkinson, B., Cohen, J. I. & Kieff, E. ( 1991; ). BHRF1, the Epstein–Barr virus gene with homology to Bc12, is dispensable for B-lymphocyte transformation and virus replication. Journal of Virology 65, 5991-6000.
    [Google Scholar]
  32. Miller, G. & Lipman, M. ( 1973; ). Release of infectious Epstein–Barr virus by transformed marmoset leukocytes. Proceedings of the National Academy of Sciences, USA 70, 190-194.[CrossRef]
    [Google Scholar]
  33. Miller, G., Shope, T., Lisco, H., Stitt, D. & Lipman, M. ( 1972; ). Epstein–Barr virus: transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proceedings of the National Academy of Sciences, USA 69, 383-387.[CrossRef]
    [Google Scholar]
  34. Miller, C. L., Lee, J. H., Kieff, E. & Longnecker, R. ( 1994; ). An integral membrane protein (LMP2) blocks reactivation of Epstein–Barr virus from latency following surface immunoglobulin crosslinking. Proceedings of the National Academy of Sciences, USA 91, 772-776.[CrossRef]
    [Google Scholar]
  35. Miller, C. L., Burkhardt, A. L., Lee, J. H., Stealey, B., Longnecker, R., Bolen, J. B. & Kieff, E. ( 1995; ). Integral membrane protein 2 of Epstein–Barr virus regulates reactivation from latency through dominant negative effects on protein-tyrosine kinases. Immunity 2, 155-166.[CrossRef]
    [Google Scholar]
  36. Miyashita, E. M., Yang, B., Babcock, G. J. & Thorley-Lawson, D. A. ( 1997; ). Identification of the site of Epstein–Barr virus persistence in vivo as a resting B cell. Journal of Virology 71, 4882-4891.
    [Google Scholar]
  37. Okano, M., Taguchi, Y., Nakamine, H., Pirruccello, S. J., Davis, J. R., Beisel, K. W., Kleveland, K. L., Sanger, W. G., Fordyce, R. R. & Purtilo, D. T. ( 1990; ). Characterization of Epstein–Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice. American Journal of Pathology 137, 517-522.
    [Google Scholar]
  38. Qu, L. & Rowe, D. ( 1992; ). Epstein–Barr virus latent gene expression in uncultured peripheral blood lymphocytes. Journal of Virology 66, 3715-3724.
    [Google Scholar]
  39. Rickinson, A. & Kieff, E. ( 1996; ). Epstein–Barr virus. In Fields Virology, pp. 2397-2446. Edited by B. Fields, D. Knipe & P. Howley. Philadelphia: Lippincott–Raven.
  40. Sample, J., Liebowitz, D. & Kieff, E. ( 1989; ). Two related Epstein–Barr virus membrane proteins are encoded by separate genes. Journal of Virology 63, 933-937.
    [Google Scholar]
  41. Sugden, B. & Mark, W. ( 1977; ). Clonal transformation of adult human leukocytes by Epstein–Barr virus. Journal of Virology 23, 503-508.
    [Google Scholar]
  42. Swaminathan, S., Tomkinson, B. & Kieff, E. ( 1991; ). Recombinant Epstein–Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro. Proceedings of the National Academy of Sciences, USA 88, 1546-1550.[CrossRef]
    [Google Scholar]
  43. Tierney, R. J., Steven, N., Young, L. S. & Rickinson, A. B. ( 1994; ). Epstein–Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. Journal of Virology 68, 7374-7385.
    [Google Scholar]
  44. Toplin, I. & Schidlovsky, G. ( 1966; ). Partial purification and electron microscopy of virus in the EB-3 cell line derived from a Burkitt lymphoma. Science 152, 1084-1085.[CrossRef]
    [Google Scholar]
  45. Vahey, M. T., Wong, M. T. & Michael, N. L. ( 1995; ). A standard PCR protocol: rapid isolation of DNA and PCR assay for β-globin. In PCR Primer: A Laboratory Manual, pp. 17-22. Edited by C. W. Dieffenbach & G. S. Dveksler. New York: Cold Spring Harbor Laboratory Press.
  46. Wang, F., Marchini, A. & Kieff, E. ( 1991; ). Epstein–Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells. Journal of Virology 65, 1701-1709.
    [Google Scholar]
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