Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Oct;70(10):7260–7263. doi: 10.1128/jvi.70.10.7260-7263.1996

Clonal propagation of Epstein-Barr virus (EBV) recombinants in EBV-negative Akata cells.

N Shimizu 1, H Yoshiyama 1, K Takada 1
PMCID: PMC190785  PMID: 8794379

Abstract

We lack a host cell supporting an efficient lytic replication of Epstein-Barr virus (EBV). Recently, we isolated EBV-negative cell clones from the Akata cell line (referred as Akata- [N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069-6073, 1994). Since the parental Akata line is one of the highest EBV producers, we examined whether Akata- cells had become a good host for EBV propagation. The parental Akata cells have about 20 copies of EBV plasmid per cell. A drug resistance gene was inserted into one of them by homologous recombination. The resultant virus preparation, a mixture of wild-type and recombinant EBV, was used to infect Akata- cells. After incubation in the selective medium, drug-resistant Akata- cell clones were isolated and proved to be infected with recombinant EBV only. By treatment of the cells with antiimmunoglobulin antibodies, a large amount of recombinant EBV (i.e., more than 10 microg/1-liter culture) was produced. In contrast, three other B-lymphoma lines, BJAB, Ramos, and Louckes, were nonpermissive for virus replication. These results indicate that Akata- cells are suitable for propagation of recombinant EBV clonally, which becomes a powerful tool for determining EBV genetics and which makes it possible to use EBV as a vector for gene therapy.

Full Text

The Full Text of this article is available as a PDF (301.0 KB).

Selected References

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

  1. 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]
  2. Bloss T. A., Sugden B. Optimal lengths for DNAs encapsidated by Epstein-Barr virus. J Virol. 1994 Dec;68(12):8217–8222. doi: 10.1128/jvi.68.12.8217-8222.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chevallier-Greco A., Manet E., Chavrier P., Mosnier C., Daillie J., Sergeant A. Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter. EMBO J. 1986 Dec 1;5(12):3243–3249. doi: 10.1002/j.1460-2075.1986.tb04635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Dolyniuk M., Pritchett R., Kieff E. Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus. J Virol. 1976 Mar;17(3):935–949. doi: 10.1128/jvi.17.3.935-949.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Klein G., Giovanella B., Westman A., Stehlin J. S., Mumford D. An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics. EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection. Intervirology. 1975;5(6):319–334. doi: 10.1159/000149930. [DOI] [PubMed] [Google Scholar]
  9. Klein G., Lindahl T., Jondal M., Leibold W., Menézes J., Nilsson K., Sundström C. Continuous lymphoid cell lines with characteristics of B cells (bone-marrow-derived), lacking the Epstein-Barr virus genome and derived from three human lymphomas. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3283–3286. doi: 10.1073/pnas.71.8.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Korman A. J., Frantz J. D., Strominger J. L., Mulligan R. C. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. doi: 10.1073/pnas.84.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lee M. A., Yates J. L. 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. J Virol. 1992 Apr;66(4):1899–1906. doi: 10.1128/jvi.66.4.1899-1906.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Marchini A., Longnecker R., Kieff E. Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants. J Virol. 1992 Aug;66(8):4972–4981. doi: 10.1128/jvi.66.8.4972-4981.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miller N., Hutt-Fletcher L. M. A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus. J Virol. 1988 Jul;62(7):2366–2372. doi: 10.1128/jvi.62.7.2366-2372.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Robertson E. S., Tomkinson B., Kieff E. An Epstein-Barr virus with a 58-kilobase-pair deletion that includes BARF0 transforms B lymphocytes in vitro. J Virol. 1994 Mar;68(3):1449–1458. doi: 10.1128/jvi.68.3.1449-1458.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shimizu N., Tanabe-Tochikura A., Kuroiwa Y., Takada K. Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV. J Virol. 1994 Sep;68(9):6069–6073. doi: 10.1128/jvi.68.9.6069-6073.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Takada K. Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines. Int J Cancer. 1984 Jan 15;33(1):27–32. doi: 10.1002/ijc.2910330106. [DOI] [PubMed] [Google Scholar]
  17. Takada K., Fujiwara S., Yano S., Osato T. Monoclonal antibody specific for capsid antigen of Epstein-Barr virus. Med Microbiol Immunol. 1983;171(4):225–231. doi: 10.1007/BF02123496. [DOI] [PubMed] [Google Scholar]
  18. Takada K., Horinouchi K., Ono Y., Aya T., Osato T., Takahashi M., Hayasaka S. An Epstein-Barr virus-producer line Akata: establishment of the cell line and analysis of viral DNA. Virus Genes. 1991 Apr;5(2):147–156. doi: 10.1007/BF00571929. [DOI] [PubMed] [Google Scholar]
  19. Takada K., Ono Y. Synchronous and sequential activation of latently infected Epstein-Barr virus genomes. J Virol. 1989 Jan;63(1):445–449. doi: 10.1128/jvi.63.1.445-449.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Takada K., Shimizu N., Sakuma S., Ono Y. trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment. J Virol. 1986 Mar;57(3):1016–1022. doi: 10.1128/jvi.57.3.1016-1022.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tomkinson B., Kieff E. Second-site homologous recombination in Epstein-Barr virus: insertion of type 1 EBNA 3 genes in place of type 2 has no effect on in vitro infection. J Virol. 1992 Feb;66(2):780–789. doi: 10.1128/jvi.66.2.780-789.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tomkinson B., Robertson E., Yalamanchili R., Longnecker R., Kieff E. Epstein-Barr virus recombinants from overlapping cosmid fragments. J Virol. 1993 Dec;67(12):7298–7306. doi: 10.1128/jvi.67.12.7298-7306.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yoshiyama H., Shimizu N., Takada K. Persistent Epstein-Barr virus infection in a human T-cell line: unique program of latent virus expression. EMBO J. 1995 Aug 1;14(15):3706–3711. doi: 10.1002/j.1460-2075.1995.tb00040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van Santen V., Cheung A., Kieff E. Epstein-Barr virus RNA VII: size and direction of transcription of virus-specified cytoplasmic RNAs in a transformed cell line. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1930–1934. doi: 10.1073/pnas.78.3.1930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. zur Hausen H., O'Neill F. J., Freese U. K., Hecker E. Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature. 1978 Mar 23;272(5651):373–375. doi: 10.1038/272373a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES