Lawrence S. Young and Alan B. Rickinson

Transcription

1 EPSTEIN BARR VIRUS: 40 YEARS ON Lawrence S. Young and Alan B. Rickinson Abstract Epstein Barr virus (EBV) was discovered 40 years ago from examining electron micrographs of cells cultured from Burkitt s lymphoma, a childhood tumour that is common in sub-saharan Africa, where its unusual geographical distribution which matches that of holoendemic malaria indicated a viral aetiology. However, far from showing a restricted distribution, EBV a γ-herpesvirus was found to be widespread in all human populations and to persist in the vast majority of individuals as a lifelong, asymptomatic infection of the B-lymphocyte pool. Despite such ubiquity, the link between EBV and endemic Burkitt s lymphoma proved consistent and became the first of an unexpectedly wide range of associations discovered between this virus and tumours. HUMAN LEUKOCYTE ANTIGEN CLASS II MOLECULES A subset of histocompatability antigens that are mainly expressed on cells of the immune system, including B cells, and are involved in the presentation of antigens to CD4 + T cells. LYTIC REPLICATION The full cycle of virus infection, leading to the production of new virus progeny and, eventually, lysis of the infected cell. Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TT, UK. Correspondence to L.S.Y. l.s.young@bham.ac.uk doi: /nrc1452 Epstein Barr virus (EBV) 1 preferentially infects B lymphocytes through the binding of the major viral envelope glycoprotein gp350 to the CD21 receptor on the surface of B cells 2, and through the binding of a second glycoprotein, gp42, to human leukocyte antigen (HLA) CLASS II MOLECULES as a co-receptor 3.Infection of other cell types (principally epithelial cells) is much less efficient and occurs through separate, as yet poorly defined, pathways 3.Cell tropism can be modified to some extent, however, by the cell type from which viral preparations are made: virions that are produced in HLA-class-II-positive B cells are relatively depleted of gp42, and therefore target the HLA-class-II co-receptor less efficiently 3. Importantly, EBV has the unique ability to transform resting B cells into permanent, latently infected lymphoblastoid cell lines (LCLs), an in vitro system that has provided an invaluable, albeit incomplete, model of the lymphomagenic potential of the virus. By contrast, infection of epithelial cells in vitro does not activate the full growth-transforming programme of the virus, and rarely if ever achieves full LYTIC REPLICATION. B-cell transformation to LCLs therefore remains the dominant in vitro model of infection. EBV latent genes and transformation In EBV-transformed LCLs, every cell carries multiple extrachromosomal copies of the viral episome (FIG. 1a) and constitutively expresses a limited set of viral gene products, the so-called latent proteins, which comprise six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and -LP) and three latent membrane proteins (LMPs 1, 2A and 2B) 4 (FIG. 1b).Transcripts from the BamHIA region of the viral genome (so-called BART transcripts; see later) are also detected in LCLs. In addition to the latent proteins, LCLs also show abundant expression of the small, non-polyadenylated (and therefore non-coding) RNAs, EBER1 and EBER2;the function of these transcripts is not clear, but they are consistently expressed in all forms of latent EBV infection 4.This pattern of latent EBV gene expression, which appears to be activated only in B-cell infections, is referred to as latency III (FIG. 1). LCLs show high levels of expression of the B-cell activation markers CD23, CD30, CD39 and CD70, and of the cell-adhesion molecules lymphocyte-function-associated antigen 1 (LFA1; also known as CD11a/18), LFA3 (also known as CD58) and intercellular cell-adhesion molecule 1 (ICAM1; also known as CD54) 5,6. These markers are usually absent or expressed at low levels on resting B cells, but are transiently induced to high levels when these cells are activated into short-term growth by antigenic or mitogenic stimulation, indicating that EBV-induced immortalization can be elicited through the constitutive activation of the same cellular pathways that drive physiological B-cell proliferation. The ability of EBNA2, EBNA3C and LMP1 to induce LCL-like phenotypic changes when expressed individually in human B-cell lines indicates that these viral proteins NATURE REVIEWS CANCER VOLUME 4 OCTOBER

