Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells

English version České info

Lytic EBV infection of differentiated oral epithelial cells results in the release of infectious viral particles and is required for efficient transmission of EBV from host to host. Lytic infection also causes a tongue lesion known as oral hairy leukoplakia (OHL). However, surprisingly little is known in regard to how EBV gene expression is regulated in epithelial cells. Using a stably EBV- infected, telomerase-immortalized normal oral keratinocyte cell line, we show here that undifferentiated basal epithelial cells support latent EBV infection, while differentiation of epithelial cells promotes lytic reactivation. Furthermore, we demonstrate that the KLF4 cellular transcription factor, which is required for normal epithelial cell differentiation and is expressed in differentiated, but not undifferentiated, normal epithelial cells, induces lytic EBV reactivation by activating transcription from the two EBV immediate-early gene promoters. We also show that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, synergistically activates lytic gene expression in epithelial cells. We confirm that KLF4 and BLIMP1 expression in normal tongue epithelium is confined to differentiated cells, and that KLF4 and BLIMP1 are expressed in a patient-derived OHL tongue lesion. These results suggest that differentiation-dependent expression of KLF4 and BLIMP1 in epithelial cells promotes lytic EBV infection.

Vyšlo v časopise: Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells. PLoS Pathog 11(10): e32767. doi:10.1371/journal.ppat.1005195
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1005195

Souhrn

Zdroje

1. Henle G, Henle W, Diehl V. Relation of Burkitt’s tumor-associated herpes-y type virus to infectious mononucleosis. Proc Natl Acad Sci U S A. 1968;59: 94–101. 5242134

2. Rickinson A. B., Kieff Elliot. Epstein-Barr Virus. Fields Virology. 5th ed. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins; 2007. pp. 2655–2700.

3. Zur Hausen H, Schulte-Holthausen H, Klein G, Henle W, Henle G, Clifford P, et al. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature. 1970;228: 1056–1058. 4320657

4. Kieff Elliot D, Rickinson A. B. Epstein-Barr Virus and Its Replication. Fields Virology. 5th ed. Philadelphia: Wolters Kluwer Health/ Lippincott Williams & Wilkins; 2007. pp. 2603–2654.

5. Souza TA, Stollar BD, Sullivan JL, Luzuriaga K, Thorley-Lawson DA. Peripheral B cells latently infected with Epstein-Barr virus display molecular hallmarks of classical antigen-selected memory B cells. Proc Natl Acad Sci U S A. 2005;102: 18093–18098. 16330748

6. Laichalk LL, Thorley-Lawson DA. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J Virol. 2005;79: 1296–1307. 15613356

7. Tovey MG, Lenoir G, Begon-Lours J. Activation of latent Epstein-Barr virus by antibody to human IgM. Nature. 1978;276: 270–272. 213727

8. Takada K. Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines. Int J Cancer J Int Cancer. 1984;33: 27–32.

9. Greenspan JS, Greenspan D, Lennette ET, Abrams DI, Conant MA, Petersen V, et al. Replication of Epstein-Barr virus within the epithelial cells of oral “hairy” leukoplakia, an AIDS-associated lesion. N Engl J Med. 1985;313: 1564–1571. 2999595

10. Walling DM, Flaitz CM, Nichols CM, Hudnall SD, Adler-Storthz K. Persistent productive Epstein-Barr virus replication in normal epithelial cells in vivo. J Infect Dis. 2001;184: 1499–1507. 11740724

11. Webster-Cyriaque J, Middeldorp J, Raab-Traub N. Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections. J Virol. 2000;74: 7610–7618. 10906215

12. Tsang CM, Yip YL, Lo KW, Deng W, To KF, Hau PM, et al. Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci. 2012;109: E3473–E3482. doi: 10.1073/pnas.1202637109 23161911

13. Young LS, Lau R, Rowe M, Niedobitek G, Packham G, Shanahan F, et al. Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia. J Virol. 1991;65: 2868–2874. 1851858

14. Gilligan K, Rajadurai P, Resnick L, Raab-Traub N. Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia. Proc Natl Acad Sci U S A. 1990;87: 8790–8794. 2174165

15. Temple RM, Zhu J, Budgeon L, Christensen ND, Meyers C, Sample CE. Efficient replication of Epstein-Barr virus in stratified epithelium in vitro. Proc Natl Acad Sci U S A. 2014;111: 16544–16549. doi: 10.1073/pnas.1400818111 25313069

16. Hummel M, Hudson JB, Laimins LA. Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. J Virol. 1992;66: 6070–6080. 1326657

17. Flores ER, Lambert PF. Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle. J Virol. 1997;71: 7167–7179. 9311789

18. Feederle R, Neuhierl B, Bannert H, Geletneky K, Shannon-Lowe C, Delecluse H-J. Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection. Int J Cancer J Int Cancer. 2007;121: 588–594.

19. Karimi L, Crawford DH, Speck S, Nicholson LJ. Identification of an epithelial cell differentiation responsive region within the BZLF1 promoter of the Epstein-Barr virus. J Gen Virol. 1995;76 (Pt 4): 759–765. 9049321

20. Li QX, Young LS, Niedobitek G, Dawson CW, Birkenbach M, Wang F, et al. Epstein-Barr virus infection and replication in a human epithelial cell system. Nature. 1992;356: 347–350. 1312681

21. Knox PG, Li QX, Rickinson AB, Young LS. In vitro production of stable Epstein-Barr virus-positive epithelial cell clones which resemble the virus:cell interaction observed in nasopharyngeal carcinoma. Virology. 1996;215: 40–50. 8553585

22. Andrei G, Duraffour S, Van den Oord J, Snoeck R. Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents. Antiviral Res. 2010;85: 431–449. doi: 10.1016/j.antiviral.2009.10.019 19883696

23. 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;61: 3672–3679. 2824806

24. 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. 1985;82: 4085–4089. 2987963

25. Ragoczy T, Heston L, Miller G. The Epstein-Barr Virus Rta Protein Activates Lytic Cycle Genes and Can Disrupt Latency in B Lymphocytes. J Virol. 1998;72: 7978–7984. 9733836

26. Takada K, Shimizu N, Sakuma S, Ono Y. trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment. J Virol. 1986;57: 1016–1022. 3005608

27. Zalani S, Holley-Guthrie E, Kenney S. Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism. Proc Natl Acad Sci U S A. 1996;93: 9194–9199. 8799177

28. Rooney CM, Rowe DT, Ragot T, Farrell PJ. The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle. J Virol. 1989;63: 3109–3116. 2542618

29. Speck SH, Chatila T, Flemington E. Reactivation of Epstein-Barr virus: regulation and function of the BZLF1 gene. Trends Microbiol. 1997;5: 399–405. 9351176

30. Kenney SC, Mertz JE. Regulation of the latent-lytic switch in Epstein-Barr virus. Semin Cancer Biol. 2014;26: 60–68. doi: 10.1016/j.semcancer.2014.01.002 24457012

31. 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;5: 3243–3249. 3028777

32. Cox MA, Leahy J, Hardwick JM. An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators. J Virol. 1990;64: 313–321. 2152819

33. Hardwick JM, Lieberman PM, Hayward SD. A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. J Virol. 1988;62: 2274–2284. 2836611

34. Holley-Guthrie EA, Quinlivan EB, Mar EC, Kenney S. The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. J Virol. 1990;64: 3753–3759. 2164595

35. Kenney S, Holley-Guthrie E, Mar EC, Smith M. The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators. J Virol. 1989;63: 3878–3883. 2548003

36. Wille CK, Nawandar DM, Panfil AR, Ko MM, Hagemeier SR, Kenney SC. Viral genome methylation differentially affects the ability of BZLF1 versus BRLF1 to activate Epstein-Barr virus lytic gene expression and viral replication. J Virol. 2013;87: 935–950. doi: 10.1128/JVI.01790-12 23135711

37. Magnúsdóttir E, Kalachikov S, Mizukoshi K, Savitsky D, Ishida-Yamamoto A, Panteleyev AA, et al. Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1. Proc Natl Acad Sci U S A. 2007;104: 14988–14993. 17846422

38. Turner CA, Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell. 1994;77: 297–306. 8168136

39. Reusch JA, Nawandar DM, Wright KL, Kenney SC, Mertz JE. Cellular Differentiation Regulator BLIMP1 Induces Epstein-Barr Virus Lytic Reactivation in Epithelial and B Cells by Activating Transcription from both the R and Z Promoters. J Virol. 2015;89: 1731–1743. doi: 10.1128/JVI.02781-14 25410866

40. Segre JA, Bauer C, Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet. 1999;22: 356–360. 10431239

41. Cordani N, Pozzi S, Martynova E, Fanoni D, Borrelli S, Alotto D, et al. Mutant p53 subverts p63 control over KLF4 expression in keratinocytes. Oncogene. 2011;30: 922–932. doi: 10.1038/onc.2010.474 20972454

42. Birdwell CE, Queen KJ, Kilgore PCSR, Rollyson P, Trutschl M, Cvek U, et al. Genome-Wide DNA Methylation as an Epigenetic Consequence of Epstein-Barr Virus Infection of Immortalized Keratinocytes. J Virol. 2014;88: 11442–11458. doi: 10.1128/JVI.00972-14 25056883

43. McMullan R, Lax S, Robertson VH, Radford DJ, Broad S, Watt FM, et al. Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway. Curr Biol CB. 2003;13: 2185–2189. 14680635

44. Chew YC, Adhikary G, Xu W, Wilson GM, Eckert RL. Protein kinase C δ increases Kruppel-like factor 4 protein, which drives involucrin gene transcription in differentiating keratinocytes. J Biol Chem. 2013;288: 17759–17768. doi: 10.1074/jbc.M113.477133 23599428

45. Zalani S, Holley-Guthrie EA, Gutsch DE, Kenney SC. The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor. J Virol. 1992;66: 7282–7292. 1331521

46. Murata T, Narita Y, Sugimoto A, Kawashima D, Kanda T, Tsurumi T. Contribution of Myocyte Enhancer Factor 2 (MEF2) Family Transcription Factors to BZLF1 Expression in Epstein-Barr virus Reactivation from Latency. J Virol. 2013.

47. Jiang W, Lobo-Ruppert SM, Ruppert JM. Bad things happen in the basal layer: KLF4 and squamous cell carcinoma. Cancer Biol Ther. 2008;7: 783–785. 18424916

48. Buettner M, Lang A, Tudor CS, Meyer B, Cruchley A, Barros MHM, et al. Lytic Epstein-Barr virus infection in epithelial cells but not in B-lymphocytes is dependent on Blimp1. J Gen Virol. 2012;93: 1059–1064. doi: 10.1099/vir.0.038661-0 22278826

49. Hu S, Wan J, Su Y, Song Q, Zeng Y, Nguyen HN, et al. DNA methylation presents distinct binding sites for human transcription factors. eLife. 2013;2: e00726. doi: 10.7554/eLife.00726 24015356

50. Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PWTC, Bauer C, et al. Dynamic Readers for 5-(Hydroxy)Methylcytosine and Its Oxidized Derivatives. Cell. 2013;152: 1146–1159. doi: 10.1016/j.cell.2013.02.004 23434322

51. Li L, Su X, Choi GC, Cao Y, Ambinder RF, Tao Q. Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins. BMC Cancer. 2012;12: 125. doi: 10.1186/1471-2407-12-125 22458933

52. Feng W, Kraus RJ, Dickerson SJ, Lim HJ, Jones RJ, Yu X, et al. ZEB1 and c-Jun Levels Contribute to the Establishment of Highly Lytic Epstein-Barr Virus Infection in Gastric AGS Cells. J Virol. 2007;81: 10113–10122. 17626078

53. Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, et al. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China. J Transl Med. 2008;6: 32. doi: 10.1186/1479-5876-6-32 18570662

54. Liu Z, Yang H, Luo W, Jiang Q, Mai C, Chen Y, et al. Loss of cytoplasmic KLF4 expression is correlated with the progression and poor prognosis of nasopharyngeal carcinoma. Histopathology. 2013;63: 362–370. doi: 10.1111/his.12176 23758499

55. Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347: 1260419. doi: 10.1126/science.1260419 25613900

56. Davis-Dusenbery BN, Chan MC, Reno KE, Weisman AS, Layne MD, Lagna G, et al. Down-regulation of Krüppel-like Factor-4 (KLF4) by MicroRNA-143/145 Is Critical for Modulation of Vascular Smooth Muscle Cell Phenotype by Transforming Growth Factor-β and Bone Morphogenetic Protein 4. J Biol Chem. 2011;286: 28097–28110. doi: 10.1074/jbc.M111.236950 21673106

57. Yori JL, Johnson E, Zhou G, Jain MK, Keri RA. Kruppel-like factor 4 (KLF4) inhibits epithelial-to-mesenchymal transition through regulation of E-cadherin gene expression. J Biol Chem. 2010; jbc.M110.114546.

58. Mahatan CS, Kaestner KH, Geiman DE, Yang VW. Characterization of the structure and regulation of the murine gene encoding gut-enriched Krüppel-like factor (Krüppel-like factor 4). Nucleic Acids Res. 1999;27: 4562–4569. 10556311

59. Chen X, Whitney EM, Gao SY, Yang VW. Transcriptional profiling of Krüppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation. J Mol Biol. 2003;326: 665–677. 12581631

60. Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol. 2005;7: 1074–1082. 16244670

61. Chen ZY, Shie J-L, Tseng C-C. Gut-enriched Kruppel-like factor represses ornithine decarboxylase gene expression and functions as checkpoint regulator in colonic cancer cells. J Biol Chem. 2002;277: 46831–46839. 12297499

62. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126: 663–676. 16904174

63. Soufi A, Donahue G, Zaret KS. Facilitators and Impediments of the Pluripotency Reprogramming Factors’ Initial Engagement with the Genome. Cell. 2012;151: 994–1004. doi: 10.1016/j.cell.2012.09.045 23159369

64. Salmon M, Gomez D, Greene E, Shankman L, Owens GK. Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22α Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo. Circ Res. 2012;111: 685–696. doi: 10.1161/CIRCRESAHA.112.269811 22811558

65. Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC, et al. Drastic down-regulation of Krüppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res. 2005;65: 2746–2754. 15805274

66. Zhang N, Zhang J, Shuai L, Zha L, He M, Huang Z, et al. Krüppel-like factor 4 negatively regulates β-catenin expression and inhibits the proliferation, invasion and metastasis of gastric cancer. Int J Oncol. 2012;40: 2038–2048. doi: 10.3892/ijo.2012.1395 22407433

67. Krstic M, Stojnev S, Jovanovic L, Marjanovic G. KLF4 expression and apoptosis-related markers in gastric cancer. J BUON Off J Balk Union Oncol. 2013;18: 695–702.

68. Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Identification of Krüppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene. 2004;23: 395–402. 14724568

69. Foster KW, Liu Z, Nail CD, Li X, Fitzgerald TJ, Bailey SK, et al. Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia. Oncogene. 2005;24: 1491–1500. 15674344

70. Liu Z, Teng L, Bailey SK, Frost AR, Bland KI, LoBuglio AF, et al. Epithelial transformation by KLF4 requires Notch1 but not canonical Notch1 signaling. Cancer Biol Ther. 2009;8: 1840–1851. 19717984

71. Tai S-K, Yang M-H, Chang S-Y, Chang Y-C, Li W-Y, Tsai T-L, et al. Persistent Krüppel-like factor 4 expression predicts progression and poor prognosis of head and neck squamous cell carcinoma. Cancer Sci. 2011;102: 895–902. doi: 10.1111/j.1349-7006.2011.01859.x 21219537

72. Evans PM, Zhang W, Chen X, Yang J, Bhakat KK, Liu C. Krüppel-like Factor 4 Is Acetylated by p300 and Regulates Gene Transcription via Modulation of Histone Acetylation. J Biol Chem. 2007;282: 33994–34002. 17908689

73. Du JX, McConnell BB, Yang VW. A small ubiquitin-related modifier-interacting motif functions as the transcriptional activation domain of Krüppel-like factor 4. J Biol Chem. 2010;285: 28298–28308. doi: 10.1074/jbc.M110.101717 20584900

74. Chen ZY, Wang X, Zhou Y, Offner G, Tseng C-C. Destabilization of Krüppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway. Cancer Res. 2005;65: 10394–10400. 16288030

75. Kim MO, Kim S-H, Cho Y-Y, Nadas J, Jeong C-H, Yao K, et al. ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4. Nat Struct Mol Biol. 2012;19: 283–290. doi: 10.1038/nsmb.2217 22307056

76. Zheng B, Bernier M, Zhang X-H, Suzuki T, Nie C-Q, Li YH, et al. miR-200c-SUMOylated KLF4 feedback loop acts as a switch in transcriptional programs that control VSMC proliferation. J Mol Cell Cardiol. 2015;82: 201–212. doi: 10.1016/j.yjmcc.2015.03.011 25791170

77. Tahmasebi S, Ghorbani M, Savage P, Yan K, Gocevski G, Xiao L, et al. Sumoylation of Krüppel-like factor 4 inhibits pluripotency induction but promotes adipocyte differentiation. J Biol Chem. 2013;288: 12791–12804. doi: 10.1074/jbc.M113.465443 23515309

78. Yusuf I, Kharas MG, Chen J, Peralta RQ, Maruniak A, Sareen P, et al. KLF4 is a FOXO target gene that suppresses B cell proliferation. Int Immunol. 2008;20: 671–681. doi: 10.1093/intimm/dxn024 18375530

79. Kharas MG, Yusuf I, Scarfone VM, Yang VW, Segre JA, Huettner CS, et al. KLF4 suppresses transformation of pre-B cells by ABL oncogenes. Blood. 2007;109: 747–755. 16954505

80. Guan H, Xie L, Leithäuser F, Flossbach L, Möller P, Wirth T, et al. KLF4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classic Hodgkin lymphoma. Blood. 2010;116: 1469–1478. doi: 10.1182/blood-2009-12-256446 20519630

81. Shaffer AL, Lin KI, Kuo TC, Yu X, Hurt EM, Rosenwald A, et al. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity. 2002;17: 51–62. 12150891

82. Lin K-I, Angelin-Duclos C, Kuo TC, Calame K. Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Mol Cell Biol. 2002;22: 4771–4780. 12052884

83. Lin Y, Wong K, Calame K. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science. 1997;276: 596–599. 9110979

84. Keller AD, Maniatis T. Identification and characterization of a novel repressor of beta-interferon gene expression. Genes Dev. 1991;5: 868–879. 1851123

85. Piboonniyom S, Duensing S, Swilling NW, Hasskarl J, Hinds PW, Münger K. Abrogation of the retinoblastoma tumor suppressor checkpoint during keratinocyte immortalization is not sufficient for induction of centrosome-mediated genomic instability. Cancer Res. 2003;63: 476–483. 12543805

86. Kanda T, Yajima M, Ahsan N, Tanaka M, Takada K. Production of High-Titer Epstein-Barr Virus Recombinants Derived from Akata Cells by Using a Bacterial Artificial Chromosome System. J Virol. 2004;78: 7004–7015. 15194777

87. Strong MJ, Baddoo M, Nanbo A, Xu M, Puetter A, Lin Z. Comprehensive high-throughput RNA sequencing analysis reveals contamination of multiple nasopharyngeal carcinoma cell lines with HeLa cell genomes. J Virol. 2014;88: 10696–10704. doi: 10.1128/JVI.01457-14 24991015

88. Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, et al. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer J Int Cancer. 1999;83: 121–126.

89. Park JG, Yang HK, Kim WH, Chung JK, Kang MS, Lee JH, et al. Establishment and characterization of human gastric carcinoma cell lines. Int J Cancer J Int Cancer. 1997;70: 443–449.

90. Molesworth SJ, Lake CM, Borza CM, Turk SM, Hutt-Fletcher LM. Epstein-Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells. J Virol. 2000;74: 6324–6332. 10864642

91. Lin C-C, Liu L-Z, Addison JB, Wonderlin WF, Ivanov AV, Ruppert JM. A KLF4–miRNA-206 Autoregulatory Feedback Loop Can Promote or Inhibit Protein Translation Depending upon Cell Context. Mol Cell Biol. 2011;31: 2513–2527. doi: 10.1128/MCB.01189-10 21518959

92. Györy I, Fejér G, Ghosh N, Seto E, Wright KL. Identification of a Functionally Impaired Positive Regulatory Domain I Binding Factor 1 Transcription Repressor in Myeloma Cell Lines. J Immunol. 2003;170: 3125–3133. 12626569

93. Klug M, Rehli M. Functional analysis of promoter CpG methylation using a CpG-free luciferase reporter vector. Epigenetics Off J DNA Methylation Soc. 2006;1: 127–130.

94. Balsitis SJ, Sage J, Duensing S, Münger K, Jacks T, Lambert PF. Recapitulation of the Effects of the Human Papillomavirus Type 16 E7 Oncogene on Mouse Epithelium by Somatic Rb Deletion and Detection of pRb-Independent Effects of E7 In Vivo. Mol Cell Biol. 2003;23: 9094–9103. 14645521

95. Ma S-D, Yu X, Mertz JE, Gumperz JE, Reinheim E, Zhou Y, et al. An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model. J Virol. 2012;86: 7976–7987. doi: 10.1128/JVI.00770-12 22623780

96. Adamson AL, Kenney S. Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J Virol. 2001;75: 2388–2399. 11160742

97. Hong GK, Delecluse H-J, Gruffat H, Morrison TE, Feng W-H, Sergeant A, et al. The BRRF1 Early Gene of Epstein-Barr Virus Encodes a Transcription Factor That Enhances Induction of Lytic Infection by BRLF1. J Virol. 2004;78: 4983–4992. 15113878

98. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11: 783–784. doi: 10.1038/nmeth.3047 25075903

99. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343: 84–87. doi: 10.1126/science.1247005 24336571