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DNA methylation and the Epstein–Barr virus
seminars in C A N C E R B I OLOG Y, Vol 9, 1999: pp. 369]375 Article No. scbi.1999.0137, available online at http:rrwww.idealibrary.com on
DNA methylation and the Epstein–Barr virus Richard F. Ambinder U , Keith D. Robertson and Qian TaoU
EBV is a ubiquitous herpesvirus associated with a variety of lymphoid and epithelial tumors. In healthy lymphocytes and in tumors immune surveillance is evaded by suppression of a family of immunodominant viral antigens. Methylation of a viral promoter plays a crucial role in this suppression. Methylation of the viral genome in the latent state over evolutionary time is believed to account for CpG suppression that distinguishes this virus from most other large DNA viruses. Pharmacologic manipulation of methylation may offer an opportunity to unmask viral antigens and expose tumors to immune surveillance.
lular genome, and a detailed characterization of the regulation of some of the viral promoters. This review will focus on the biology of the virus, what is known about patterns of methylation and their importance for gene regulation, and potential therapeutic implications.
Biology of EBV Lytic versus latent infection
Key words: Epstein-Barr virus r Burkitt’s lymphoma r Hodgkin’s disease r nasopharyngeal carcinoma r EBNA r methylation
There are two alternative states of EBV infection: lytic and latent.2 Virions are only produced in lytic infection. These are icosahedral capsids that carry a large Ž171 kb. genome of linear double-stranded DNA.3 Latent infection is associated with persistence of the viral genome most commonly as an episome. In vitro in tissue culture systems and in vivo in most settings including neoplasia, latent rather than lytic infection predominates.
Q1999 Academic Press
Introduction EPSTEIN ]BARR VIRUS ŽEBV. is a large DNA virus that is ubiquitous in human populations.1 It is associated with a variety of lymphoid, epithelial and smooth muscle tumors. It encodes 80 q proteins, but infected cells may express only one or a very few proteins. Methylation of the viral genome appears to play a crucial role in the regulation of viral gene expression in normal and neoplastic tissues, in the escape of infected cells from immune surveillance, and in the resistance of infected tumor cells to antiviral nucleoside analogues. Several aspects of EBV biology make it an especially attractive model system for studying the effects of DNA methylation on gene regulation. These include knowledge of the complete sequence of the viral genome, its persistence in cell lines as a mini-chromosome independent of the cel-
Transmission and viral infection The virus is transmitted in the saliva.1 ] 4 Primary infection in childhood is usually asymptomatic, but when delayed to adolescence or young adulthood is often associated with the syndrome of infectious mononucleosis.5 Serologic studies suggest that more than 90% of the world’s adult population is infected by the virus.1 Once infection occurs it is generally life long, with viral episomes persisting in B cells. Infection of B cells B cells become infected, perhaps first in the oral mucosa, and are driven to proliferate.1 Viral episomes are passed on to progeny B cells and thus the pool of latently infected cells expands. Early in infection, as many as several percent of lymphocytes may be infected by virus and as many as 2 = 10 4 copies of the genome per 10 4 peripheral blood mononuclear cells may be present.6 ] 9 The capacity to proliferate and
U
From the Department of Oncology, The Johns Hopkins University, School of Medicine, 418 N. Bond St., Baltimore, MD 21231, USA and Johns Hopkins Singapore Q1999 Academic Press 1044-579Xr 99 r 050369q 07 $30.00r 0
369
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expand the infected cell pool by several orders of magnitude without further cycles of infection is a feature that distinguishes EBV from other herpesviruses and may help to explain the association of the virus with proliferative or neoplastic diseases.
expression is driven from the Q promoter.33,34 When all of the EBNAs are expressed, their expression is driven from the C promoter by alternative splicing from a primary transcript of over 100 kb.35
Immortalization and persistence
DNA methylation and EBV gene regulation
The proliferation of the infected lymphocytes is driven by latency viral proteins such that these cells become immortalized, meaning that they will proliferate indefinitely in culture.2 They will also form human B cell tumors in immunodeficient ŽSCID. mice.10 At least six nuclear proteins and three membrane proteins are expressed. In addition to driving the proliferation of lymphocytes, a subset of these viral proteins are also target antigens for a massive cellular immune response that controls the proliferation of EBV infected lymphocytes.11,12 EBV infected cells that persist in healthy persons can be demonstrated by spontaneous transformation assays.13 The frequency of infected B cells has been estimated at 10 4 ]10 6 B cells.11,14,15
Methylation of the EBV C promoter Methylation of the EBV genome, rather than functioning as part of a genome-wide defense system that inactivates proviral DNA, transposable elements and other parasitic foreign sequences, may ensure that a proportion of EBV infected cells survive cytotoxic T cell immune surveillance by silencing the C promoter and thus the transcription of the immunodominant EBNA proteins ŽFigure 2..36 ] 38 C promoter transcription is activated by a combination of viral and cellular proteins. EBNA1 binds to the family of tandem repeats upstream of the C promoter leading to function as a transcriptional enhancer.39 ] 41 Two C promoter binding f actors of cellular origin ŽCBF 1 and 2. bind proximally to the EBNA2 response region. CBF1, also known as RBP-Jk , is a ubiquitous sequence specific DNA binding protein that functions as a transcriptional repressor. EBNA-2 binds to CBF1 masking the CBF1 repression domain while providing its own acidic transactivation domain.42,43 CBF2 binds immediately downstream of CBF1 and functions as a transcriptional activator. Methylation of the middle ‘C’ within the sequence . . . CAGTGCGTCG . . . ’ blocks CBF2 binding.44,45 We used a methylation cassette reporter assay in which regions of the C promoter were methylated in vitro and ligated back into their natural context to assess the effect of methylation on expression of a chloramphenicol acetyl transferase reporter.46 Methylation of the EBNA-2 response element was sufficient to abolish EBNA-2 mediated C promoter activity. Evidence that methylation of the C promoter is critical in regulating transcription is both circumstantial and experimental. The EBNA2 responsive region of the C promoter is methylated in normal lymphocytes in healthy seropositive individuals as well as in tumors in which the C promoter is silent ŽBurkitt’s lymphoma, primary nasopharyngeal carcinoma, nasal lymphoma, Hodgkin’s disease, and gastric carcinoma..47 ] 51 Several experimental studies suggest that alterations in chromatin rather than the presence or absence of transcription factors ensures the silence of the C promoter in tumor cell lines.52,53 The impor-
Expression of the EBNA family of genes A family of viral genes, the EBNAs, plays a critical role in maintenance of the viral episome, immortalization and immune recognition. EBNA1 is required for maintenance of the viral episome.16,17 In resting lymphocytes and in many tumors, EBNA1 may be the only member of the EBNA family of genes expressed ŽFigure 1..11,18,19 Tumors with this pattern of expression include Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma.20 ] 24 EBNAs 1, 2, 3A and 3C are required for immortalization and are all expressed in EBV-immortalized lymphocytes as well as in most of the EBV-associated lymphomas arising in the immunosuppressed.25 ] 29 EBNAs 3A, 3B, 3C are the immunodominant targets for cytotoxic T cells.30,31 Cytotoxic T cells targeting EBV latency antigens occur with a frequency as high as 0.5% of peripheral blood mononuclear cells in healthy volunteers.32 These T cells limit the proliferation of EBVinfected B cells in healthy EBV-seropositive individuals. As such their expression is functionally incompatible with persistence in an immunologically competent seropositive. These antigens are only expressed early after primary infection when the cytotoxic T cell response has not yet developed or in profoundly immunocompromised patients. When EBNA1 is the only EBNA expressed, its 370
DNA methylation and EBV
Figure 1. Top. Expression of the EBNAs in tumors and infected lymphocytes. Only EBNA1 is expressed in most tumors and in latently infected lymphocytes from immunocompetent healthy donors. All of the EBNAs are expressed in a subset of lymphoproliferative tumors arising in immunocompromised patients such as those immunosuppressed for organ transplantation. All of the EBNAs are also expressed in lymphocytes following primary infection before and immune response has been generated and in lymphocytes immortalized in vitro. Bottom. Expression of the EBNAs from the C promoter and the Q promoter.
tance of methylation in the regulation of the C promoter has been directly demonstrated by treatment of a Burkitt’s cell line with a DNA methyltransferase
inhibitors Ž5-azacytidine. leading to activation of the C promoter and expression of this family of proteins.44,54 371
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Figure 2. Methylation-specific PCR of the C promoter. ŽTop. Schema showing the C promoter and neighboring regions in the EBV genome. The location of the EBER genes Žyielding two polymerase III transcripts., the origin of replication ŽoriP. which consists of the family of tandem repeats ŽFR. and the region of dyad symmetry ŽDS. both of which have binding sites for EBNA1, and the EBNA2 response element ŽRE. are shown. Also shown are the location of primers used on bisulfite treated DNA to distinguish methylated from unmethylated sequences. The procedure used to characterize methylation of this region and results have been presented in detail.55 ŽBottom. Agarose gel stained with ethidium showing PCR amplification products using primers that selectively amplify unmethylated Žu. DNA or methylated Žm. DNA.
The C promoter is silent in virtually all tumors except for lymphoid tumors arising in patients with profound immunocompromise. In order to characterize the status of the C promoter with regard to methylation, we adapted a technique previously used to characterize CpG islands of tumor suppressor genes.55,56 This PCR-based assay detects the CpG methylation status of the PCR primer annealing regions and only represents an average of the methylation status of the studied promoter. To validate the methylation-specific PCR assay of the C promoter, we compared the results of this technique with those of bisulfite genomic sequencing and RT-PCR previously carried out on a variety of EBVŽq. cell lines and tumor tissues. Results of the different assays were consistent, indicating that it is appropriate to draw inferences from the methylation-specific PCR assay about the C promoter. In lymphoproliferative disease specimens, C promoter methylation was the exception being detected in only three of 11 cases. In the two cases where methylated DNA but not unmethylated viral DNA was detected, EBNA2 expression was not detected.55 In contrast to several of the cases of post-transplant lymphoma in the series, these cases
did not regress with withdrawal of immunosuppression. CpG suppression of the EBV genome Further evidence to support the hypothesis that EBV has subverted the methylation defense system comes from analysis of the CpG content of the viral genome.51 Most CpG sequences in vertebrate DNA are methylated and the frequency of the CpG dinucleotide in vertebrate DNA is much lower than would be predicted by chance, probably as a consequence of enhanced spontaneous deamination of methylcytosine over evolutionary time.38 EBV and its lymphotropic simian cousin, herpesvirus saimiri, are CpG suppressed with a relative CpG abundances of 0.60 of 0.33, respectively.57 The presence of CpG suppression in EBV is consistent with the hypothesis that methylated EBV genomes detected in peripheral blood mononuclear cells are susceptible to mutagenesis by deamination of methylcytosines and important in perpetuating the virus over evolutionary time. In contrast, it is interesting to note that in other large DNA viruses, there is no significant CpG suppression, 372
DNA methylation and EBV
References
presumably because methylation plays no role in perpetuation of the virus.
1. Rickinson AB, Kieff E Ž1996. Epstein]Barr virus, in Fields Virology ŽFields BN, Knipe DM, Howley PM, eds., pp. 2397]2446. Lippincott-Raven Publishers, Philadelphia 2. Kieff E Ž1996. Epstein]Barr virus and its replication, in Fields Virology ŽFields BN, Knipe DM, Howley PM, et al, eds., pp. 2343]2396. Raven, Philadelphia 3. Baer B, Bankier A, Biggin MD, Deininger P, Farrell PJ, Gibson T, Hudson G, Satchwell S, Seguin C, Tuffnell P, Barrel B Ž1984. DNA sequence and expression of the B95-8 Epstein]Barr virus genome. Nature 310:207]211 4. Yao Q, Rickinson AB, Epstein M Ž1988. Oropharyngeal shedding of infectious Epstein]Barr virus in healthy virus immune donors: a prospective study. Chin Med J 98:191]196 5. Evans AS, Niederman JC, McCollum RW Ž1968. Seroepidemiologic studies of infectious mononucleosis with EB virus. N Engl J Med 279:1121]1127 6. Ryon JJ, Hayward SD, MacMahon EME, Mann RB, Ling Y, Charache P, Phelan JA, Miller G, Ambinder RF Ž1993. In situ detection of lytic Epstein]Barr virus infection: Expression of the Not1 early gene and vIL-10 late gene in clinical specimens. J Infect Dis 168:345]351 7. Reynolds DJ, Banks PM, Gulley ML Ž1995. New characterization of infectious mononucleosis and a phenotypic comparison with Hodgkin’s disease. Am J Pathol 146:379]388 8. Klein G, Svedmyr E, Jondal M, Persson PO Ž1976. EBV-determined nuclear antigen ŽEBNA.-positive cells in the peripheral blood of infectious mononucleosis patients. Int J Cancer 17:21]26 9. Laroche C, Drouet EB, Brousset P, Pain C, Boibieux A, Biron F, Icart J, Denoyel GA, Niveleau A Ž1995. Measurement by the polymerase chain reaction of the Epstein]Barr virus load in infectious mononucleosis and AIDS-related non-Hodgkin’s lymphomas. J Med Virol 46:66]74 10. Rowe M, Young LS, Crocker J, Stokes H, Henderson S, Rickinson AB Ž1991. Epstein]Barr virus ŽEBV.-associated lymphoproliferative disease in the SCID mouse model: implications for the pathogenesis of EBV-positive lymphomas in man. J Exp Med 173:147]158 11. Tierney RJ, Steven N, Young LS, Rickinson AB Ž1994. Epstein]Barr virus latency in blood mononuclear cells}analysis of viral gene transcription during primary infection and in the carrier state. J Virol 68:7374]7385 12. Callan MF, Steven N, Krausa P, Wilson JD, Moss PA, Gillespie GM, Bell JI, Rickinson AB, McMichael AJ Ž1996. Large clonal expansions of CD8q T cells in acute infectious mononucleosis. Nat Med 2:906]911 13. Rickinson AB, Rowe M, Hart IJ, Yao QY, Henderson LE, Rabin H, Epstein MA Ž1984. T-cell-mediated regression of ‘spontaneous’ and of Epstein]Barr virus-induced B-cell transformation in vitro: Studies with Cyclosporin A. Cell Immunol 87:646]658 14. Yao QY, Rickinson AB, Epstein MA Ž1985. A re-examination of the Epstein]Barr virus carrier state in healthy seropositive individuals. Int J Cancer 35:35]43 15. Miyashita EM, Yang B, Lam KM, Crawford DH, Thorley-Lawson DA Ž1995. A novel form of Epstein]Barr virus latency in normal B cells in vivo. Cell 80:593]601 16. Yates JL, Warren N, Sugden B Ž1985. Stable replication of plasmids derived from Epstein]Barr virus in various mannalian cells. Nature 313:812]815 17. Rawlins DR, Milman G, Hayward SD, Hayward GS Ž1985. Sequence-specific DNA binding of the Epstein]Barr virus nuclear antigen ŽEBNA-1. to clustered sites in the plasmid maintenance region. Cell 42:859]868 18. Qu L, Rowe DT Ž1992. Epstein]Barr virus latent gene expression in uncultured peripheral blood lymphocytes. J Virol 66:3715]3724
Regions of the viral genome protected from methylation Although methylation silences much of the EBV genome and thus cloaks the presence of the virus so as to facilitate immune evasion, the patterns of methylation are highly regulated and select viral regions appear never to be methylated. This is true of the Q promoter and the EBER region. The Q promoter is required for EBNA1 expression in the most restricted forms of latency. EBNA1 expression, in turn, must be maintained in order to maintain the viral episome during cellular proliferation. A TATA-less promoter residing within a CG-rich region, the Q promoter has many features of the promoters of housekeeping genes. By methylation cassette assay, we tested the effects of methylation on Qp and showed marked inhibition by methylation.58 Thus it is crucial that Qp be excluded from the global methylation of the viral genome in tumors and tumor cell lines. Exclusion from methylation may be a function of Sp1 binding sites in the region. The EBERs are two genes transcribed by RNA polymerase III. Their functions remain ill-defined but they are expressed in high abundance in Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinomas and in peripheral blood mononuclear cells from healthy EBV seropositive individuals.59 In contrast to the neighboring C promoter region, the EBER region shows no methylation.51,60 Therapeutic implications of Cp hypermethylation The strong cellular immune response directed against antigens expressed from the C promoter and the ability to modulate C promoter activity in Burkitt’s cell lines by the use of DNA methyltransferase inhibitors paves the way for a unique therapeutic approach aimed at up-regulating genes silenced by methylation and thus allowing immune recognition of tumor antigens.44,54,61,62 In a pilot trial, we have used 5-azacytidine to treat patients with such EBV malignancies that have failed conventional treatments. Although EBNA antigen expression was not achieved, doses of 5-azacytidine associated with minimal toxicity did lead to substantial demethylation of the viral genome ŽAmbinder, in preparation.. Achieving at least partial demethylation appears to be a prerequisite for transcriptional activation, and may represent a first step. 373
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19. Chen F, Zou JZ, di Renzo L, Winberg G, Hu LF, Klein E, Klein G, Ernberg I Ž1995. A subpopulation of normal B cells latently infected with Epstein]Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1. J Virol 69:3752]3758 20. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF Ž1998. Epstein]Barr virus ŽEBV. in endemic Burkitt’s lymphoma: molecular analysis of primary tumor tissue. Blood 91:1373]1381 21. Deacon EM, Pallesen G, Niedobitek G, Crocker J, Brooks L, Rickinson AB, Young LS Ž1993. Epstein]Barr virus and Hodgkin’s disease: Transcriptional analysis of virus latency in the malignant cells. J Exp Med 177:339]349 22. Grasser FA, Murray PG, Kremmer E, Klein K, Remberger K, Feiden W, Reynolds G, Niedobitek G, Young LS, Mueller]Lantzsch N Ž1994. Monoclonal antibodies directed against the Epstein]Barr virus-encoded nuclear antigen 1 ŽEBNA1.: immunohistologic detection of EBNA1 in the malignant cells of Hodgkin’s disease. Blood 84:3792]3798 23. Niedobitek G, Kremmer E, Herbst H, Whitehead L, Dawson CW, Niedobitek E, von Ostau C, Rooney N, Grasser FA, Young LS Ž1997. Immunohistochemical detection of the Epstein]Barr virus-encoded latent membrane protein 2A in Hodgkin’s disease and infectious mononucleosis. Blood 90:1664]1672 24. Niedobitek G, Agathanggelou A, Rowe M, Jones EL, Jones DB, Turyaguma P, Oryema J, Wright DH, Young LS Ž1995. Heterogeneous expression of Epstein]Barr virus latent proteins in endemic Burkitt’s lymphoma. Blood 86:659]665 25. Marchini A, Kieff E, Longnecker R Ž1993. Marker rescue of a transformation-negative Epstein]Barr virus recombinant from an infected Burkitt lymphoma cell line: a method useful for analysis of genes essential for transformation. J Virol 67:606]609 26. Kempkes B, Pich D, Zeidler R, Hammerschmidt W Ž1995. Immortalization of human primary B lymphocytes in vitro with DNA. Proc Natl Acad Sci USA 92:5875]5879 27. Hammerschmidt W, Sugden B Ž1989. Genetic analysis of immortalizing functions of Epstein]Barr virus in human B lymphocytes. Nature 340:393]398 28. Young L, Alfieri C, Hennessy K, Evans H, O’Hara C, Anderson KC, Ritz J, Shapiro RS, Rickinson AB, Kieff E, Cohen JI Ž1989. Expression of Epstein]Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. N Engl J Med 321:1080]1085 29. Ambinder RF, Lemas MV, Moore S, Yang J, Fabian D, Krone C Ž1999. Epstein]Barr virus and lymphoma. Cancer Treat Res 99:27]45 30. Khanna R, Burrows SR, Moss DJ Ž1995. Immune regulation in Epstein]Barr virus-associated diseases. wReviewx. Microbiol Rev 59:387]405 31. Rickinson AB, Moss DJ Ž1997. Human cytotoxic T lymphocyte responses to Epstein]Barr virus infection. Annu Rev Immunol 15:405]431 32. Yang J, Lemas V, Flinn I, Krone C, Ambinder RF Ž1999. Application of the ELISPOT assay to the characterization of CD8 responses to Epstein]Barr virus antigens. ŽUnPub. 33. Nonkwelo C, Skinner J, Bell A, Rickinson AB, Sample J Ž1996. Transcription start sites downstream of the Epstein]Barr virus ŽEBV. Fp promoter in early-passage Burkitt lymphoma cells define a fourth promoter for expression of the EBV EBNA-1 protein. J Virol 70:623]627 34. Schaefer BC, Strominger JL, Speck SH Ž1995. Redefining the Epstein]Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group 1 Burkitt lymphoma cell lines. Proc Natl Acad Sci USA 92:10565]10569 35. Bodescot M, Perricaudet M, Farrell PJ Ž1987. A promoter for the highly spliced EBNA family of RNAs of Epstein]Barr Virus. J Virol 61:3424]3430
36. Jahner D, Stuhlmann H, Stewart CL et al Ž1982. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298:623]628 37. Bestor TH, Tycko B Ž1996. Creation of genomic methylation patterns. Nat Genet 12:363]367 38. Bestor TH, Coxon A Ž1993. The pros and cons of cytosine methylation. Curr Biol 3:384]386 39. Reisman D, Sugden B Ž1986. Trans activation of an Epstein ] Barr viral transcriptional enhancer by the Epstein]Barr viral nuclear antigen 1. Mol Cell Biol 6:3838]3846 40. Sample J, Brooks L, Sample C, Young L, Rowe M, Gregory C, Rickinson AB, Kieff E Ž1991. 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 USA 88:6343]6347 41. Sugden B, Warren N Ž1989. A promoter of Epstein]Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection. J Virol 63:2644]2649 42. Dou S, Zeng X, Cortes P, Erdjument-Bromage H, Tempst P, Honjo T, Vales LD Ž1994. The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor. Mol Cell Biol 14:3310]3319 43. Hsieh JJ, Hayward SD Ž1995. Masking of the CBF1rRBPJ kappa transcriptional repression domain by Epstein]Barr virus EBNA2. Science 268:560]563 44. Robertson KD, Hayward DJ, Ling PD, Samid D, Ambinder RF Ž1995. Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: Evidence that demethylation at a single CpG site is crucial. Mol Cell Biol 15:6150]6159 45. Fuentes-Panana EM, Ling PD Ž1998. Characterization of the CBF2 binding site within the Epstein]Barr virus latency C promoter and its role in modulating EBNA2-mediated transactivation. J Virol 72:693]700 46. Robertson KD, Ambinder RF Ž1997. Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: Application of the methylation cassette assay to the Epstein]Barr virus major latency promoter. J Virol 71:6445]6454 47. Minarovits J, Hu LF, Minarovits-Kormuta S, Klein G, Ernberg I Ž1994. Sequence-specific methylation inhibits the activity of the Epstein]Barr virus LMP 1 and BCR2 enhancer-promoter regions. Virology 200:661]667 48. Altiok E, Minarovits J, Hu LF, Contreras-Brodin B, Klein G, Ernberg I Ž1992. Host-cell-phenotype-dependent control of the BCR2rBWR1 promoter complex regulates the expression of Epstein]Barr virus nuclear antigens 2-6. Proc Natl Acad Sci USA 89:905]909 49. Imai S, Koizumi S, Sugiura M et al Ž1994. Gastric carcinoma: m onoclonal epithelial m alignant cells expressing Epstein]Barr virus latent infection protein. Proc Natl Acad Sci USA 91:9131]9135 50. Robertson KD, Manns A, Swinnen LJ, Zong JC, Gulley ML, Ambinder RF Ž1996. CpG methylation of the major Epstein]Barr virus latency promoter in Burkitt’s lymphoma and Hodgkin’s disease. Blood 88:3129]3136 51. Robertson KD, Ambinder RF Ž1997. Methylation of the Epstein]Barr virus genome in normal lymphocytes. Blood 90:4480]4484 52. Evans TJ, Jacquemin MG, Farrell PJ Ž1995. Efficient EBV superinfection of group I Burkitt’s lymphoma cells distinguishes requirements for expression of the Cp viral promoter and can activate the EBV productive cycle. Virology 206:866]877 53. Schaefer BC, Strominger JL, Speck SH Ž1997. Host-cell-determined methylation of specific Epstein]Barr virus pro-
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moters regulates the choice between distinct viral latency programs. Mol Cell Biol 17:364]377 Masucci MG, Contreras-Salazar B, Ragnar E, Falk K, Minarovits J, Ernberg I, Klein G Ž1989. 5-Azacytidine up regulates the expression of Epstein]Barr virus nuclear antigen 2 ŽEBNA-2. through EBNA-6 and latent membrane protein in the Burkitt’s lymphoma line Rael. J Virol 63:3135]3141 Tao Q, Swinnen LJ, Yang J, Srivastava G, Robertson KD, Ambinder RF Ž1999. Methylation status of the Epstein]Barr virus major latent promoter C in iatrogenic B cell lymphoproliferative disease: application of PCR-based analysis. Am J Pathol 155:619]625 Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB Ž1996. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821]9826 Karlin S, Doerfler W, Cardon LR Ž1994. Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? J Virol 68:2889]2897
58. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF Ž1998. The Epstein]Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive. J Virol 72:7075]7083 59. Ambinder RF, Mann R Ž1994. Epstein]Barr-encoded RNA in situ hybridization. Hum Pathol 25:602]605 60. Minarovits J, Hu LF, Marcsek Z, Minarovits-Kormuta S, Klein G, Ernberg I Ž1992. RNA polymerase III-transcribed EBER 1 and 2 transcription units are expressed and hypomethylated in the major Epstein]Barr virus-carrying cell types. J Gen Virol 73:1687]1692 61. Ambinder RF, Robertson KD, Moore SM, Yang J Ž1996. Epstein]Barr virus as a therapeutic target in Hodgkin’s disease and nasopharyngeal carcinoma. Semin Cancer Biol 7. 62. Robertson KD, Barletta J, Samid D, Ambinder RF Ž1995. Pharmacologic activation of expression of immunodominant viral antigens: a new strategy for the treatment of Epstein]Barr-virus-associated malignancies. Curr Top Microbiol Immunol 194:145]154
375
DNA methylation and the Epstein–Barr virus Richard F. Ambinder U , Keith D. Robertson and Qian TaoU
EBV is a ubiquitous herpesvirus associated with a variety of lymphoid and epithelial tumors. In healthy lymphocytes and in tumors immune surveillance is evaded by suppression of a family of immunodominant viral antigens. Methylation of a viral promoter plays a crucial role in this suppression. Methylation of the viral genome in the latent state over evolutionary time is believed to account for CpG suppression that distinguishes this virus from most other large DNA viruses. Pharmacologic manipulation of methylation may offer an opportunity to unmask viral antigens and expose tumors to immune surveillance.
lular genome, and a detailed characterization of the regulation of some of the viral promoters. This review will focus on the biology of the virus, what is known about patterns of methylation and their importance for gene regulation, and potential therapeutic implications.
Biology of EBV Lytic versus latent infection
Key words: Epstein-Barr virus r Burkitt’s lymphoma r Hodgkin’s disease r nasopharyngeal carcinoma r EBNA r methylation
There are two alternative states of EBV infection: lytic and latent.2 Virions are only produced in lytic infection. These are icosahedral capsids that carry a large Ž171 kb. genome of linear double-stranded DNA.3 Latent infection is associated with persistence of the viral genome most commonly as an episome. In vitro in tissue culture systems and in vivo in most settings including neoplasia, latent rather than lytic infection predominates.
Q1999 Academic Press
Introduction EPSTEIN ]BARR VIRUS ŽEBV. is a large DNA virus that is ubiquitous in human populations.1 It is associated with a variety of lymphoid, epithelial and smooth muscle tumors. It encodes 80 q proteins, but infected cells may express only one or a very few proteins. Methylation of the viral genome appears to play a crucial role in the regulation of viral gene expression in normal and neoplastic tissues, in the escape of infected cells from immune surveillance, and in the resistance of infected tumor cells to antiviral nucleoside analogues. Several aspects of EBV biology make it an especially attractive model system for studying the effects of DNA methylation on gene regulation. These include knowledge of the complete sequence of the viral genome, its persistence in cell lines as a mini-chromosome independent of the cel-
Transmission and viral infection The virus is transmitted in the saliva.1 ] 4 Primary infection in childhood is usually asymptomatic, but when delayed to adolescence or young adulthood is often associated with the syndrome of infectious mononucleosis.5 Serologic studies suggest that more than 90% of the world’s adult population is infected by the virus.1 Once infection occurs it is generally life long, with viral episomes persisting in B cells. Infection of B cells B cells become infected, perhaps first in the oral mucosa, and are driven to proliferate.1 Viral episomes are passed on to progeny B cells and thus the pool of latently infected cells expands. Early in infection, as many as several percent of lymphocytes may be infected by virus and as many as 2 = 10 4 copies of the genome per 10 4 peripheral blood mononuclear cells may be present.6 ] 9 The capacity to proliferate and
U
From the Department of Oncology, The Johns Hopkins University, School of Medicine, 418 N. Bond St., Baltimore, MD 21231, USA and Johns Hopkins Singapore Q1999 Academic Press 1044-579Xr 99 r 050369q 07 $30.00r 0
369
R. F. Ambinder et al
expand the infected cell pool by several orders of magnitude without further cycles of infection is a feature that distinguishes EBV from other herpesviruses and may help to explain the association of the virus with proliferative or neoplastic diseases.
expression is driven from the Q promoter.33,34 When all of the EBNAs are expressed, their expression is driven from the C promoter by alternative splicing from a primary transcript of over 100 kb.35
Immortalization and persistence
DNA methylation and EBV gene regulation
The proliferation of the infected lymphocytes is driven by latency viral proteins such that these cells become immortalized, meaning that they will proliferate indefinitely in culture.2 They will also form human B cell tumors in immunodeficient ŽSCID. mice.10 At least six nuclear proteins and three membrane proteins are expressed. In addition to driving the proliferation of lymphocytes, a subset of these viral proteins are also target antigens for a massive cellular immune response that controls the proliferation of EBV infected lymphocytes.11,12 EBV infected cells that persist in healthy persons can be demonstrated by spontaneous transformation assays.13 The frequency of infected B cells has been estimated at 10 4 ]10 6 B cells.11,14,15
Methylation of the EBV C promoter Methylation of the EBV genome, rather than functioning as part of a genome-wide defense system that inactivates proviral DNA, transposable elements and other parasitic foreign sequences, may ensure that a proportion of EBV infected cells survive cytotoxic T cell immune surveillance by silencing the C promoter and thus the transcription of the immunodominant EBNA proteins ŽFigure 2..36 ] 38 C promoter transcription is activated by a combination of viral and cellular proteins. EBNA1 binds to the family of tandem repeats upstream of the C promoter leading to function as a transcriptional enhancer.39 ] 41 Two C promoter binding f actors of cellular origin ŽCBF 1 and 2. bind proximally to the EBNA2 response region. CBF1, also known as RBP-Jk , is a ubiquitous sequence specific DNA binding protein that functions as a transcriptional repressor. EBNA-2 binds to CBF1 masking the CBF1 repression domain while providing its own acidic transactivation domain.42,43 CBF2 binds immediately downstream of CBF1 and functions as a transcriptional activator. Methylation of the middle ‘C’ within the sequence . . . CAGTGCGTCG . . . ’ blocks CBF2 binding.44,45 We used a methylation cassette reporter assay in which regions of the C promoter were methylated in vitro and ligated back into their natural context to assess the effect of methylation on expression of a chloramphenicol acetyl transferase reporter.46 Methylation of the EBNA-2 response element was sufficient to abolish EBNA-2 mediated C promoter activity. Evidence that methylation of the C promoter is critical in regulating transcription is both circumstantial and experimental. The EBNA2 responsive region of the C promoter is methylated in normal lymphocytes in healthy seropositive individuals as well as in tumors in which the C promoter is silent ŽBurkitt’s lymphoma, primary nasopharyngeal carcinoma, nasal lymphoma, Hodgkin’s disease, and gastric carcinoma..47 ] 51 Several experimental studies suggest that alterations in chromatin rather than the presence or absence of transcription factors ensures the silence of the C promoter in tumor cell lines.52,53 The impor-
Expression of the EBNA family of genes A family of viral genes, the EBNAs, plays a critical role in maintenance of the viral episome, immortalization and immune recognition. EBNA1 is required for maintenance of the viral episome.16,17 In resting lymphocytes and in many tumors, EBNA1 may be the only member of the EBNA family of genes expressed ŽFigure 1..11,18,19 Tumors with this pattern of expression include Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma.20 ] 24 EBNAs 1, 2, 3A and 3C are required for immortalization and are all expressed in EBV-immortalized lymphocytes as well as in most of the EBV-associated lymphomas arising in the immunosuppressed.25 ] 29 EBNAs 3A, 3B, 3C are the immunodominant targets for cytotoxic T cells.30,31 Cytotoxic T cells targeting EBV latency antigens occur with a frequency as high as 0.5% of peripheral blood mononuclear cells in healthy volunteers.32 These T cells limit the proliferation of EBVinfected B cells in healthy EBV-seropositive individuals. As such their expression is functionally incompatible with persistence in an immunologically competent seropositive. These antigens are only expressed early after primary infection when the cytotoxic T cell response has not yet developed or in profoundly immunocompromised patients. When EBNA1 is the only EBNA expressed, its 370
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Figure 1. Top. Expression of the EBNAs in tumors and infected lymphocytes. Only EBNA1 is expressed in most tumors and in latently infected lymphocytes from immunocompetent healthy donors. All of the EBNAs are expressed in a subset of lymphoproliferative tumors arising in immunocompromised patients such as those immunosuppressed for organ transplantation. All of the EBNAs are also expressed in lymphocytes following primary infection before and immune response has been generated and in lymphocytes immortalized in vitro. Bottom. Expression of the EBNAs from the C promoter and the Q promoter.
tance of methylation in the regulation of the C promoter has been directly demonstrated by treatment of a Burkitt’s cell line with a DNA methyltransferase
inhibitors Ž5-azacytidine. leading to activation of the C promoter and expression of this family of proteins.44,54 371
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Figure 2. Methylation-specific PCR of the C promoter. ŽTop. Schema showing the C promoter and neighboring regions in the EBV genome. The location of the EBER genes Žyielding two polymerase III transcripts., the origin of replication ŽoriP. which consists of the family of tandem repeats ŽFR. and the region of dyad symmetry ŽDS. both of which have binding sites for EBNA1, and the EBNA2 response element ŽRE. are shown. Also shown are the location of primers used on bisulfite treated DNA to distinguish methylated from unmethylated sequences. The procedure used to characterize methylation of this region and results have been presented in detail.55 ŽBottom. Agarose gel stained with ethidium showing PCR amplification products using primers that selectively amplify unmethylated Žu. DNA or methylated Žm. DNA.
The C promoter is silent in virtually all tumors except for lymphoid tumors arising in patients with profound immunocompromise. In order to characterize the status of the C promoter with regard to methylation, we adapted a technique previously used to characterize CpG islands of tumor suppressor genes.55,56 This PCR-based assay detects the CpG methylation status of the PCR primer annealing regions and only represents an average of the methylation status of the studied promoter. To validate the methylation-specific PCR assay of the C promoter, we compared the results of this technique with those of bisulfite genomic sequencing and RT-PCR previously carried out on a variety of EBVŽq. cell lines and tumor tissues. Results of the different assays were consistent, indicating that it is appropriate to draw inferences from the methylation-specific PCR assay about the C promoter. In lymphoproliferative disease specimens, C promoter methylation was the exception being detected in only three of 11 cases. In the two cases where methylated DNA but not unmethylated viral DNA was detected, EBNA2 expression was not detected.55 In contrast to several of the cases of post-transplant lymphoma in the series, these cases
did not regress with withdrawal of immunosuppression. CpG suppression of the EBV genome Further evidence to support the hypothesis that EBV has subverted the methylation defense system comes from analysis of the CpG content of the viral genome.51 Most CpG sequences in vertebrate DNA are methylated and the frequency of the CpG dinucleotide in vertebrate DNA is much lower than would be predicted by chance, probably as a consequence of enhanced spontaneous deamination of methylcytosine over evolutionary time.38 EBV and its lymphotropic simian cousin, herpesvirus saimiri, are CpG suppressed with a relative CpG abundances of 0.60 of 0.33, respectively.57 The presence of CpG suppression in EBV is consistent with the hypothesis that methylated EBV genomes detected in peripheral blood mononuclear cells are susceptible to mutagenesis by deamination of methylcytosines and important in perpetuating the virus over evolutionary time. In contrast, it is interesting to note that in other large DNA viruses, there is no significant CpG suppression, 372
DNA methylation and EBV
References
presumably because methylation plays no role in perpetuation of the virus.
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Regions of the viral genome protected from methylation Although methylation silences much of the EBV genome and thus cloaks the presence of the virus so as to facilitate immune evasion, the patterns of methylation are highly regulated and select viral regions appear never to be methylated. This is true of the Q promoter and the EBER region. The Q promoter is required for EBNA1 expression in the most restricted forms of latency. EBNA1 expression, in turn, must be maintained in order to maintain the viral episome during cellular proliferation. A TATA-less promoter residing within a CG-rich region, the Q promoter has many features of the promoters of housekeeping genes. By methylation cassette assay, we tested the effects of methylation on Qp and showed marked inhibition by methylation.58 Thus it is crucial that Qp be excluded from the global methylation of the viral genome in tumors and tumor cell lines. Exclusion from methylation may be a function of Sp1 binding sites in the region. The EBERs are two genes transcribed by RNA polymerase III. Their functions remain ill-defined but they are expressed in high abundance in Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinomas and in peripheral blood mononuclear cells from healthy EBV seropositive individuals.59 In contrast to the neighboring C promoter region, the EBER region shows no methylation.51,60 Therapeutic implications of Cp hypermethylation The strong cellular immune response directed against antigens expressed from the C promoter and the ability to modulate C promoter activity in Burkitt’s cell lines by the use of DNA methyltransferase inhibitors paves the way for a unique therapeutic approach aimed at up-regulating genes silenced by methylation and thus allowing immune recognition of tumor antigens.44,54,61,62 In a pilot trial, we have used 5-azacytidine to treat patients with such EBV malignancies that have failed conventional treatments. Although EBNA antigen expression was not achieved, doses of 5-azacytidine associated with minimal toxicity did lead to substantial demethylation of the viral genome ŽAmbinder, in preparation.. Achieving at least partial demethylation appears to be a prerequisite for transcriptional activation, and may represent a first step. 373
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