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. 1991 Mar;65(3):1245–1254. doi: 10.1128/jvi.65.3.1245-1254.1991

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

E A Hurley 1, S Agger 1, J A McNeil 1, J B Lawrence 1, A Calendar 1, G Lenoir 1, D A Thorley-Lawson 1
PMCID: PMC239896  PMID: 1847452

Abstract

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.

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Selected References

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  1. Adams A., Lindahl T., Klein G. Linear association between cellular DNA and Epstein-Barr virus DNA in a human lymphoblastoid cell line. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2888–2892. doi: 10.1073/pnas.70.10.2888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aman P., Ehlin-Henriksson B., Klein G. Epstein-Barr virus susceptibility of normal human B lymphocyte populations. J Exp Med. 1984 Jan 1;159(1):208–220. doi: 10.1084/jem.159.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andersson-Anvret M., Lindahl T. Integrated viral DNA sequences in Epstein-Barr virus-converted human lymphoma lines. J Virol. 1978 Mar;25(3):710–718. doi: 10.1128/jvi.25.3.710-718.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Azim T., Allday M. J., Crawford D. H. Immortalization of Epstein-Barr virus-infected CD23-negative B lymphocytes by the addition of B cell growth factor. J Gen Virol. 1990 Mar;71(Pt 3):665–671. doi: 10.1099/0022-1317-71-3-665. [DOI] [PubMed] [Google Scholar]
  5. Brandsma J., Miller G. Nucleic acid spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6851–6855. doi: 10.1073/pnas.77.11.6851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Calender A., Billaud M., Aubry J. P., Banchereau J., Vuillaume M., Lenoir G. M. Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8060–8064. doi: 10.1073/pnas.84.22.8060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gardella T., Medveczky P., Sairenji T., Mulder C. Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. J Virol. 1984 Apr;50(1):248–254. doi: 10.1128/jvi.50.1.248-254.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gregory C. D., Tursz T., Edwards C. F., Tetaud C., Talbot M., Caillou B., Rickinson A. B., Lipinski M. Identification of a subset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype. J Immunol. 1987 Jul 1;139(1):313–318. [PubMed] [Google Scholar]
  9. Harris A., Young B. D., Griffin B. E. Random association of Epstein-Barr virus genomes with host cell metaphase chromosomes in Burkitt's lymphoma-derived cell lines. J Virol. 1985 Oct;56(1):328–332. doi: 10.1128/jvi.56.1.328-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henderson A., Ripley S., Heller M., Kieff E. Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1987–1991. doi: 10.1073/pnas.80.7.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hurley E. A., Thorley-Lawson D. A. B cell activation and the establishment of Epstein-Barr virus latency. J Exp Med. 1988 Dec 1;168(6):2059–2075. doi: 10.1084/jem.168.6.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaschka-Dierich C., Adams A., Lindahl T., Bornkamm G. W., Bjursell G., Klein G., Giovanella B. C., Singh S. Intracellular forms of Epstein-Barr virus DNA in human tumour cells in vivo. Nature. 1976 Mar 25;260(5549):302–306. doi: 10.1038/260302a0. [DOI] [PubMed] [Google Scholar]
  13. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [DOI] [PubMed] [Google Scholar]
  14. Matsuo T., Heller M., Petti L., O'Shiro E., Kieff E. Persistence of the entire Epstein-Barr virus genome integrated into human lymphocyte DNA. Science. 1984 Dec 14;226(4680):1322–1325. doi: 10.1126/science.6095452. [DOI] [PubMed] [Google Scholar]
  15. Murray R. J., Young L. S., Calender A., Gregory C. D., Rowe M., Lenoir G. M., Rickinson A. B. Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains. J Virol. 1988 Mar;62(3):894–901. doi: 10.1128/jvi.62.3.894-901.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nonoyama M., Pagano J. S. Detection of Epstein-Barr viral genome in nonproductive cells. Nat New Biol. 1971 Sep 22;233(38):103–106. doi: 10.1038/newbio233103a0. [DOI] [PubMed] [Google Scholar]
  17. Raab-Traub N., Flynn K. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell. 1986 Dec 26;47(6):883–889. doi: 10.1016/0092-8674(86)90803-2. [DOI] [PubMed] [Google Scholar]
  18. Robinson J., Frank A., Henderson E., Schweitzer J., Miller G. Surface markers and size of lymphocytes in human umbilical cord blood stimulated into deoxyribonucleic acid synthesis by Epstein-Barr Virus. Infect Immun. 1979 Oct;26(1):225–231. doi: 10.1128/iai.26.1.225-231.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rooney C. M., Gregory C. D., Rowe M., Finerty S., Edwards C., Rupani H., Rickinson A. B. Endemic Burkitt's lymphoma: phenotypic analysis of tumor biopsy cells and of derived tumor cell lines. J Natl Cancer Inst. 1986 Sep;77(3):681–687. doi: 10.1093/jnci/77.3.681. [DOI] [PubMed] [Google Scholar]
  20. Sixbey J. W., Pagano J. S. Biotin-labeled DNA probes for detection of Epstein-Barr virus by in-situ cytohybridization. Clin Lab Med. 1985 Sep;5(3):503–512. [PubMed] [Google Scholar]
  21. Sugden B., Phelps M., Domoradzki J. Epstein-Barr virus DNA is amplified in transformed lymphocytes. J Virol. 1979 Sep;31(3):590–595. doi: 10.1128/jvi.31.3.590-595.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thorley-Lawson D. A., Mann K. P. Early events in Epstein-Barr virus infection provide a model for B cell activation. J Exp Med. 1985 Jul 1;162(1):45–59. doi: 10.1084/jem.162.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Yates J. L., Guan N. Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. J Virol. 1991 Jan;65(1):483–488. doi: 10.1128/jvi.65.1.483-488.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]

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