2 Summary Epstein Barr virus (EBV) infection is implicated in the aetiology of several different lymphoid and epithelial malignancies. EBV-encoded latent genes induce B-cell transformation in vitro by altering cellular gene transcription and constitutively activating key cell-signalling pathways. EBV exploits the physiology of normal B-cell differentiation to persist within the memory-b-cell pool of the immunocompetent host. Immunosuppressed transplant patients are at risk of developing fatal EBVtransformed B-cell proliferations, presenting as post-transplant lymphomas. Other EBV-associated tumours show more restricted forms of latent gene expression, reflecting a more complex pathogenesis that involves additional cofactors. Pharmacological and immunotherapeutic approaches are being developed to treat or prevent EBV-associated tumours. HODGKIN S AND REED STERNBERG CELLS The malignant cells of Hodgkin s lymphoma, named after the pathologists who first identified them as characteristic markers of this particular tumour. In EBVassociated Hodgkin s lymphoma lesions, only these malignant cells express EBV latent-cycle antigens. GERMINAL CENTRES Structures in peripheral lymphoid tissues that arise through clonal proliferation of antigen-stimulated B cells (germinal centroblasts) whose immunoglobulin genes undergo somatic hypermutation. A small fraction of cells expressing immunoglobulins of higher affinity for antigen are selected as memory B cells. are key effectors of the immortalization process 7.The role of EBV latent genes in the in vitro transformation of B cells has been confirmed more recently by the generation of recombinant forms of EBV that lack individual latent genes. Studies using these viruses have confirmed the absolute requirement for EBNA2 and LMP1 in the transformation process, and have highlighted a crucial role for EBNA1, EBNA-LP, EBNA3A and EBNA3C 4. The EBV-encoded nuclear antigens EBV-infected cells express a group of nuclear proteins that influence both viral and cellular transcription. EBNA1 is expressed in all virus-infected cells, in which its role in the maintenance and replication of the episomal EBV genome is achieved through sequence-specific binding to the plasmid origin of viral replication, OriP 4 (FIG. 1b,c). EBNA1 can also interact with certain viral promoters, thereby contributing to the transcriptional regulation of the EBNAs (including EBNA1 itself) and of LMP1. EBNA1 is separated into amino- and carboxy-terminal domains by a Gly-Ala repeat sequence, the main function of which seems to be to stabilize the mature protein preventing its proteasomal breakdown 8 rather than functioning in its originally suggested role as an immune-evasion domain 9 11.Gene-knockout studies indicate that EBNA1 does not have a crucial function in in vitro B-cell transformation beyond the maintenance of the viral genome 12 ;on the other hand, a more direct involvement in oncogenesis is indicated by the ability of B-cell-directed EBNA1 expression to produce B-cell lymphomas in transgenic mice 13, and by its possible contribution to the survival of Burkitt s lymphoma cells in vitro 14. The inability of one EBV strain P3HR-1, which carries a deletion of the gene that encodes EBNA2 and the last two exons of that for EBNA-LP to transform B cells in vitro was the first indication of the crucial role of EBNA2 in the transformation process 4.Restoration of the EBNA2 gene in P3HR-1 has unequivocally confirmed the importance of EBNA2 in B-cell transformation and has allowed the functionally relevant domains of EBNA2 to be identified 15,16. EBNA2 interacts with a sequence-specific DNA-binding protein, Jκ-recombination-binding protein (RBP-Jκ), to transcriptionally activate cellular genes such as CD23 and the key viral genes LMP1 and LMP2A 4,17,18 (FIG. 2). EBNA-LP interacts with EBNA2 and is required for the efficient outgrowth of virus-transformed B cells in vitro 19,20.The transcriptional activation that is mediated by EBNA2 in conjunction with EBNA-LP is modulated by the EBNA3 family of proteins, which repress transactivation 21,22 (FIG. 2).An essential role for EBNA3A and EBNA3C in B-cell transformation in vitro has been shown using EBV recombinants 23. EBNA3C can cooperate with RAS in rodent-fibroblast transformation assays and disrupt cell-cycle checkpoints 24,25.These effects are partly explained by the interaction of EBNA3C with factors that modulate transcription (for example, histone deacetylase 1, nonmetastatic protein 23-homologue 1 and C-terminal binding protein) or influence cell-cycle progression (for example, cyclin A) 26. The EBV-encoded latent membrane proteins LMP1 is the main transforming protein of EBV; it functions as a classic oncogene in rodent-fibroblast transformation assays and is essential for EBV-induced B-cell transformation in vitro 27,28. LMP1 has pleiotropic effects when it is expressed in cells, resulting in the induction of cell-surface adhesion molecules and activation antigens 7, and upregulation of anti-apoptotic proteins (for example, BCL2 and A20) 31,32. LMP1 functions as a constitutively activated member of the tumour necrosis factor receptor (TNFR) superfamily, and activates several signalling pathways in a ligand-independent manner (FIG. 3).Functionally, LMP1 resembles CD40 another member of the TNFR superfamily and can partially substitute for CD40 in vivo,providing both growth and differentiation signals to B cells 36.The LMP1 protein activates several downstream signalling pathways that contribute to the many phenotypic consequences of LMP1 expression, including the induction of various genes that encode anti-apoptotic proteins and cytokines 37 (see the legend for FIG. 3 for details). The LMP2 proteins, LMP2A and LMP2B, are not essential for EBV-induced B-cell transformation in vitro 38 (FIG. 4). However, expression of LMP2A in B cells in transgenic mice abrogates normal B-cell development, allowing immunoglobulin (Ig)-negative cells to colonize peripheral lymphoid organs 39.This indicates that LMP2A can drive the proliferation and survival of B cells in the absence of signalling through the B-cell receptor (BCR). LMP2A can transform epithelial cells and enhance their adhesion and motility, effects that might be mediated by the activation of the phosphatidylinositol-3-kinase AKT pathway 40.Repressive effects of LMP2A expression have recently been reported in human and murine B cells, and many of these target B-cell-specific factors, resulting in a phenotype that is similar to those of malignant HODGKIN S AND REED STERNBERG (HRS) CELLS in Hodgkin s lymphoma and GERMINAL-CENTRE B cells 41,42.In addition to these effects, LMP2A was found to induce expression of a range of genes that are involved in cell-cycle induction, inhibition of apoptosis and suppression of cell-mediated immunity. 758 OCTOBER 2004 VOLUME 4

3 a EBV electron micrograph BamHIA region EBNA1 BARF0 LMP2A BARF1 EBNA3C EBNA3B EBNA3A LMP1 LMP2B TR EBER1 OriP EBER2 Cp or Wp Double-stranded DNA episome EBNA-LP EBNA2 OriP N a S e1 e3 Z R c bt d het N het CW W W W W W Y H F Q U P O M L E K B G D X V I A TR W W W W W W b EBV genome: latent genes c Open reading frames for the EBV latent proteins EBNA-LP EBNA2 EBNA3A EBNA3B EBNA3C EBNA1 LMP1 Figure 1 The Epstein Barr virus genome. a Electron micrograph of the Epstein Barr virus (EBV) virion. b Diagram showing the location and transcription of the EBV latent genes on the double-stranded viral DNA episome. The origin of plasmid replication (OriP) is shown in orange. The large green solid arrows represent exons encoding each of the latent proteins, and the arrows indicate the direction in which the genes encoding these proteins are transcribed. The latent proteins include the six nuclear antigens (EBNAs 1, 2, 3A, 3B and 3C, and EBNA-LP) and the three latent membrane proteins (LMPs 1, 2A and 2B). EBNA-LP is transcribed from a variable number of repetitive