Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Apr;66(4):1899–1906. doi: 10.1128/jvi.66.4.1899-1906.1992

BHRF1 of Epstein-Barr virus, which is homologous to human proto-oncogene bcl2, is not essential for transformation of B cells or for virus replication in vitro.

M A Lee 1, J L Yates 1
PMCID: PMC288977  PMID: 1312610

Abstract

The Epstein-Barr virus (EBV) genome contains an open reading frame, BHRF1, that encodes a presumptive membrane protein with sequence similarity to the proto-oncogene bcl2, which is linked to human B-cell follicular lymphoma. Potential roles for BHRF1 in EBV's ability to growth transform human B cells and to replicate in B cells in culture were investigated by generating EBV mutants that lack most of the open reading frame. This was accomplished by recombination of plasmids carrying mutations in BHRF1 with the transformation-defective EBV strain P3HR1. Because BHRF1 resides close to the deletion in P3HR1 that renders this strain transformation defective, B-cell transformation could be used to select for recombination events in the region. B-cell clones were established by recombinants which lacked most of the BHRF1 open reading frame, although most of these initial B-cell transformants also carried nonrecombinant (BHRF1+) P3HR1 genomes, at levels ranging from a fraction of a copy to four copies per cell. Secondary B-cell transformants that lacked BHRF1+ EBV at detectable levels were found to release transforming, BHRF1-deficient EBV at levels that were within the normal range for EBV-immortalized B-cell clones. These studies demonstrate that BHRF1 is nonessential for growth transformation of B cells and for virus replication and release from these cells in culture.

