Spontaneous lymphoblastoid cell lines from patients with Epstein-Barr virus infection show highly variable proliferation characteristics that correlate with the expression levels of viral microRNAs

Autoři: Susanne Delecluse aff001;  Jiyang Yu aff001;  Katharina Bernhardt aff001;  Janina Haar aff001;  Remy Poirey aff001;  Ming-Han Tsai aff001;  Rama Kiblawi aff001;  Annette Kopp-Schneider aff005;  Paul Schnitzler aff006;  Martin Zeier aff004;  Peter Dreger aff007;  Patrick Wuchter aff007;  Olcay Cem Bulut aff009;  Uta Behrends aff003;  Henri-Jacques Delecluse aff001
Působiště autorů: German Cancer Research Centre (DKFZ) Unit F100, Heidelberg, Germany aff001;  Institut National de la Santé et de la Recherche Médicale (INSERM) Unit U1074, Heidelberg, Germany aff002;  German Centre for Infection Research (DZIF), Braunschweig, Germany aff003;  Nierenzentrum Heidelberg, Heidelberg, Germany aff004;  German Cancer Research Centre (DKFZ), Unit C060, Heidelberg, Germany aff005;  Center for Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany aff006;  Department of Medicine V, University of Heidelberg, Heidelberg, Germany aff007;  Institute of Transfusion Medicine and Immunology, German Red Cross Blood Donor Service Baden-Württemberg–Hessen, Medical Faculty Mannheim, Heidelberg University, Germany aff008;  Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Heidelberg, Heidelberg, Germany aff009;  Children’s Hospital Klinikum Rechts der Isar, Technische Universitaet Muenchen, Munich, Germany aff010
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222847


The Epstein-Barr virus (EBV) induces B-cell proliferation with high efficiency through expression of latent proteins and microRNAs. This process takes place in vivo soon after infection, presumably to expand the virus reservoir, but can also induce pathologies, e.g. an infectious mononucleosis (IM) syndrome after primary infection or a B-cell lymphoproliferation in immunosuppressed individuals. In this paper, we investigated the growth characteristics of EBV-infected B-cells isolated from transplant recipients or patients with IM. We found that these cells grew and withstood apoptosis at highly variable rates, suggesting that the expansion rate of the infected B-cells widely varies between individuals, thereby influencing the size of the B-cell reservoir and the ability to form tumors in infected individuals. All viruses investigated were type 1 and genetically close to western strains. EBV-infected B-cells expressed the transforming EBV latent genes and microRNAs (miRNAs) at variable levels. We found that the B-cell growth rates positively correlated with the BHRF1 miRNA levels. Comparative studies showed that infected B-cells derived from transplant recipients with iEBVL on average expressed higher levels of EBV miR-BHRF1 miRNAs and grew more rapidly than B-cells from IM patients, suggesting infection by more transforming viruses. Altogether, these findings suggest that EBV infection has a highly variable impact on the B-cell compartment that probably reflects the genetic diversity of both the virus and the host. It also demonstrates the unexpected finding that B-cells from different individuals can grow at different speed under the influence of the same virus infection.

Klíčová slova:

Apoptosis – B cells – Epstein-Barr virus – MicroRNAs – Multiple alignment calculation – Protein expression – Sequence alignment – Stem cell transplantation


1. Taylor GS, Long HM, Brooks JM, Rickinson AB, Hislop AD. The immunology of Epstein-Barr virus-induced disease. Annu Rev Immunol. 2015;33:787–821. doi: 10.1146/annurev-immunol-032414-112326 25706097.

2. Luzuriaga K, Sullivan JL. Infectious mononucleosis. N Engl J Med. 2010;362(21):1993–2000. doi: 10.1056/NEJMcp1001116 20505178.

3. Longnecker EK R.M., Cohen J.I. Fields Virology. 6 ed. USA: Lippincott Williams & Wilkins; 2013. 2455 p.

4. Klinke O, Feederle R, Delecluse HJ. Genetics of Epstein-Barr virus microRNAs. Semin Cancer Biol. 2014;26:52–9. doi: 10.1016/j.semcancer.2014.02.002 24602823.

5. Skalsky RL, Cullen BR. EBV Noncoding RNAs. Curr Top Microbiol Immunol. 2015;391:181–217. doi: 10.1007/978-3-319-22834-1_6 26428375.

6. Feederle R, Linnstaedt SD, Bannert H, Lips H, Bencun M, Cullen BR, et al. A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog. 2011;7(2):e1001294. doi: 10.1371/journal.ppat.1001294 21379335; PubMed Central PMCID: PMC3040666.

7. Seto E, Moosmann A, Gromminger S, Walz N, Grundhoff A, Hammerschmidt W. Micro RNAs of Epstein-Barr virus promote cell cycle progression and prevent apoptosis of primary human B cells. PLoS Pathog. 2010;6(8):e1001063. doi: 10.1371/journal.ppat.1001063 20808852; PubMed Central PMCID: PMC2924374.

8. Lin X, Tsai MH, Shumilov A, Poirey R, Bannert H, Middeldorp JM, et al. The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells. PLoS Pathog. 2015;11(12):e1005344. doi: 10.1371/journal.ppat.1005344 26694854; PubMed Central PMCID: PMC4691206.

9. Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. EBV persistence in memory B cells in vivo. Immunity. 1998;9(3):395–404. doi: 10.1016/s1074-7613(00)80622-6 9768759.

10. Dharnidharka VR, Webster AC, Martinez OM, Preiksaitis JK, Leblond V, Choquet S. Post-transplant lymphoproliferative disorders. Nat Rev Dis Primers. 2016;2:15088. Epub 2016/05/18. doi: 10.1038/nrdp.2015.88 27189056.

11. Bamoulid J, Courivaud C, Coaquette A, Chalopin JM, Gaiffe E, Saas P, et al. Subclinical Epstein-Barr virus viremia among adult renal transplant recipients: incidence and consequences. Am J Transplant. 2013;13(3):656–62. doi: 10.1111/ajt.12009 23331474.

12. Fink S, Tsai MH, Schnitzler P, Zeier M, Dreger P, Wuchter P, et al. The Epstein-Barr virus DNA load in the peripheral blood of transplant recipients does not accurately reflect the burden of infected cells. Transpl Int. 2017;30(1):57–67. Epub 2016/10/08. doi: 10.1111/tri.12871 27717030.

13. Evens AM, Choquet S, Kroll-Desrosiers AR, Jagadeesh D, Smith SM, Morschhauser F, et al. Primary CNS posttransplant lymphoproliferative disease (PTLD): an international report of 84 cases in the modern era. Am J Transplant. 2013;13(6):1512–22. doi: 10.1111/ajt.12211 23721553.

14. Trappe R, Oertel S, Leblond V, Mollee P, Sender M, Reinke P, et al. Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial. Lancet Oncol. 2012;13(2):196–206. doi: 10.1016/S1470-2045(11)70300-X 22173060.

15. Shannon-Lowe C, Baldwin G, Feederle R, Bell A, Rickinson A, Delecluse HJ. Epstein-Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth. J Gen Virol. 2005;86(Pt 11):3009–19. doi: 10.1099/vir.0.81153-0 16227222.

16. Pulvertaft JV. Cytology of Burkitt's Tumour (African Lymphoma). Lancet. 1964;1(7327):238–40. Epub 1964/02/01. doi: 10.1016/s0140-6736(64)92345-1 14086209.

17. Rowe M, Rooney CM, Rickinson AB, Lenoir GM, Rupani H, Moss DJ, et al. Distinctions between endemic and sporadic forms of Epstein-Barr virus-positive Burkitt's lymphoma. Int J Cancer. 1985;35(4):435–41. Epub 1985/04/15. doi: 10.1002/ijc.2910350404 2985508.

18. Kelly G, Bell A, Rickinson A. Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2. Nat Med. 2002;8(10):1098–104. Epub 2002/09/10. doi: 10.1038/nm758 12219084.

19. Neuhierl B, Feederle R, Hammerschmidt W, Delecluse HJ. Glycoprotein gp110 of Epstein-Barr virus determines viral tropism and efficiency of infection. Proc Natl Acad Sci U S A. 2002;99(23):15036–41. doi: 10.1073/pnas.232381299 12409611; PubMed Central PMCID: PMC137540.

20. Delecluse HJ, Bartnizke S, Hammerschmidt W, Bullerdiek J, Bornkamm GW. Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines. J Virol. 1993;67(3):1292–9. Epub 1993/03/01. 8382295; PubMed Central PMCID: PMC237496.

21. Haar J, Contrant M, Bernhardt K, Feederle R, Diederichs S, Pfeffer S, et al. The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation. Nucleic Acids Res. 2016;44(3):1326–41. doi: 10.1093/nar/gkv1330 26635399; PubMed Central PMCID: PMC4756819.

22. Cosmopoulos K, Pegtel M, Hawkins J, Moffett H, Novina C, Middeldorp J, et al. Comprehensive profiling of Epstein-Barr virus microRNAs in nasopharyngeal carcinoma. J Virol. 2009;83(5):2357–67. doi: 10.1128/JVI.02104-08 19091858; PubMed Central PMCID: PMC2643705.

23. Tsai MH, Raykova A, Klinke O, Bernhardt K, Gartner K, Leung CS, et al. Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in carcinomas. Cell Rep. 2013;5(2):458–70. doi: 10.1016/j.celrep.2013.09.012 24120866.

24. Montecucco A, Zanetta F, Biamonti G. Molecular mechanisms of etoposide. EXCLI J. 2015;14:95–108. Epub 2015/11/26. doi: 10.17179/excli2015-561 26600742; PubMed Central PMCID: PMC4652635.

25. Belmokhtar CA, Hillion J, Segal-Bendirdjian E. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene. 2001;20(26):3354–62. Epub 2001/06/26. doi: 10.1038/sj.onc.1204436 11423986.

26. Gil-Parrado S, Fernandez-Montalvan A, Assfalg-Machleidt I, Popp O, Bestvater F, Holloschi A, et al. Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members. J Biol Chem. 2002;277(30):27217–26. Epub 2002/05/10. doi: 10.1074/jbc.M202945200 12000759.

27. Tzellos S, Correia PB, Karstegl CE, Cancian L, Cano-Flanagan J, McClellan MJ, et al. A single amino acid in EBNA-2 determines superior B lymphoblastoid cell line growth maintenance by Epstein-Barr virus type 1 EBNA-2. J Virol. 2014;88(16):8743–53. Epub 2014/05/23. doi: 10.1128/JVI.01000-14 24850736; PubMed Central PMCID: PMC4136291.

28. Correia S, Bridges R, Wegner F, Venturini C, Palser A, Middeldorp JM, et al. Sequence Variation of Epstein-Barr Virus: Viral Types, Geography, Codon Usage, and Diseases. J Virol. 2018;92(22). Epub 2018/08/17. doi: 10.1128/JVI.01132-18 30111570; PubMed Central PMCID: PMC6206488.

29. Sculley TB, Sculley DG, Pope JH, Bornkamm GW, Lenoir GM, Rickinson AB. Epstein-Barr virus nuclear antigens 1 and 2 in Burkitt lymphoma cell lines containing either 'A'- or 'B'-type virus. Intervirology. 1988;29(2):77–85. doi: 10.1159/000150032 2842274.

30. Mainou BA, Raab-Traub N. LMP1 strain variants: biological and molecular properties. J Virol. 2006;80(13):6458–68. Epub 2006/06/16. doi: 10.1128/JVI.00135-06 16775333; PubMed Central PMCID: PMC1488979.

31. Carpenter B, Haque T, Dimopoulou M, Atkinson C, Roughton M, Grace S, et al. Incidence and dynamics of Epstein-Barr virus reactivation after alemtuzumab-based conditioning for allogeneic hematopoietic stem-cell transplantation. Transplantation. 2010;90(5):564–70. Epub 2010/06/18. doi: 10.1097/TP.0b013e3181e7a3bf 20555307.

32. Tsai MH, Lin X, Shumilov A, Bernhardt K, Feederle R, Poirey R, et al. The biological properties of different Epstein-Barr virus strains explain their association with various types of cancers. Oncotarget. 2017;8(6):10238–54. Epub 2017/01/05. doi: 10.18632/oncotarget.14380 28052012; PubMed Central PMCID: PMC5354655.

33. Poirel HA, Bernheim A, Schneider A, Meddeb M, Choquet S, Leblond V, et al. Characteristic pattern of chromosomal imbalances in posttransplantation lymphoproliferative disorders: correlation with histopathological subcategories and EBV status. Transplantation. 2005;80(2):176–84. Epub 2005/07/26. doi: 10.1097/01.tp.0000163288.98419.0d 16041261.

34. Pratt ZL, Kuzembayeva M, Sengupta S, Sugden B. The microRNAs of Epstein-Barr Virus are expressed at dramatically differing levels among cell lines. Virology. 2009;386(2):387–97. Epub 2009/02/17. doi: 10.1016/j.virol.2009.01.006 19217135; PubMed Central PMCID: PMC2763627.

35. Cai X, Schafer A, Lu S, Bilello JP, Desrosiers RC, Edwards R, et al. Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog. 2006;2(3):e23. Epub 2006/03/25. doi: 10.1371/journal.ppat.0020023 16557291; PubMed Central PMCID: PMC1409806.

36. Bernhardt K, Haar J, Tsai MH, Poirey R, Feederle R, Delecluse HJ. A Viral microRNA Cluster Regulates the Expression of PTEN, p27 and of a bcl-2 Homolog. PLoS Pathog. 2016;12(1):e1005405. doi: 10.1371/journal.ppat.1005405 26800049; PubMed Central PMCID: PMC4723338.

37. Feederle R, Haar J, Bernhardt K, Linnstaedt SD, Bannert H, Lips H, et al. The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocytes. J Virol. 2011;85(19):9801–10. Epub 2011/07/15. doi: 10.1128/JVI.05100-11 21752900; PubMed Central PMCID: PMC3196389.

38. Shumilov A, Tsai MH, Schlosser YT, Kratz AS, Bernhardt K, Fink S, et al. Epstein-Barr virus particles induce centrosome amplification and chromosomal instability. Nat Commun. 2017;8:14257. Epub 2017/02/12. doi: 10.1038/ncomms14257 28186092; PubMed Central PMCID: PMC5309802.

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