The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

English version České info

Epstein-Barr virus (EBV) is a gammaherpesvirus implicated in the pathogenesis of multiple malignancies, including Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma, and gastric carcinoma. EBV infection of resting B-lymphocytes drives them to proliferate as lymphoblastoid cell lines (LCLs), an in vitro model of PTLD. LCLs express a limited EBV gene repertoire, including six nuclear proteins (EBNA1, 2, 3A, 3B, 3C, and LP), three integral membrane proteins (LMP1, 2A, and 2B), and more than 30 micro RNAs. EBNA2 and the EBNA3 proteins are transcription factors that regulate viral and cell gene expression through the cell DNA binding protein RBPJ. In this study, we established LCLs transformed by recombinant EBV genomes in which a Flag-HA epitope tag is fused in-frame to the C-terminus of EBNA3A, EBNA3B or EBNA3C. Using these LCLs, we purified endogenous EBNA3 complexes and identified the USP46 deubiquitinating enzyme (DUB) and its associated chaperones WDR48 and WDR20 as EBNA3 binding proteins. We find that EBNA3s interact primarily with the WDR48 protein and that loss of WDR48 interaction with EBNA3A or EBNA3C impairs LCL growth. This study represents the first characterization of EBNA3 complexes from LCLs and implicates the USP46 DUB complex in EBNA3 mediated gene regulation.

Vyšlo v časopise: The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth. PLoS Pathog 11(4): e32767. doi:10.1371/journal.ppat.1004822
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004822

Souhrn

Zdroje

1. Rickinson AB, Kieff E (2007) Epstein-Barr Virus. Philadelphia: Lippincott Williams and Wilkins. 2655–2700 p.

2. Henle W, Diehl V, Kohn G, Zur Hausen H, Henle G (1967) Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157: 1064–1065. 6036237

3. Pope JH, Horne MK, Scott W (1968) Transformation of foetal human keukocytes in vitro by filtrates of a human leukaemic cell line containing herpes-like virus. International journal of cancer 3: 857–866. 4894385

4. Rooney CM, Smith CA, Ng CY, Loftin SK, Sixbey JW, et al. (1998) Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92: 1549–1555. 9716582

5. Thorley-Lawson DA, Hawkins JB, Tracy SI, Shapiro M (2013) The pathogenesis of Epstein-Barr virus persistent infection. Current opinion in virology 3: 227–232. doi: 10.1016/j.coviro.2013.04.005 23683686

6. Floettmann JE, Rowe M (1997) Epstein-Barr virus latent membrane protein-1 (LMP1) C-terminus activation region 2 (CTAR2) maps to the far C-terminus and requires oligomerisation for NF-kappaB activation. Oncogene 15: 1851–1858. 9362452

7. Izumi KM, Kieff ED (1997) The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. Proceedings of the National Academy of Sciences of the United States of America 94: 12592–12597. 9356494

8. Hatzivassiliou E, Miller WE, Raab-Traub N, Kieff E, Mosialos G (1998) A fusion of the EBV latent membrane protein-1 (LMP1) transmembrane domains to the CD40 cytoplasmic domain is similar to LMP1 in constitutive activation of epidermal growth factor receptor expression, nuclear factor-kappa B, and stress-activated protein kinase. Journal of immunology 160: 1116–1121.

9. Kilger E, Kieser A, Baumann M, Hammerschmidt W (1998) Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. The EMBO journal 17: 1700–1709. 9501091

10. Mosialos G, Birkenbach M, Yalamanchili R, VanArsdale T, Ware C, et al. (1995) The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80: 389–399. 7859281

11. Longnecker R, Miller CL, Miao XQ, Marchini A, Kieff E (1992) The only domain which distinguishes Epstein-Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential. Journal of virology 66: 6461–6469. 1328675

12. Longnecker R, Druker B, Roberts TM, Kieff E (1991) An Epstein-Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase. Journal of virology 65: 3681–3692. 1710288

13. Miller CL, Lee JH, Kieff E, Longnecker R (1994) An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking. Proceedings of the National Academy of Sciences of the United States of America 91: 772–776. 8290598

14. Grossman SR, Johannsen E, Tong X, Yalamanchili R, Kieff E (1994) The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein. Proceedings of the National Academy of Sciences of the United States of America 91: 7568–7572. 8052621

15. Harada S, Kieff E (1997) Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. Journal of virology 71: 6611–6618. 9261383

16. Johannsen E, Koh E, Mosialos G, Tong X, Kieff E, et al. (1995) Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1. Journal of virology 69: 253–262. 7983717

17. Zhao B, Zou J, Wang H, Johannsen E, Peng CW, et al. (2011) Epstein-Barr virus exploits intrinsic B-lymphocyte transcription programs to achieve immortal cell growth. Proceedings of the National Academy of Sciences of the United States of America 108: 14902–14907. doi: 10.1073/pnas.1108892108 21746931

18. Tomkinson B, Kieff E (1992) 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. Journal of virology 66: 2893–2903. 1313908

19. Tomkinson B, Robertson E, Kieff E (1993) Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. Journal of virology 67: 2014–2025. 8445720

20. Kempkes B, Pich D, Zeidler R, Sugden B, Hammerschmidt W (1995) Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA. Journal of virology 69: 231–238. 7983714

21. Lee S, Sakakibara S, Maruo S, Zhao B, Calderwood MA, et al. (2009) Epstein-Barr virus nuclear protein 3C domains necessary for lymphoblastoid cell growth: interaction with RBP-Jkappa regulates TCL1. Journal of virology 83: 12368–12377. doi: 10.1128/JVI.01403-09 19776126

22. Maruo S, Johannsen E, Illanes D, Cooper A, Kieff E (2003) Epstein-Barr Virus nuclear protein EBNA3A is critical for maintaining lymphoblastoid cell line growth. Journal of virology 77: 10437–10447. 12970429

23. Maruo S, Johannsen E, Illanes D, Cooper A, Zhao B, et al. (2005) Epstein-Barr virus nuclear protein 3A domains essential for growth of lymphoblasts: transcriptional regulation through RBP-Jkappa/CBF1 is critical. Journal of virology 79: 10171–10179. 16051810

24. Maruo S, Wu Y, Ishikawa S, Kanda T, Iwakiri D, et al. (2006) Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells. Proceedings of the National Academy of Sciences of the United States of America 103: 19500–19505. 17159137

25. Maruo S, Wu Y, Ito T, Kanda T, Kieff ED, et al. (2009) Epstein-Barr virus nuclear protein EBNA3C residues critical for maintaining lymphoblastoid cell growth. Proceedings of the National Academy of Sciences of the United States of America 106: 4419–4424. doi: 10.1073/pnas.0813134106 19237563

26. Parker GA, Touitou R, Allday MJ (2000) Epstein-Barr virus EBNA3C can disrupt multiple cell cycle checkpoints and induce nuclear division divorced from cytokinesis. Oncogene 19: 700–709. 10698515

27. Jha HC, Lu J, Saha A, Cai Q, Banerjee S, et al. (2013) EBNA3C-mediated regulation of aurora kinase B contributes to Epstein-Barr virus-induced B-cell proliferation through modulation of the activities of the retinoblastoma protein and apoptotic caspases. Journal of virology 87: 12121–12138. doi: 10.1128/JVI.02379-13 23986604

28. Parker GA, Crook T, Bain M, Sara EA, Farrell PJ, et al. (1996) Epstein-Barr virus nuclear antigen (EBNA)3C is an immortalizing oncoprotein with similar properties to adenovirus E1A and papillomavirus E7. Oncogene 13: 2541–2549. 9000128

29. Bajaj BG, Murakami M, Cai Q, Verma SC, Lan K, et al. (2008) Epstein-Barr virus nuclear antigen 3C interacts with and enhances the stability of the c-Myc oncoprotein. Journal of virology 82: 4082–4090. doi: 10.1128/JVI.02500-07 18256156

30. Saha A, Murakami M, Kumar P, Bajaj B, Sims K, et al. (2009) Epstein-Barr virus nuclear antigen 3C augments Mdm2-mediated p53 ubiquitination and degradation by deubiquitinating Mdm2. Journal of virology 83: 4652–4669. doi: 10.1128/JVI.02408-08 19244339

31. Saha A, Halder S, Upadhyay SK, Lu J, Kumar P, et al. (2011) Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1. PLoS pathogens 7: e1001275. doi: 10.1371/journal.ppat.1001275 21347341

32. Maruo S, Zhao B, Johannsen E, Kieff E, Zou J, et al. (2011) Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16INK4A and p14ARF expression. Proceedings of the National Academy of Sciences of the United States of America 108: 1919–1924. doi: 10.1073/pnas.1019599108 21245331

33. Skalska L, White RE, Parker GA, Sinclair AJ, Paschos K, et al. (2013) Induction of p16(INK4a) is the major barrier to proliferation when Epstein-Barr virus (EBV) transforms primary B cells into lymphoblastoid cell lines. PLoS pathogens 9: e1003187. doi: 10.1371/journal.ppat.1003187 23436997

34. Zhao B, Mar JC, Maruo S, Lee S, Gewurz BE, et al. (2011) Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines. Proceedings of the National Academy of Sciences of the United States of America 108: 337–342. doi: 10.1073/pnas.1017419108 21173222

35. Anderton E, Yee J, Smith P, Crook T, White RE, et al. (2008) Two Epstein-Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma. Oncogene 27: 421–433. 17653091

36. White RE, Groves IJ, Turro E, Yee J, Kremmer E, et al. (2010) Extensive co-operation between the Epstein-Barr virus EBNA3 proteins in the manipulation of host gene expression and epigenetic chromatin modification. PloS one 5: e13979. doi: 10.1371/journal.pone.0013979 21085583

37. Zhao B, Marshall DR, Sample CE (1996) A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa. Journal of virology 70: 4228–4236. 8676443

38. Robertson ES, Grossman S, Johannsen E, Miller C, Lin J, et al. (1995) Epstein-Barr virus nuclear protein 3C modulates transcription through interaction with the sequence-specific DNA-binding protein J kappa. Journal of virology 69: 3108–3116. 7707539

39. Marshall D, Sample C (1995) Epstein-Barr virus nuclear antigen 3C is a transcriptional regulator. Journal of virology 69: 3624–3630. 7745710

40. Krauer KG, Kienzle N, Young DB, Sculley TB (1996) Epstein-Barr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J kappa in B lymphocytes. Virology 226: 346–353. 8955054

41. Calderwood MA, Lee S, Holthaus AM, Blacklow SC, Kieff E, et al. (2011) Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; only the NTD interaction is essential for lymphoblastoid cell growth. Virology 414: 19–25. doi: 10.1016/j.virol.2011.02.018 21440926

42. Jiang S, Willox B, Zhou H, Holthaus AM, Wang A, et al. (2014) Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A. Proceedings of the National Academy of Sciences of the United States of America 111: 421–426. doi: 10.1073/pnas.1321704111 24344258

43. McClellan MJ, Wood CD, Ojeniyi O, Cooper TJ, Kanhere A, et al. (2013) Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming. PLoS pathogens 9: e1003636. doi: 10.1371/journal.ppat.1003636 24068937

44. Hickabottom M, Parker GA, Freemont P, Crook T, Allday MJ (2002) Two nonconsensus sites in the Epstein-Barr virus oncoprotein EBNA3A cooperate to bind the co-repressor carboxyl-terminal-binding protein (CtBP). The Journal of biological chemistry 277: 47197–47204. 12372828

45. Touitou R, Hickabottom M, Parker G, Crook T, Allday MJ (2001) Physical and functional interactions between the corepressor CtBP and the Epstein-Barr virus nuclear antigen EBNA3C. Journal of virology 75: 7749–7755. 11462050

46. Skalska L, White RE, Franz M, Ruhmann M, Allday MJ (2010) Epigenetic repression of p16(INK4A) by latent Epstein-Barr virus requires the interaction of EBNA3A and EBNA3C with CtBP. PLoS pathogens 6: e1000951. doi: 10.1371/journal.ppat.1000951 20548956

47. Chen A, Divisconte M, Jiang X, Quink C, Wang F (2005) Epstein-Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro. Journal of virology 79: 4506–4509. 15767450

48. Chen A, Zhao B, Kieff E, Aster JC, Wang F (2006) EBNA-3B- and EBNA-3C-regulated cellular genes in Epstein-Barr virus-immortalized lymphoblastoid cell lines. Journal of virology 80: 10139–10150. 17005691

49. Johannsen E, Miller CL, Grossman SR, Kieff E (1996) EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein-Barr virus-transformed B lymphocytes. Journal of virology 70: 4179–4183. 8648764

50. Lin J, Johannsen E, Robertson E, Kieff E (2002) Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2. Journal of virology 76: 232–242. 11739688

51. Park J, Lee BS, Choi JK, Means RE, Choe J, et al. (2002) Herpesviral protein targets a cellular WD repeat endosomal protein to downregulate T lymphocyte receptor expression. Immunity 17: 221–233. 12196293

52. Cohn MA, Kowal P, Yang K, Haas W, Huang TT, et al. (2007) A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway. Molecular cell 28: 786–797. 18082604

53. Lehoux M, Gagnon D, Archambault J (2014) E1-mediated recruitment of a UAF1-USP deubiquitinase complex facilitates human papillomavirus DNA replication. Journal of virology 88: 8545–8555. doi: 10.1128/JVI.00379-14 24850727

54. Portal D, Zhao B, Calderwood MA, Sommermann T, Johannsen E, et al. (2011) EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding. Proceedings of the National Academy of Sciences of the United States of America 108: 7808–7813. doi: 10.1073/pnas.1104991108 21518914

55. Yang K, Moldovan GL, Vinciguerra P, Murai J, Takeda S, et al. (2011) Regulation of the Fanconi anemia pathway by a SUMO-like delivery network. Genes & development 25: 1847–1858.

56. Li R, Wang L, Liao G, Guzzo CM, Matunis MJ, et al. (2012) SUMO binding by the Epstein-Barr virus protein kinase BGLF4 is crucial for BGLF4 function. Journal of virology 86: 5412–5421. doi: 10.1128/JVI.00314-12 22398289

57. McClellan MJ, Khasnis S, Wood CD, Palermo RD, Schlick SN, et al. (2012) Downregulation of integrin receptor-signaling genes by Epstein-Barr virus EBNA 3C via promoter-proximal and-distal binding elements. Journal of virology 86: 5165–5178. doi: 10.1128/JVI.07161-11 22357270

58. Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, et al. (2008) High-quality binary protein interaction map of the yeast interactome network. Science 322: 104–110. doi: 10.1126/science.1158684 18719252

59. Waltzer L, Perricaudet M, Sergeant A, Manet E (1996) Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA. Journal of virology 70: 5909–5915. 8709211

60. Radkov SA, Bain M, Farrell PJ, West M, Rowe M, et al. (1997) Epstein-Barr virus EBNA3C represses Cp, the major promoter for EBNA expression, but has no effect on the promoter of the cell gene CD21. Journal of virology 71: 8552–8562. 9343213

61. Cooper A, Johannsen E, Maruo S, Cahir-McFarland E, Illanes D, et al. (2003) EBNA3A association with RBP-Jkappa down-regulates c-myc and Epstein-Barr virus-transformed lymphoblast growth. Journal of virology 77: 999–1010. 12502816

62. Paschos K, Parker GA, Watanatanasup E, White RE, Allday MJ (2012) BIM promoter directly targeted by EBNA3C in polycomb-mediated repression by EBV. Nucleic acids research 40: 7233–7246. doi: 10.1093/nar/gks391 22584624

63. Arnett KL, Hass M, McArthur DG, Ilagan MX, Aster JC, et al. (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nature structural & molecular biology 17: 1312–1317.

64. Kee Y, Yang K, Cohn MA, Haas W, Gygi SP, et al. (2010) WDR20 regulates activity of the USP12 x UAF1 deubiquitinating enzyme complex. The Journal of biological chemistry 285: 11252–11257. doi: 10.1074/jbc.M109.095141 20147737

65. Quesada Vc, Dı́az-Perales A, Gutiérrez-Fernández A, Garabaya C, Cal S, et al. (2004) Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochemical and Biophysical Research Communications 314: 54–62. 14715245

66. Cohn MA, Kee Y, Haas W, Gygi SP, D'Andrea AD (2009) UAF1 is a subunit of multiple deubiquitinating enzyme complexes. The Journal of biological chemistry 284: 5343–5351. doi: 10.1074/jbc.M808430200 19075014

67. Gangula NR, Maddika S (2013) WD repeat protein WDR48 in complex with deubiquitinase USP12 suppresses Akt-dependent cell survival signaling by stabilizing PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1). The Journal of biological chemistry 288: 34545–34554. doi: 10.1074/jbc.M113.503383 24145035

68. Li X, Stevens PD, Yang H, Gulhati P, Wang W, et al. (2013) The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer. Oncogene 32: 471–478. doi: 10.1038/onc.2012.66 22391563

69. Molina JR, Agarwal NK, Morales FC, Hayashi Y, Aldape KD, et al. (2012) PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma. Oncogene 31: 1264–1274. doi: 10.1038/onc.2011.324 21804599

70. Sowa ME, Bennett EJ, Gygi SP, Harper JW (2009) Defining the human deubiquitinating enzyme interaction landscape. Cell 138: 389–403. doi: 10.1016/j.cell.2009.04.042 19615732

71. Dahlberg CL, Juo P (2014) The WD40-repeat proteins WDR-20 and WDR-48 bind and activate the deubiquitinating enzyme USP-46 to promote the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of Caenorhabditis elegans. The Journal of biological chemistry 289: 3444–3456. doi: 10.1074/jbc.M113.507541 24356955

72. Moretti J, Chastagner P, Liang CC, Cohn MA, Israel A, et al. (2012) The ubiquitin-specific protease 12 (USP12) is a negative regulator of notch signaling acting on notch receptor trafficking toward degradation. The Journal of biological chemistry 287: 29429–29441. doi: 10.1074/jbc.M112.366807 22778262

73. Joo HY, Jones A, Yang C, Zhai L, Smith ADt, et al. (2011) Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46. The Journal of biological chemistry 286: 7190–7201. doi: 10.1074/jbc.M110.158311 21183687

74. Banerjee S, Lu J, Cai Q, Saha A, Jha HC, et al. (2013) The EBV Latent Antigen 3C Inhibits Apoptosis through Targeted Regulation of Interferon Regulatory Factors 4 and 8. PLoS pathogens 9: e1003314. doi: 10.1371/journal.ppat.1003314 23658517

75. Cai Q, Guo Y, Xiao B, Banerjee S, Saha A, et al. (2011) Epstein-Barr virus nuclear antigen 3C stabilizes Gemin3 to block p53-mediated apoptosis. PLoS pathogens 7: e1002418. doi: 10.1371/journal.ppat.1002418 22174681

76. Lebkowski JS, Clancy S, Calos MP (1985) Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression. Nature 317: 169–171. 2993921

77. Kavathas P, Bach FH, DeMars R (1980) Gamma ray-induced loss of expression of HLA and glyoxalase I alleles in lymphoblastoid cells. Proceedings of the National Academy of Sciences of the United States of America 77: 4251–4255. 6933474

78. Verma D, Ling C, Johannsen E, Nagaraja T, Swaminathan S (2009) Negative autoregulation of Epstein-Barr virus (EBV) replicative gene expression by EBV SM protein. Journal of virology 83: 8041–8050. doi: 10.1128/JVI.00382-09 19515786

79. Hatzivassiliou E, Cardot P, Zannis VI, Mitsialis SA (1997) Ultraspiracle, a Drosophila retinoic X receptor alpha homologue, can mobilize the human thyroid hormone receptor to transactivate a human promoter. Biochemistry 36: 9221–9231. 9230055

80. Young L, Alfieri C, Hennessy K, Evans H, O'Hara C, et al. (1989) Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. The New England journal of medicine 321: 1080–1085. 2552313

81. Maunders MJ, Petti L, Rowe M (1994) Precipitation of the Epstein-Barr virus protein EBNA 2 by an EBNA 3c-specific monoclonal antibody. The Journal of general virology 75 (Pt 4): 769–778. 7512118

82. Mann KP, Staunton D. & Thorley-Lawson D. A. (1985) Epstein-Barr virus-encoded protein found in plasma membranes of transformed cells. Journal of virology 55: 710–720. 2991591

83. Kanamori M, Watanabe S, Honma R, Kuroda M, Imai S, et al. (2004) Epstein-Barr virus nuclear antigen leader protein induces expression of thymus- and activation-regulated chemokine in B cells. Journal of virology 78: 3984–3993. 15047814

84. Mali P, Yang L, Esvelt KM, Aach J, Guell M, et al. (2013) RNA-guided human genome engineering via Cas9. Science 339: 823–826. doi: 10.1126/science.1232033 23287722

85. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343: 80–84. doi: 10.1126/science.1246981 24336569

86. Cong L, Ran FA, Cox D, Lin S, Barretto R, et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823. doi: 10.1126/science.1231143 23287718

87. Heilmann AM, Calderwood MA, Portal D, Lu Y, Johannsen E (2012) Genome-wide analysis of Epstein-Barr virus Rta DNA binding. Journal of virology 86: 5151–5164. doi: 10.1128/JVI.06760-11 22379087