Full text

PDF
1899

Images in this article

1902Image
on p.1902
1904Image
on p.1904

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Austin P. J., Flemington E., Yandava C. N., Strominger J. L., Speck S. H. Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3678–3682. doi: 10.1073/pnas.85.11.3678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Baichwal V. R., Sugden B. The multiple membrane-spanning segments of the BNLF-1 oncogene from Epstein-Barr virus are required for transformation. Oncogene. 1989 Jan;4(1):67–74. [PubMed] [Google Scholar]
  4. Bodescot M., Perricaudet M. Epstein-Barr virus mRNAs produced by alternative splicing. Nucleic Acids Res. 1986 Sep 11;14(17):7103–7114. doi: 10.1093/nar/14.17.7103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cleary M. L., Smith S. D., Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 1986 Oct 10;47(1):19–28. doi: 10.1016/0092-8674(86)90362-4. [DOI] [PubMed] [Google Scholar]
  6. Cohen J. I., Wang F., Mannick J., Kieff E. Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9558–9562. doi: 10.1073/pnas.86.23.9558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Countryman J., Miller G. Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4085–4089. doi: 10.1073/pnas.82.12.4085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hammerschmidt W., Sugden B. Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes. Nature. 1989 Aug 3;340(6232):393–397. doi: 10.1038/340393a0. [DOI] [PubMed] [Google Scholar]
  9. Hammerschmidt W., Sugden B. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell. 1988 Nov 4;55(3):427–433. doi: 10.1016/0092-8674(88)90028-1. [DOI] [PubMed] [Google Scholar]
  10. Hardwick J. M., Lieberman P. M., Hayward S. D. A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. J Virol. 1988 Jul;62(7):2274–2284. doi: 10.1128/jvi.62.7.2274-2284.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henderson S., Rowe M., Gregory C., Croom-Carter D., Wang F., Longnecker R., Kieff E., Rickinson A. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell. 1991 Jun 28;65(7):1107–1115. doi: 10.1016/0092-8674(91)90007-l. [DOI] [PubMed] [Google Scholar]
  12. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  13. Jeang K. T., Hayward S. D. Organization of the Epstein-Barr virus DNA molecule. III. Location of the P3HR-1 deletion junction and characterization of the NotI repeat units that form part of the template for an abundant 12-O-tetradecanoylphorbol-13-acetate-induced mRNA transcript. J Virol. 1983 Oct;48(1):135–148. doi: 10.1128/jvi.48.1.135-148.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaplan M. E., Clark C. An improved rosetting assay for detection of human T lymphocytes. J Immunol Methods. 1974 Jul;5(2):131–135. doi: 10.1016/0022-1759(74)90003-9. [DOI] [PubMed] [Google Scholar]
  15. McDonnell T. J., Deane N., Platt F. M., Nunez G., Jaeger U., McKearn J. P., Korsmeyer S. J. bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell. 1989 Apr 7;57(1):79–88. doi: 10.1016/0092-8674(89)90174-8. [DOI] [PubMed] [Google Scholar]
  16. McDonnell T. J., Nunez G., Platt F. M., Hockenberry D., London L., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population. Mol Cell Biol. 1990 May;10(5):1901–1907. doi: 10.1128/mcb.10.5.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nunez G., Seto M., Seremetis S., Ferrero D., Grignani F., Korsmeyer S. J., Dalla-Favera R. Growth- and tumor-promoting effects of deregulated BCL2 in human B-lymphoblastoid cells. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4589–4593. doi: 10.1073/pnas.86.12.4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pearson G. R., Luka J., Petti L., Sample J., Birkenbach M., Braun D., Kieff E. Identification of an Epstein-Barr virus early gene encoding a second component of the restricted early antigen complex. Virology. 1987 Sep;160(1):151–161. doi: 10.1016/0042-6822(87)90055-9. [DOI] [PubMed] [Google Scholar]
  19. Rabson M., Heston L., Miller G. Identification of a rare Epstein-Barr virus variant that enhances early antigen expression in Raji cells. Proc Natl Acad Sci U S A. 1983 May;80(9):2762–2766. doi: 10.1073/pnas.80.9.2762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reed J. C., Cuddy M., Haldar S., Croce C., Nowell P., Makover D., Bradley K. BCL2-mediated tumorigenicity of a human T-lymphoid cell line: synergy with MYC and inhibition by BCL2 antisense. Proc Natl Acad Sci U S A. 1990 May;87(10):3660–3664. doi: 10.1073/pnas.87.10.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reed J. C., Haldar S., Cuddy M. P., Croce C., Makover D. Deregulated BCL2 expression enhances growth of a human B cell line. Oncogene. 1989 Sep;4(9):1123–1127. [PubMed] [Google Scholar]
  22. Rooney C., Howe J. G., Speck S. H., Miller G. Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus. J Virol. 1989 Apr;63(4):1531–1539. doi: 10.1128/jvi.63.4.1531-1539.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  24. Stinski M. F., Roehr T. J. Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. J Virol. 1985 Aug;55(2):431–441. doi: 10.1128/jvi.55.2.431-441.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Strasser A., Harris A. W., Bath M. L., Cory S. Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature. 1990 Nov 22;348(6299):331–333. doi: 10.1038/348331a0. [DOI] [PubMed] [Google Scholar]
  26. Sugden B., Marsh K., Yates J. A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985 Feb;5(2):410–413. doi: 10.1128/mcb.5.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Swaminathan S., Tomkinson B., Kieff E. Recombinant Epstein-Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1546–1550. doi: 10.1073/pnas.88.4.1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsujimoto Y. Stress-resistance conferred by high level of bcl-2 alpha protein in human B lymphoblastoid cell. Oncogene. 1989 Nov;4(11):1331–1336. [PubMed] [Google Scholar]
  29. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  30. Wang D., Liebowitz D., Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell. 1985 Dec;43(3 Pt 2):831–840. doi: 10.1016/0092-8674(85)90256-9. [DOI] [PubMed] [Google Scholar]
  31. Wang D., Liebowitz D., Kieff E. The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts. J Virol. 1988 Jul;62(7):2337–2346. doi: 10.1128/jvi.62.7.2337-2346.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang F., Gregory C. D., Rowe M., Rickinson A. B., Wang D., Birkenbach M., Kikutani H., Kishimoto T., Kieff E. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23. Proc Natl Acad Sci U S A. 1987 May;84(10):3452–3456. doi: 10.1073/pnas.84.10.3452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang F., Gregory C., Sample C., Rowe M., Liebowitz D., Murray R., Rickinson A., Kieff E. Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol. 1990 May;64(5):2309–2318. doi: 10.1128/jvi.64.5.2309-2318.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang F., Marchini A., Kieff E. Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells. J Virol. 1991 Apr;65(4):1701–1709. doi: 10.1128/jvi.65.4.1701-1709.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang F., Tsang S. F., Kurilla M. G., Cohen J. I., Kieff E. Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. J Virol. 1990 Jul;64(7):3407–3416. doi: 10.1128/jvi.64.7.3407-3416.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson G., Miller G. Recovery of Epstein-Barr virus from nonproducer neonatal human lymphoid cell transformants. Virology. 1979 Jun;95(2):351–358. doi: 10.1016/0042-6822(79)90490-2. [DOI] [PubMed] [Google Scholar]
  37. Wysokenski D. A., Yates J. L. Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus. J Virol. 1989 Jun;63(6):2657–2666. doi: 10.1128/jvi.63.6.2657-2666.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yates J. L., Warren N., Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. 1985 Feb 28-Mar 6Nature. 313(6005):812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES