Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Nov 2;17(21):6394–6403. doi: 10.1093/emboj/17.21.6394

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

A Aiyar 1, C Tyree 1, B Sugden 1
PMCID: PMC1170964  PMID: 9799247

Abstract

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.

Full Text

The Full Text of this article is available as a PDF (384.6 KB).

Selected References

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

  1. Adams A. Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol. 1987 May;61(5):1743–1746. doi: 10.1128/jvi.61.5.1743-1746.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiyar A., Hindmarsh P., Skalka A. M., Leis J. Concerted integration of linear retroviral DNA by the avian sarcoma virus integrase in vitro: dependence on both long terminal repeat termini. J Virol. 1996 Jun;70(6):3571–3580. doi: 10.1128/jvi.70.6.3571-3580.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ambinder R. F., Mullen M. A., Chang Y. N., Hayward G. S., Hayward S. D. Functional domains of Epstein-Barr virus nuclear antigen EBNA-1. J Virol. 1991 Mar;65(3):1466–1478. doi: 10.1128/jvi.65.3.1466-1478.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ambinder R. F., Shah W. A., Rawlins D. R., Hayward G. S., Hayward S. D. Definition of the sequence requirements for binding of the EBNA-1 protein to its palindromic target sites in Epstein-Barr virus DNA. J Virol. 1990 May;64(5):2369–2379. doi: 10.1128/jvi.64.5.2369-2379.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bacchetti S., Graham F. L. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1590–1594. doi: 10.1073/pnas.74.4.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Biamonti G., Della Valle G., Talarico D., Cobianchi F., Riva S., Falaschi A. Fate of exogenous recombinant plasmids introduced into mouse and human cells. Nucleic Acids Res. 1985 Aug 12;13(15):5545–5561. doi: 10.1093/nar/13.15.5545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bochkarev A., Barwell J. A., Pfuetzner R. A., Bochkareva E., Frappier L., Edwards A. M. Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA. Cell. 1996 Mar 8;84(5):791–800. doi: 10.1016/s0092-8674(00)81056-9. [DOI] [PubMed] [Google Scholar]
  9. Bruckner R. C., Crute J. J., Dodson M. S., Lehman I. R. The herpes simplex virus 1 origin binding protein: a DNA helicase. J Biol Chem. 1991 Feb 5;266(4):2669–2674. [PubMed] [Google Scholar]
  10. Delecluse H. J., Bartnizke S., Hammerschmidt W., Bullerdiek J., Bornkamm G. W. Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines. J Virol. 1993 Mar;67(3):1292–1299. doi: 10.1128/jvi.67.3.1292-1299.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  12. Frappier L., O'Donnell M. Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus. J Biol Chem. 1991 Apr 25;266(12):7819–7826. [PubMed] [Google Scholar]
  13. Gahn T. A., Schildkraut C. L. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell. 1989 Aug 11;58(3):527–535. doi: 10.1016/0092-8674(89)90433-9. [DOI] [PubMed] [Google Scholar]
  14. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  15. Grogan E. A., Summers W. P., Dowling S., Shedd D., Gradoville L., Miller G. Two Epstein-Barr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7650–7653. doi: 10.1073/pnas.80.24.7650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harrison S., Fisenne K., Hearing J. Sequence requirements of the Epstein-Barr virus latent origin of DNA replication. J Virol. 1994 Mar;68(3):1913–1925. doi: 10.1128/jvi.68.3.1913-1925.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hearing J., Mülhaupt Y., Harper S. Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication. J Virol. 1992 Feb;66(2):694–705. doi: 10.1128/jvi.66.2.694-705.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  19. Hsieh D. J., Camiolo S. M., Yates J. L. Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection. EMBO J. 1993 Dec 15;12(13):4933–4944. doi: 10.1002/j.1460-2075.1993.tb06187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kirchmaier A. L., Sugden B. Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus. J Virol. 1997 Mar;71(3):1766–1775. doi: 10.1128/jvi.71.3.1766-1775.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kirchmaier A. L., Sugden B. Plasmid maintenance of derivatives of oriP of Epstein-Barr virus. J Virol. 1995 Feb;69(2):1280–1283. doi: 10.1128/jvi.69.2.1280-1283.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kirchmaier A. L., Sugden B. Rep*: a viral element that can partially replace the origin of plasmid DNA synthesis of Epstein-Barr virus. J Virol. 1998 Jun;72(6):4657–4666. doi: 10.1128/jvi.72.6.4657-4666.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Knutson J. C., Yee D. Electroporation: parameters affecting transfer of DNA into mammalian cells. Anal Biochem. 1987 Jul;164(1):44–52. doi: 10.1016/0003-2697(87)90365-4. [DOI] [PubMed] [Google Scholar]
  24. Krysan P. J., Haase S. B., Calos M. P. Isolation of human sequences that replicate autonomously in human cells. Mol Cell Biol. 1989 Mar;9(3):1026–1033. doi: 10.1128/mcb.9.3.1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lacks S. A. Purification and properties of the complementary endonucleases DpnI and DpnII. Methods Enzymol. 1980;65(1):138–146. doi: 10.1016/s0076-6879(80)65019-8. [DOI] [PubMed] [Google Scholar]
  26. Laine A., Frappier L. Identification of Epstein-Barr virus nuclear antigen 1 protein domains that direct interactions at a distance between DNA-bound proteins. J Biol Chem. 1995 Dec 29;270(52):30914–30918. doi: 10.1074/jbc.270.52.30914. [DOI] [PubMed] [Google Scholar]
  27. Lehman C. W., Botchan M. R. Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4338–4343. doi: 10.1073/pnas.95.8.4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li J. J., Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993 Dec 17;262(5141):1870–1874. doi: 10.1126/science.8266075. [DOI] [PubMed] [Google Scholar]
  29. Lupton S., Levine A. J. Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. Mol Cell Biol. 1985 Oct;5(10):2533–2542. doi: 10.1128/mcb.5.10.2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mackey D., Middleton T., Sugden B. Multiple regions within EBNA1 can link DNAs. J Virol. 1995 Oct;69(10):6199–6208. doi: 10.1128/jvi.69.10.6199-6208.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mackey D., Sugden B. Studies on the mechanism of DNA linking by Epstein-Barr virus nuclear antigen 1. J Biol Chem. 1997 Nov 21;272(47):29873–29879. doi: 10.1074/jbc.272.47.29873. [DOI] [PubMed] [Google Scholar]
  32. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  33. Masukata H., Satoh H., Obuse C., Okazaki T. Autonomous replication of human chromosomal DNA fragments in human cells. Mol Biol Cell. 1993 Nov;4(11):1121–1132. doi: 10.1091/mbc.4.11.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meuth M., Green H. Induction of a deoxycytidineless state in cultured mammalian cells by bromodeoxyuridine. Cell. 1974 Jun;2(2):109–112. doi: 10.1016/0092-8674(74)90099-3. [DOI] [PubMed] [Google Scholar]
  35. Middleton T., Sugden B. EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication. J Virol. 1992 Jan;66(1):489–495. doi: 10.1128/jvi.66.1.489-495.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Middleton T., Sugden B. Retention of plasmid DNA in mammalian cells is enhanced by binding of the Epstein-Barr virus replication protein EBNA1. J Virol. 1994 Jun;68(6):4067–4071. doi: 10.1128/jvi.68.6.4067-4071.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Piirsoo M., Ustav E., Mandel T., Stenlund A., Ustav M. Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator. EMBO J. 1996 Jan 2;15(1):1–11. [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Reisman D., Yates J., Sugden B. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol. 1985 Aug;5(8):1822–1832. doi: 10.1128/mcb.5.8.1822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sandberg M., Hammerschmidt W., Sugden B. Characterization of LMP-1's association with TRAF1, TRAF2, and TRAF3. J Virol. 1997 Jun;71(6):4649–4656. doi: 10.1128/jvi.71.6.4649-4656.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schleiss M. R., Degnin C. R., Geballe A. P. Translational control of human cytomegalovirus gp48 expression. J Virol. 1991 Dec;65(12):6782–6789. doi: 10.1128/jvi.65.12.6782-6789.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shimizu N., Itoh N., Utiyama H., Wahl G. M. Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J Cell Biol. 1998 Mar 23;140(6):1307–1320. doi: 10.1083/jcb.140.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Simpson K., McGuigan A., Huxley C. Stable episomal maintenance of yeast artificial chromosomes in human cells. Mol Cell Biol. 1996 Sep;16(9):5117–5126. doi: 10.1128/mcb.16.9.5117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Simpson R. T. Nucleosome positioning can affect the function of a cis-acting DNA element in vivo. Nature. 1990 Jan 25;343(6256):387–389. doi: 10.1038/343387a0. [DOI] [PubMed] [Google Scholar]
  45. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sugden B., Warren N. Plasmid origin of replication of Epstein-Barr virus, oriP, does not limit replication in cis. Mol Biol Med. 1988 Apr;5(2):85–94. [PubMed] [Google Scholar]
  47. Thompson J. F., Hayes L. S., Lloyd D. B. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene. 1991 Jul 22;103(2):171–177. doi: 10.1016/0378-1119(91)90270-l. [DOI] [PubMed] [Google Scholar]
  48. Veit B. E., Fangman W. L. Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators. Mol Cell Biol. 1988 Nov;8(11):4949–4957. doi: 10.1128/mcb.8.11.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Von Hoff D. D., McGill J. R., Forseth B. J., Davidson K. K., Bradley T. P., Van Devanter D. R., Wahl G. M. Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8165–8169. doi: 10.1073/pnas.89.17.8165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wen L. T., Lai P. K., Bradley G., Tanaka A., Nonoyama M. Interaction of Epstein-Barr viral (EBV) origin of replication (oriP) with EBNA-1 and cellular anti-EBNA-1 proteins. Virology. 1990 Sep;178(1):293–296. doi: 10.1016/0042-6822(90)90407-i. [DOI] [PubMed] [Google Scholar]
  51. Wu L. C., Fisher P. A., Broach J. R. A yeast plasmid partitioning protein is a karyoskeletal component. J Biol Chem. 1987 Jan 15;262(2):883–891. [PubMed] [Google Scholar]
  52. 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]
  53. Yang L., Mohr I., Fouts E., Lim D. A., Nohaile M., Botchan M. The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5086–5090. doi: 10.1073/pnas.90.11.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. 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]
  57. Yee C., Krishnan-Hewlett I., Baker C. C., Schlegel R., Howley P. M. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol. 1985 Jun;119(3):361–366. [PMC free article] [PubMed] [Google Scholar]
  58. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES