Epstein-Barr virus genes and cancer cells

Biorned

Dossier “Virus

virus

PJ Farrell’.‘, Institute

for

1997;51:258-267 0 Elsevier. Paris

and Cancer”

Epstein-Barr

‘Ludwig

& Pharmacother

Cancer

Research

genes and cancer cells I Cludts’,

and ‘Deportment St Mary’s, IVorfolk

A Stiihler’

of Medical Microbiology, Place, London W2 IPG.

Imperial UK

College

School

of Medicine

at

Summary - Human B lymphocytes infected with Epstein-Barr virus (EBV) express II viral genes, of which six are essential for efficient transformation. The protein products of these genes appear to cause cell growth by modifying cell signal transduction pathways. For example, EBNA-2 mimics the Notch 1 pathway and LMP-1 interacts with the signalling from CD40/CD40-L, which promotes growth in normal B cells. In the human cancers linked to EBV, most of the viral transforming genes are not expressed. It is likely that growth of these cells is controlled by a combination of the EBV genes whose expression continues with altered cell proto-oncogenes and tumour suppressor genes. but other explanations of the role of EBV in cancer cells are also possible. The presence of the virus in the tumour cells of EBV-associated cancers constitutes a potenti,ally useful tumour specific marker that might be used to direct therapy to the tumour cells. Burkitt’s

lymphoma

I nasopharyngeal

carcinoma

/ cancer

INTRODUCTION Most of the world’s population is thought to be infected with the human herpesvirus, Epstein-Barr virus (EBV). The virus is transmitted through infected saliva early in life and is carried thereafter as a life-long infection in B lymphoid cells. The number of EBV infected cells in a normal virus carrier is very low but there is intermittent viral shedding into the saliva from viral replication that may have occurred in the oropharyngeal lymphoid or epithelial tissue. When EBV infects a B lymphocyte, the virus follows a programme of gene expression that takes it into a latent non-productive infection; the acid viral double stranded deoxyribonucleic (DNA) is maintained as a circular multicopy episome in the nucleus of the infected cell but no virus is produced. This infection of B lymphocytes also causes growth of the B cell so that permanent cell lines are readily established in culture. These cells, in which growth is clearly being driven by EBV proteins, are called lymphoblastoid cell lines (LCLs) and their appearance and cell surface markers are very similar to B cells stimulated to grow in response to antigen. Cells similar to LCLs are observed in vivo during the disease, infectious mononucleosis. All normal virus carriers maintain cytotoxic T cell memory

therapy

directed against various EBV proteins expressed in LCLs that results in killing of LCL-type cells; normal virus persistence is thought to occur in long-lived (memory) B cells through an alternative pattern of latent viral gene expression in which very few viral genes are expressed. Many of these aspects of EBV biology have been reviewed recently [58]. Certain types of human cancer are closely associated with EBV and in an EBV positive tumour, all the tumour cells carry EBV. Examples of such cancers are Burkitt’s lymphoma ([BL] a B cell lymphoma), nasopharyngeal carcinoma (NPC), immunoblastic lymphomas in immunosuppressed people and some rare T cell lymphomas. EBV

GENE

EXPRESSION

EBV has a large and complex DN.4 genome with at least 85 genes [ 121. Eleven of the viral genes are expressed in LCLs but genetic analysis indicates that only six of these are essential for efficient transformation and permanent growth of B cells in response to EBV [59] (table I). The genes comprise several nuclear proteins (the EpsteinBarr nuclear antigens, [EBNAs])., some plasma membrane proteins (the latent membrane proteins,

Epstein-Barr

Table (EBV)

I. Alternative genes.

Nomenclature

nomenclatures

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virus

Alternative

cells

259

but there is no direct evidence for this mechanism Recently EBNA-1 transgenic mice were generated [77] with expression targeted to the B cell compartment (using the mouse immunoglobulin [Ig] heavy chain intron enhancer, Ep). There was an enhanced incidence of B cell lymphoma in these mice suggesting that EBNA-1 could be oncogenic and might have a further, as yet undiscoveyed, biochemical function which was responsible for its tumourigenicity in the mice.

nomenclature

EBNAS EBNA4 EBNA6 EBNAS LMP TPl TP2 -

EBV nuclear EBV-encoded

genes and cancer

WI.

used here

EBNA- 1 EBNA-2 EBNA-3A EBNA-3B EBNA-3C EBNA-LP LMP- 1 LMP-2A LMP-2B EBER I EBER2 EBNA, EBER,

for

virus

antigen; LMP, latent membrane RNA; TP, terminal protein.

protein;

[LMPs]) and some very abundant small untranslated ribonucleic acids ([RNAs] - the EpsteinBarr virus encoded RNAs [EBERs]). The genes that are essential for efficient transformation are EBNA-1, 2, 3A, 3C, leader protein (LP) and LMP-1. The virus can use different transcription programmes depending on the cell type in which it resides. The three main gene expression programmes are frequently referred to as Latency I, III and the lytic cycle. The layout of the EBNA, LMP and EBER genes on the viral genome and transcripts encoding them are shown schematically in figure 1 for Latency I and III. An intermediate pattern of gene expression (Latency II) in which Qp driven EBNA-1 expression is combined with LMP-1 expression is also found in certain diseases (see below). In vivo, a further pattern in which only LMP-2 is expressed has also been reported [55]. FUNCTIONS

OF EBV GENES

EBNA-1 EBNA-1 is a sequence specific DNA binding protein, which binds to the origin of replication oriP [57]. This binding is essential for the maintenance replication of the viral episome by the cell DNA polymerase [79]. In addition to DNA replication, EBNA-1 binding to oriP also acts as a transcription enhancer for the Cp EBNA promoter [44, 701 and the promoter for LMP-1 [ 141. It is possible that the DNA binding site sequence for EBNA-1 might also occur in the cell genome near genes so that it could affect expression of cell genes,

EBNA-2 EBNA-2 is a transcriptional activator that regulates expression of viral latency genes (EBNAs and LMPs) and activates expression of cell genes (CD21, CD23, c-fgr and probably many more) [66]. Although it does not bind DNA directly, EBNA-2 achieves promoter specificity through interaction with other sequence specific DNA binding proteins, particularly the transcription factors CBFl/RBP-Jk [20] and PU. 1 [31, 381, and EBNA-2 contains a strong transcription activation domain [7]. CBFl/RBP-Jk is a key component of the Notch signalling pathway present in cells and it seems likely [23] that a major role of EBNA-2 is to mimic Notch signal transduction (fig 2A). EBNA-LP The function of this protein is still unknown but it and EBNA-2 are the earliest EBV genes expressed upon infection of B lymphocytes. Transfection of plasmids expressing EBNA-LP and EBNA-2 is sufficient to drive resting B lymphocytes into the Gl phase of the cell cycle [67]. EBNA-LP is phosphorylated and this varies during the cell cycle [35]. EBNA-3A,

3B, 3C

These related genes are adjacent in the viral genome. EBNA-3C will co-operate with Ha-ras assays and displays in cell transformation functional properties similar to El A, a transforming protein of adenovirus [52]. This activity of EBNA-3C overcomes the pRB checkpoint in the Gl phase of the cell cycle, which is a major control point of cell growth. EBNA-3A and 3C have also been found to repress transcription [2, 39,

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et al

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I

I

I

I

I

I

I

I

1

-

Lymphoblastoid

cell line

kb

(LCL)

CP b b

LMP-2. 1-1 P\

II EBERs

Latency

EBNA-LP

ABC EBNA-3

EBNA-2

EBNA-1

I w LMP-1

I ori P

I - Burkitt’s

lymphoma

QP P EBNA-1 EBERs

ori P

Fig 1. Schematic layout of EBV genes expressed in LCLs (latency III) compared to Burkitt’s lymphoma cells (latency I). In both cases, genes expressed are illustrated as filled boxes. In LCLs the full set of EBNAs (EBNA-LP, -1, -2, -3A, -3B, -3C), LMPs and EBER RNAs is expressed from the Cp promotor. In EBV positive BL only EBNA-1 and the EBER R.NAs are expressed; transcription of EBNA-1 is from the Qp promotor. In the virus particle the genome is linearised as shown but in the infected cell, the ends of the viral genome are joined to give the circular DNA episome.

45, 741 and EBNA-3A, 3B and 3C can all bind to the CBFl/RBP-Jk transcription factor [60, 811, but the significance of this binding is unclear. LMP-1 LMP-1 is an integral membrane protein with six membrane spanning hydrophobic segments and a C terminal cytoplasmic domain, which seems to contain the effector part of the molecule. No extracellular ligand has been identified for LMP-1.

Part of the cytoplasmic domain (amino acids 187231) binds directly to the TRAF2 signalling protein [33, 481 which is involved in the signal transduction from CD40 and tumour necrosis factor receptor (TNF-R) (fig 2B). This is considered to be a key part of the mechanism by which EBV causes cell growth, presumably substituting for the signal that in normal B cell growth would result from CD40-L binding CD40 1821.One result of TRAF interaction is the activation of the NFkB transcription factor, which is important for B cell growth but there are likely to be other sig-

Epstein-Barr

virus

NOTCH pathway

genes and cancer

cells

261

EBV mfection

LMF’-l

NF-KB

EGFR

7

Fig 2. EBNA-2 and LMP-1 signal transduction A : EBNA-2 is targeted to its responsive promoters through interaction with a cellular DNA binding protein CBFl/RBP-Jk. CBFl/RBP-Jk is by itself a transcriptional repressor, probably acting with a unidentified co-repressor (?). CBFl/RBP-Jk is a participant in the Notch-regulated signal transduction pathway. The Notch receptor can be activated by ligand stimulation or by removal of its extracellular domain. This leads to a constitutively active form of Notch (Notch IC) but the mechanism underlying the activation of CBFl/RBP-Jk by Notch is still unclear. + or - refer to the transcriptional activity of the cofactors. B : LMP-1 acts like a constitutively activated receptor 01‘ the tumour necrosis factor receptor (TNF-R) family. There is a similarity between the effects of LMP-I and those induced by CD40 activation. The CD40 cytoplasmic domain interacts with TRAFZ and TRAF3. There is a differential association of TRAFl, 2 and 3 with the LMP-1 cytoplasmic domain. TRAFl and TRAF2 are both implicated in LMP-1 (amino acids 187 to 231).induced NF-kB activation. TRAFS represses this LMP-1 (amino acids 187 to 231).mediated NF-kB activation. LMP-1 also induces set of genes through a TRAF-independent activation of NF-kB, mediated by amino acids 351 to 386. TRAF signalling from LMPl and CD40 activates a downstream transcription pathway distinct from NF-kB that induces expression of the EGFR.

nificant targets too [47]. A further part of LMP-1 (amino acids 351-386) seems to activate NF-kB independently through an unknown mechanism [26]. These two effector pathways may account for the ability of LMP-1 to transform Rat- 1 fibroblasts in culture [75] and induce the expression of activation markers in lymphocytes [76].

EBER

RNAs

These are the most abundantly expressed EBV genes but the function of the EBER RNAs is not known. Current thinking is based on the ability of the EBER RNAs to prevent activation of the interferon inducible dsRNA dependent protein ki-

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nase (PK)R [6], which can be activated by dsRNA in virus infected cells. In addition to its well established effects on protein synthesis, PKR has been shown to phosphorylate IkB, the regulator subunit bound to NF-kB [37]. So it is possible that the EBER RNAs also affect the regulation of NF-kB. LMP-2 LMP-2A,B are generated by alternate splicing and differ only at the N terminus of the protein. LMP-2A is a substrate for src family tyrosine kinases and acts as a dominant negative regulator of surface immunoglobulin receptor signalling through its effects on Lyn, Syk or regulators of these kinases [46]. The effects of LMP-2A on src family kinases and on calcium mobilisation enable LMP-2A to block the switch from latent infection to the lytic cycle. Its role may thus be prevention of accidental reactivation of the virus during latency. BARFO

and CST

The complementary strand transcripts (CSTs) are a family of highly spliced RNAs present in various latently infected cells. They contain several open reading frames but proteins from these RNAs have not yet been characterised [68]. The entire region of EBV encoding BARFO and the CSTs can be deleted from EBV without losing the ability to transform B cells [61]. GENE EXPRESSION IN EBV ASSOCIATED CANCERS In post-transplant lymphoproliferative disease (PTLD) and other immunoblastic B lymphomas (eg, in AIDS), the tumour cells are very similar to LCLs [58]. In each case a defect in immune surveillance results in a loss of normal cytotoxic T cell surveillance of LCLs. In PTLD it is because of immunosuppressive drugs used after in acquired immunodeficiency transplantation, syndrome (AIDS) it is due to general destruction of the immune response by the human immunodeficiency virus (HIV). In these diseases the full EBV LCL (Latency III) set of EBNAs, LMPs and EBER RNAs is expressed and EBV genes appear to be driving cell growth.

et al

Burkitt’s

lymphoma

In EBV positive BL, only EBNA-1 and the EBER RNAs are expressed (Latency I) [63]. The CSTs are also expressed variably and at low level. Transcription of EBNA-1 is from the Qp promoter [50, 641. A low incidence of BL occurs world-wide in which the tumour cells are EBV negative although most of the patients will be carriers of EBV (sporadic BL). Superimposed on this in parts of Africa, New Guinea and South America where malaria is hyperendemic, a much higher incidence of ‘endemic’ BL is observed in which every tumour cell carries EBV. In cemral Africa 95% of cases are of this EBV positive type [29]. The tumour cells of all BLs are characterised by translocation of the c-myc gene to one of the immunoglobulin loci, resulting in altered regulation of c-myc. Additionally c-myc is frequently found to be mutated in the region which normally binds the ~107 repressor of myc function [17]. Some of the lymphomas found in AIDS are of the BL type and about 50% of them are EBV positive. Other oncogene changes that have been less well studied but are sometimes found in BL are in pim1, c-fps/c-fes and ~53 [29]. About 30% of BL tumours have been reported to have ~53 mutations [15]. Nasopharyngeal

carcinoma

All the tumour cells of undifferentiated NPC are infected with EBV [56]. This cancer shows remarkable variation in incidence, being 100 times more frequent in Southeast Asian Chinese populations than in Western peoples. The difference may be accounted for by genetic or environmental factors and possibly by different strains of EBV [30]. EBV gene expression in NPC consists of EBNA-1 (from Qp) with the EBER RNAs, LMP2A,B and (in about 65% of cases) LMP-1 [3, 11, 801. Transcripts of the BamHI A region (BARFO, CSTs) have also been reported [S, 161. Alteration of the p16 cyclin dependent kinase inhibitor gene (9q21-22) is frequently found in NPC [25, 43, 711. Other chromosome regions reported to be changed are 3~13-14.3 [42], llq13.3-22 and llq22-24 [24, 271, but affected genes at these loci have not yet been identified. Studies on ~53 usually showed no mutation in NPC tumours, although the rare tumours that could successfully be established in mice as xeno-

Epstein-Barr

virus

grafts showed ~53 mutations in the xenograft [9, 691. Another carcinoma, gastric carcinoma is also found to be EBV positive in about 7% of cases

Lw. Hodgkin’s

Disease

(HD)

and T cell lymphomas

EBV is also found to be present in the ReedSternberg cells of HD in about 40% of cases, mostly of the mixed cellularity type [72]. No consistent oncogene changes have been described in these cells but EBV gene expression has been studied by reverse transcriptase polymerase chain reaction (RT-PCR). A latency II pattern of EBNA1, LMP- 1 and 2 and EBER RNAs has been found [8, 21, 51, 54, 781. Certain rare forms of T cell lymphoma such as midline granuloma [19] and T cell lymphomas in immunodeficiency [32, 341 have also been reported to be EBV positive, with EBNA-1 and EBER RNAs expressed. Recently it has been suggested that a substantial proportion of T cell lymphomas may be EBV positive in far eastern countries in contrast to Europe or the USA. ROLE

OF EBV

IN CANCER

For all of the EBV associated cancers, the very large proportion of tumour cells in an EBV associated case (usually 100%) that carry EBV in contrast with the very low number of non-tumour cells infected with EBV suggests that the virus has a causative role in the cancer. It is difficult to absolutely exclude the notion that the virus is simply an inconsequential passenger in the tumour cells but it would be surprising if the episomal virus genome were retained if it were doing nothing for the cell growth and survival. It is also noteworthy that some of the tumour cell types (BL, NPC) have only very low or undetectable levels of CD21, the known EBV receptor, and are difficult or impossible to infect with the virus in culture so EBV seems unlikely to be present in the tumour cells simply by chance. Clonality of the tumour cells with respect to cell and viral polymorphic markers [4, 491 indicates that the virus was present in the tumour cells at a very early stage. Genetic and biochemical evidence demonstrates that in LCLs, EBV proteins are directly causing the cells to grow and preliminary mechanisms for the action of these proteins are emerging (see

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and cancer

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above). In the lymphoid tumours in which latency III gene expression is present (PTLD and immunoblastic lymphoma)l it is likely that E,BV gene expression is causing the tumour cell growth, as in an LCL. There may be further oncogenie changes present in these tumour cells but the causative role of EBV in the latency III tumours seems to be clear. The picture is not so simple in the other tumours (BL, NPC, HD) which have latency I or II EBV gene expression. The expectation would be that viral genes that are expressed may complement oncogene changes in the tumour cells to result in the loss of normal cell growth control. The difficulty with this type of model is that no biochemical function that would readily fulfil this role is known at present for EBNA-I or the EBER RNAs. The tumourigenic effect of EBNA-1 in transgenic mice [77] suggests that there might be such a function. In nasopharyngeal carcinoma the LMP-1 gene is expressed in about 65% of cases; perhaps there is a heterogeneity of these tumours in terms of their cell genetic changes but noI relationship between the loss of p16 function and LMP-1 expression has been reported. Another type of explanation proposes that the role of EBV is to expand the number of cells in a target population which is prone to the true oncogenic change, after which EBV might be of less importance. This model has been proposed for BL [36], although an alternative version in which the virus infects after the initial oncogenic change and enhances the probability of the early cancer cell growing out into a tumour is equally plausible [40]. An analogous argument has been made for NPC [53], in which it was reported that all early precancerous nasopharyngeal lesions were positive for LMP-1 but in later stage tumours LMP-1 was not always expressed, perhaps as other cell mutations accumulated. In most cancer types, particularly carcinomas, the ~53 gene is frequently found to be mutated. One exception to this is cervical cancer, where p53 is normally wild-type, presumably because the papillomavirus E6 gene which is expressed in the cancer causes degradation of ~53, obviating the need for mutation to cause loss of ~53 f’unction in the tumour [73]. In this context it is interesting to note that NPC does not normally have ~53 mutations [9, 691, perhaps suggesting that an EBV function might inactivate ~53. In lymphocytes (LCLs) there was no evidence for loss of

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~53 function when they are immortalised by EBV [l] but p.53 has not been tested functionally in NPC. Further evidence for a role for EBV in BL comes from differences in the pattern of c-myc translocations in EBV positive and negative tumours [65]. In EBV negative tumours, the breakpoint of the c-myc gene is always very close to the beginning of the gene, either just upstream or in the first, non-coding exon. In contrast, breakpoints can be far away in the EBV positive turnours, frequently more than 20kb. Since the chromosome translocation presurnably occurs at random, a much wider variety of myc chromosome translocations appears to be sufficient to contribute to tumourigenicity in the presence of EBV than in the absence of EBV. An alternative interpretation of the same phenomena would be that EBV positive BL is generated in a different way from EBV negative BL, even though there are so many similarities between the diseases. One aspect that has received considerable attention recently is the possibility that specific subtypes of EBV might relate to particular diseases, perhaps contributing to some of the large geographic variations observed in incidence of EBV associated diseases [30]. The established virus types are called A and B (or I and II respectively); there is no evidence to link either of those types preferentially to disease, although the A type is a more efficient immortalising virus in vitro. Considerable attention has focused recently on strains that contain an LMP-1 gene with a 30bp deletion near the C terminus. This LMP-1 gene was reported to be more transforming in cell culture assays [41] and is frequently detected in NPC biopsies. Most studies indicate that virus with this type of LMP-1 is widespread throughout the far east [30] and is found equally in NPC tumours and normal control patients in the far east. It is, however, an interesting possibility that the endemic strain in the far east is more transforming for epithelial cells and this might be a contributing factor to the high incidence of NPC there. NOVEL STRATEGIES FOR TARGETING THERAPY TO EBV ASSOCIATED CANCERS The presence of the virus in the tumour cells of EBV associated cancers constitutes a potentially

et al

useful tumour specific marker that might be used to direct therapy to the tumour cells. For example, the ubiquitous expression of EESNA-1 in the tumour cells could be used to activate expression constructs containing oriP. Such constructs expressing the thymidine kinase gene cause cells to become sensitive to killing by ga.nciclovir. The ability of an oriP/EBNA-1 dependent construct to persist over time by replication could also be used to increase the differential expression between cells containing or lacking EBNA-1. In a model system, a 108-fold differential wa:s achieved between such cells [lo], which might provide sufficient specificity for a successful in vivo therapeutic strategy. Another sirnilar proposal using oriP based vectors has suggested using the BZLFl gene of EBV as the toxic agent to be activated in tumour cells [ 181 and an analogous approach has been advocated using EBNA-2 activation of the EBV Cp promoter to selectively activate a thymidine kinase gene in tumour cells of EBV positive lymphoblastoid tumours in AIDS and transplant patients [ 131. An alternative therapeutic strategy depends on reactivating viral gene expression in tumour cells from the latency I state so as to make the cells targets for immune surveillance by cytotoxic T lymphocytes. One approach to this will exploit the effect of 5-azacytidine on the EBV Cp promoter [62]. This promoter is silent in tumours such as BL and NPC and is methylated. 5-azacytidine can cause demethylation of Cp and activation of the expression of EBNAs which is expected to sensitise the cell to immune control. Understanding the roles and mechanisms of EBV genes in human cancers thus provides attractive new approaches to killing the cancer cells. REFERENCES Allday MJ, Sinclair A, Parker G, Crawford DH, Farrell PJ. Epstein-Barr virus efficiently immortalizes human B cells without neutralizing the function of ~53. EMBO J 1995;14:1382-91 Bain M, Watson RJ, Farrell PJ, Allday MJ. Epstein-Barr virus Nuclear Antigen 3C is a Powerful Repressor of Transcription when Tethered to DNA. J Viral 1996;70: 2481-9 Brooks L, Yao QY, Rickinson AB. Young: LS. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNAl, LMPI. and LMP-2 transcripts. J Vim! 1992;66:2689-97 Brown NA, Liu CR, Wane YF. Garcia CR. B-cell lvmphoproliferation and lymphomagenesis are associated with

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22 Homer DS, Lewis M, Farrell PJ. Novel hypotheses for the roles of EBNA-1 and BHRFl in EBV related cancers. Infervirology 1995;38:195-205 23 Hsieh JJ, Henkel T, Salmon P. Robey E, Peterson MG, Hayward SD. Truncated mammalian Notch1 activates CBFl/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mel Cell Biol 1996;16:952-9 24 Huang DP. Lo KW, Choi PH, Ng AY, Tsao SY, Yiu GK, Lee JC. Loss of heterozygosity on the short arm of chromosome 3 in nasopharyngeal carcinoma. Cancer Genet Cytogenet 1991;54:91-9 25 Huang DP, Lo KW, van-Hasselt CA, Woo JK, Choi PH, Leung SF, Cheung ST, Cairns P, Sidransky D, Lee JC. A region of homozygous deletion on chromosome 9~21-22 in primary nasopharyngeal carcinoma. Cancer Res 1994;54:4003-6 26 Huen DS, Henderson SA. Croom-Carter D, Rowe M. The Epstein-Barr virus latent membrane protein-l (LMPl) mediates activation of NF-kappa B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Onco~ene 1995: 10:549-60 27 Hui A, Lo K, Leung S, Choi P, Fong Y, Lee J, Huang D. Loss of heterozygosity on the long arm of chromosome 11 in nasopharyngeal carcinoma. Cancer Res 1996;56: 3225-9 28 Imai S, Koizumi S, Sugiura M, Tokunaga M, Uemura Y, Yamamoto N, Tanaka S, Sato E, Osato T. Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc Natl Acad Sci USA 1994;91:9131-5 29 Jacquemin M, Sinclair A, Farrell P. Gene expression in Burkitt’s Lymphoma cells. In: Becker Y and Darai Ci, ed. Frontiers in Virology. Berlin: Springer-Verlag, 1994;283-97 30 Jenkins P, Farrell P. Are particular Epstein-Barr virus strains linked to disease? Sem Cancer Biol 1996;7:209-15 31 Johannsen E, Koh E, Mosialos G, Tong X, Kieff E, Grossman SR. Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1. J Viral 1995;69:253-62 32 Jones JF, Shurin S, Abramowsky C, Tubbs RR, Sciotto CG, Wahl R, Sands J, Gottman D, Katz BZ, Sklar J. T-cell lymphomas containing Epstein-Barr viral DNA in patients with chronic Epstein-Barr virus infections. N Engl J Med 1988;318:733-41 33 Kaye K, Devergne 0, Harada J, Izumi K. Yalamanchili R, Kieff E, Mosialos G. Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kB activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein. Proc Nat1 Acad Sci USA 1996;93: 11085-90 34 Kikuta H. Taguchi Y, Tomizawa K, Kojima K, Kawamura N, Ishizaka A, Sakiyama Y, Matsumoto S, Imai S, Kinoshita T, et al. Epstein-Barr virus genome-positive T lymphocytes in a boy with chronic active EBV infection associated with Kawasaki-like disease. Nature 1988;333: 455-7 35 Kitay M. Rowe D. Cell cycle stage-specific phosphorylation of the Epstein-Barr virus immortalization protein EBNA-LP. J Viral 1996;70:7885-93 36 Klein G. In defence of the ‘old’ Burkitt lymphoma scenario. In: Klein G, ed. Advances in Viral Oncology. New York: Raven, 1987;207-211 37 Kumar A, Haque J, Lacoste J, Hiscott J, Williams BR. Double-stranded RNA-dependent protein kinase activates

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