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. 1992 Aug;66(8):4972–4981. doi: 10.1128/jvi.66.8.4972-4981.1992

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

A Marchini 1, R Longnecker 1, E Kieff 1
PMCID: PMC241347  PMID: 1321281

Abstract

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.

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Selected References

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

  1. Ahearn J. M., Hayward S. D., Hickey J. C., Fearon D. T. Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9307–9311. doi: 10.1073/pnas.85.23.9307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Baichwal V. R., Sugden B. Transformation of Balb 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene. 1988 May;2(5):461–467. [PubMed] [Google Scholar]
  5. 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]
  6. Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cohen J. I., Wang F., Kieff E. Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation. J Virol. 1991 May;65(5):2545–2554. doi: 10.1128/jvi.65.5.2545-2554.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cordier M., Calender A., Billaud M., Zimber U., Rousselet G., Pavlish O., Banchereau J., Tursz T., Bornkamm G., Lenoir G. M. 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. J Virol. 1990 Mar;64(3):1002–1013. doi: 10.1128/jvi.64.3.1002-1013.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Countryman J., Jenson H., Seibl R., Wolf H., Miller G. Polymorphic proteins encoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency. J Virol. 1987 Dec;61(12):3672–3679. doi: 10.1128/jvi.61.12.3672-3679.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  15. Grogan E., Jenson H., Countryman J., Heston L., Gradoville L., Miller G. Transfection of a rearranged viral DNA fragment, WZhet, stably converts latent Epstein-Barr viral infection to productive infection in lymphoid cells. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1332–1336. doi: 10.1073/pnas.84.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Heller M., Dambaugh T., Kieff E. Epstein-Barr virus DNA. IX. Variation among viral DNAs from producer and nonproducer infected cells. J Virol. 1981 May;38(2):632–648. doi: 10.1128/jvi.38.2.632-648.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hurley E. A., Agger S., McNeil J. A., Lawrence J. B., Calendar A., Lenoir G., Thorley-Lawson D. A. When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates. J Virol. 1991 Mar;65(3):1245–1254. doi: 10.1128/jvi.65.3.1245-1254.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hurley E. A., McNeil J. A., Lawrence J. B., Thorley-Lawson D. A. Genomic integration as a novel mechanism of EBV persistence. Curr Top Microbiol Immunol. 1990;166:367–374. doi: 10.1007/978-3-642-75889-8_45. [DOI] [PubMed] [Google Scholar]
  22. Kaschka-Dierich C., Falk L., Bjursell G., Adams A., Lindahl T. Human lymphoblastoid cell lines derived from individuals without lymphoproliferative disease contain the same latent forms of Epstein-Barr virus DNA as those found in tumor cells. Int J Cancer. 1977 Aug 15;20(2):173–180. doi: 10.1002/ijc.2910200203. [DOI] [PubMed] [Google Scholar]
  23. King W., Thomas-Powell A. L., Raab-Traub N., Hawke M., Kieff E. Epstein-Barr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line. J Virol. 1980 Nov;36(2):506–518. doi: 10.1128/jvi.36.2.506-518.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Klein G., Zeuthen J., Terasaki P., Billing R., Honig R., Jondal M., Westman A., Clements G. Inducibility of the Epstein-Barr virus (EBV) cycle and surface marker properties of EBV-negative lymphoma lines and their in vitro EBV-converted sublines. Int J Cancer. 1976 Nov 15;18(5):639–652. doi: 10.1002/ijc.2910180513. [DOI] [PubMed] [Google Scholar]
  26. Knutson J. C. The level of c-fgr RNA is increased by EBNA-2, an Epstein-Barr virus gene required for B-cell immortalization. J Virol. 1990 Jun;64(6):2530–2536. doi: 10.1128/jvi.64.6.2530-2536.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kocache M. M., Pearson G. R. Protein kinase activity associated with a cell cycle regulated, membrane-bound Epstein-Barr virus induced early antigen. Intervirology. 1990;31(1):1–13. doi: 10.1159/000150128. [DOI] [PubMed] [Google Scholar]
  28. Longnecker R., Druker B., Roberts T. M., Kieff E. An Epstein-Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase. J Virol. 1991 Jul;65(7):3681–3692. doi: 10.1128/jvi.65.7.3681-3692.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mannick J. B., Cohen J. I., Birkenbach M., Marchini A., Kieff E. The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. J Virol. 1991 Dec;65(12):6826–6837. doi: 10.1128/jvi.65.12.6826-6837.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marchini A., Cohen J. I., Wang F., Kieff E. A selectable marker allows investigation of a nontransforming Epstein-Barr virus mutant. J Virol. 1992 May;66(5):3214–3219. doi: 10.1128/jvi.66.5.3214-3219.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marchini A., Tomkinson B., Cohen J. I., Kieff E. BHRF1, the Epstein-Barr virus gene with homology to Bc12, is dispensable for B-lymphocyte transformation and virus replication. J Virol. 1991 Nov;65(11):5991–6000. doi: 10.1128/jvi.65.11.5991-6000.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Masucci M. G., Contreras-Salazar B., Ragnar E., Falk K., Minarovits J., Ernberg I., Klein G. 5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael. J Virol. 1989 Jul;63(7):3135–3141. doi: 10.1128/jvi.63.7.3135-3141.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Menezes J., Leibold W., Klein G., Clements G. Establishment and characterization of an Epstein-Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt's lymphoma. Biomedicine. 1975 Jul;22(4):276–284. [PubMed] [Google Scholar]
  35. Miller G., Robinson J., Heston L., Lipman M. Differences between laboratory strains of Epstein-Barr virus based on immortalization, abortive infection, and interference. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4006–4010. doi: 10.1073/pnas.71.10.4006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Nemerow G. R., Houghten R. A., Moore M. D., Cooper N. R. Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2). Cell. 1989 Feb 10;56(3):369–377. doi: 10.1016/0092-8674(89)90240-7. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Rawlins D. R., Milman G., Hayward S. D., Hayward G. S. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell. 1985 Oct;42(3):859–868. doi: 10.1016/0092-8674(85)90282-x. [DOI] [PubMed] [Google Scholar]
  40. Reisman D., Sugden B. trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. Mol Cell Biol. 1986 Nov;6(11):3838–3846. doi: 10.1128/mcb.6.11.3838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rosenfeld M. A., Yoshimura K., Trapnell B. C., Yoneyama K., Rosenthal E. R., Dalemans W., Fukayama M., Bargon J., Stier L. E., Stratford-Perricaudet L. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992 Jan 10;68(1):143–155. doi: 10.1016/0092-8674(92)90213-v. [DOI] [PubMed] [Google Scholar]
  42. Rowe M., Lear A. L., Croom-Carter D., Davies A. H., Rickinson A. B. Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes. J Virol. 1992 Jan;66(1):122–131. doi: 10.1128/jvi.66.1.122-131.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rowe M., Rowe D. T., Gregory C. D., Young L. S., Farrell P. J., Rupani H., Rickinson A. B. Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. EMBO J. 1987 Sep;6(9):2743–2751. doi: 10.1002/j.1460-2075.1987.tb02568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sample J., Brooks L., Sample C., Young L., Rowe M., Gregory C., Rickinson A., Kieff E. Restricted Epstein-Barr virus protein expression in Burkitt lymphoma is due to a different Epstein-Barr nuclear antigen 1 transcriptional initiation site. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6343–6347. doi: 10.1073/pnas.88.14.6343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Tomkinson B., Kieff E. Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro. J Virol. 1992 May;66(5):2893–2903. doi: 10.1128/jvi.66.5.2893-2903.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. 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]
  54. 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]
  55. 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]
  56. 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]

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