exons. LMP2A and LMP2B are composed of multiple exons, which are located on either side of the terminal repeat (TR) region, which is formed during the circularization of the linear DNA to produce the viral episome. The blue arrows at the top represent the highly transcribed nonpolyadenylated RNAs EBER1 and EBER2; their transcription is a consistent feature of latent EBV infection. The long outer green arrow represents EBV transcription during a form of latency known as latency III (Lat III), in which all the EBNAs are transcribed from either the Cp or Wp promoter; the different EBNAs are encoded by individual mrnas that are generated by differential splicing of the same long primary transcript. The inner, shorter red arrow represents the EBNA1 transcript, which originates from the Qp promoter during Lat I and Lat II. Transcripts from the BamHIA region can be detected during latent infection, but no protein arising from this region has been definitively identified. The locations of the BARF0 and BARF1 coding regions are shown here. c Location of open reading frames for the EBV latent proteins on the BamHI restriction-endonuclease map of the prototype B95.8 genome. The BamHI fragments are named according to size, with A being the largest. Lowercase letters indicate the smallest fragments. Note that the LMP2 proteins are produced from mrnas that splice across the terminal repeats (TRs) in the circularized EBV genome. This region is referred to as N het, to denote the heterogeneity in this region due to the variable number of TRs in different virus isolates and in different clones of EBV-infected cells. b and c modified with permission from REF. 138 (2003) Nature Publishing Group. INFECTIOUS MONONUCLEOSIS A transient illness, associated with hyperactivation of the CD8 + T-cell response, that occurs in some individuals whose primary EBV infection is delayed until the second or third decade of life. Primary infection during childhood is almost always asymptomatic. Other EBV latent transcripts In addition to the latent proteins, the two small nonpolyadenylated (non-coding) RNAs EBER1 and EBER2 are expressed in all forms of latency. However, the EBERs are not essential for the EBVinduced transformation of primary B lymphocytes 4. The EBERs assemble into stable ribonucleoprotein particles with the autoantigen La and ribosomal Qp TR protein L22, and bind the interferon-inducible, doublestranded-rna-activated protein kinase PKR 43. PKR has a role in mediating the antiviral effects of the interferons, and it has been suggested that EBER-mediated inhibition of PKR function might be important for viral persistence 44.Expression of the EBERs in Burkitt s lymphoma cell lines has been found to increase tumorigenicity, promote cell survival and induce interleukin-10 (IL-10) expression 43,45.Such studies indicate that EBV genes that were previously shown to be dispensable for transformation in B-cell systems might make more important contributions to the pathogenesis of some EBV-associated malignancies, and to EBV persistence, than was previously appreciated. A group of abundantly expressed RNAs that are encoded by the BamHIA region of the EBV genome were originally identified in nasopharyngeal carcinoma (NPC), but were subsequently found to be expressed in other EBV-associated malignancies, such as Burkitt s lymphoma, Hodgkin s lymphoma and nasal T-cell lymphoma, as well as in the peripheral blood of healthy individuals These highly spliced transcripts are commonly referred to as either BamHIA rightward transcripts (BARTs) or complementary-strand transcripts (CSTs) 49,50.The protein products of these open reading frames remain to be conclusively identified. Another transcript that is generated from the BamHIA region is BARF1, which encodes a 31-kDa protein that was originally identified as an early antigen expressed on induction of the EBV lytic cycle. Recent studies have shown that BARF1 is a secreted protein that is expressed as a latent protein in EBV-associated NPC and gastric carcinoma 51,52.BARF1 shares limited homology with the human colony-stimulating factor 1 receptor (the FMS oncogene) and displays oncogenic activity when it is expressed in rodent fibroblasts and simian primary epithelial cells 53. EBV infections in immunocompetent hosts In contrast to in vitro studies of EBV infection and latent-gene function, our understanding of the biology of EBV infection in vivo (FIG. 5) is still rudimentary. Primary infection (by oral transmission) is usually asymptomatic, but if it is delayed until adolescence it occasionally presents as INFECTIOUS MONONUCLEOSIS (IM). Patients with acute IM shed high titres of infectious virus in the throat from lytic infection at oropharyngeal sites. It is possible that this occurs in local mucosal B cells but, from evidence of virus replicative lesions that are seen in the oral mucosa of immunocompromised patients, it is likely that this also involves the oropharyngeal epithelium. At the same time, large numbers of latently infected B cells at least some of which represent transformed EBNA2 +, LMP1 + lymphoblasts 54,55 appear in tonsillar (and possibly other) lymphoid tissues. In vitro,both naive and MEM- ORY B CELLS seem equally susceptible to EBV infection. However, although some of the infected tonsillar cells in IM tonsils have a naive Ig genotype, the expanding clones preferentially involve cells with mutated Ig NATURE REVIEWS CANCER VOLUME 4 OCTOBER

4 MEMORY B CELLS B cells that have experienced antigen stimulation and, usually, somatic hypermutation and germinal-centre transit, before subsequent selection into a pool of long-lived recirculating cells. These cells rapidly respond to a later re-challenge with their specific antigen, mounting an efficient secondary antibody response. LYMPHOCYTOSIS A marked expansion of lymphocyte numbers in the blood, caused by proliferation of EBV-specific CD8 + T cells in the blood of patients with infectious mononucleosis. T-CELL IMMUNOCOMPROMISED A state of immune T-cell impairment seen, for instance, in transplant patients receiving high doses of T-cell-suppressive drugs to prevent rejection of the transplant and in late-stage AIDS patients; in both situations, immune control over persistent viral infections, such as EBV, is impaired. a Repression b Activation EBNA3A EBNA3B EBNA3C SIN3A SMRT HDAC1/2 SAP30 SKIP CIR RBP-Jκ GTGGGAA EBNA2 SKIP EBNA2 RBP-Jκ GTGGGAA BTM SIN3A SMRT HDAC1/2 SAP30 CIR EBNA-LP Figure 2 The EBV-encoded nuclear antigens. a Epstein Barr virus (EBV)-encoded nuclear antigen 2 (EBNA2) functions as a transcriptional activator by interacting with the DNA-binding Jκ-recombination-binding protein (RBP-Jκ) and relieving the transcriptional repression that is mediated by a large multiprotein complex consisting of SMAT, SIN3A, histone deacetylase 1 (HDAC1) and HDAC2 (REFS 4, 17, 18). SKIP (Ski interacting protein) is another RBP-Jκinteracting protein that also interacts with the SMRT HDAC corepressor complex. EBNA2 abolishes RBP-Jκ mediated repression by competing for the SMRT HDAC corepressor complex through binding to both RBP-Jκ and SKIP 130. b The acidic domain of EBNA2 then recruits the basal transcription machinery (TFIIB, TFIIH and p300; not shown) to activate transcription. EBNA-LP cooperates with EBNA2 in RBP-Jκ-mediated transcriptional activation by interacting with the acidic activation domain of EBNA2 (REF. 4). The EBNA3 family of proteins modulate EBNA2-mediated RBP-Jκ activation by interacting with RBP-Jκ and competing for binding and activation by EBNA2. The RBP-Jκ homologue in Drosophila is involved in signal transduction from the Notch receptor, a pathway that is important in cell-fate determination in Drosophila and has also been implicated in the development of T-cell tumours in humans 131. EBNA2 can functionally replace the intracellular region of Notch 132. BTM, basal transcription machinery; CIR, CBFI (RBP-Jκ)-interacting corepressor; SAP30, SIN3-associated protein 30. sequences that are typical of antigen-selected memory cells. This is consistent with the important finding that, in the blood of patients with acute IM and subsequently of long-term virus carriers, EBV-infected cells are concentrated in the IgD CD27 + memory-b-cell subset 56,57.Furthermore these cells have by that time returned to a resting state and have downregulated the expression of most, and possibly all, viral proteins 58. The precise route of entry into memory is still a subject of much debate (FIG. 5). This reservoir of infected cells is then stably maintained, and seems to be subject to the same physiological controls as the general mucosa-associated memory-b-cell pool 59.Such a strategy brings with it the possibility of fortuitous, antigen-driven recruitment of Off On infected cells into germinal centres, leading to progeny that either re-enter the circulating memory pool or differentiate to become plasma cells that might migrate to mucosal sites. The different forms of latency that are seen in virus-associated malignancies might represent latency programmes that have evolved to accommodate such changes in host-cell physiology. Germinal-centre transit therefore seems to activate a latency programme in which only the genome-maintenance protein EBNA1 is expressed, whereas exit from germinal centres is possibly linked to the transient activation of LMP1 and LMP2 expression 58.Similarly, a commitment to plasmacytoid differentiation is thought to trigger these cells to undergo lytic viral replication, providing a source of low-level virus shedding into the oropharynx. There might also be circumstances in which infected cells in the reservoir can become reactivated to produce further proliferative latency III infections. Primary EBV infection elicits strong cellular immune responses that then bring the infection under control. The LYMPHOCYTOSIS that typifies acute IM therefore reflects the hyperexpansion of cytotoxic CD8 + T cells that are reactive to both lytic- and latent-cycle viral antigens, reactivities that are subsequently maintained in the CD8 + T-cell memory at levels that, collectively, might constitute up to 5% of the total circulating CD8 + T-cell pool 60.This level of commitment to a single virus, which is apparent even in EBV carriers who have no prior history of IM, implies a crucial role for immune T-cell surveillance in controlling persistent EBV infection. Virus-associated B-cell lymphomas There are three histologically and clinically distinct types of EBV-associated B-cell lymphoma that show different patterns of latent gene expression and seem, from Ig-gene sequencing, to derive from cells at different positions in the B-cell differentiation pathway. Here, we describe the main features of these tumours and discuss the role of EBV in their pathogenesis. Lymphomas in immunosuppressed individuals. T-CELL- IMMUNOCOMPROMISED patients are at high risk of developing B-cell lymphomas, and those that arise in transplant patients are the best studied of these lymphomas. Most post-transplant lymphomas (PTLs) arise as polyclonal or monoclonal lesions within the first year of allografting, when immunosuppression is most severe. Almost all of these early-onset tumours are EBV-positive and (on the basis of positive EBNA2 and LMP1 staining) express the latency III programme, which identifies them as virus-transformed B cells that grow out in the absence of effective T-cell surveillance 61.Some of the lymphomas that are seen in highly immunocompromised AIDS patients, particularly central nervous system (CNS) lesions, show essentially the same phenotype. Transplant cohorts continue to show a significant, albeit lower, risk of developing lymphomas well beyond the first year, but these late-onset tumours are a more heterogeneous group and as with the non-cns lymphomas of 760 OCTOBER 2004 VOLUME 4

5 EBV LMP1 NH 2 JNK, ERK? CTAR1 CTAR2 MAPKKK TRAFs Lipid raft TRADD TRAF2 PI3K MAPKKK IKKα NIK p100 p65 p52 IKKγ AKT RHO GTPase p38, ERK? IKKα IKKβ IκB p65 p50 p65 p50 NF-κB 2 NF-κB 1 Figure 3 Structure and function of LMP1. The Epstein Barr virus latent membrane protein 1 (LMP1) is an integral membrane protein of 63 kda and can be subdivided into three domains: first, an amino-terminal cytoplasmic tail (amino acids 1 23), which tethers LMP1 to the plasma membrane and orientates the protein; second, six hydrophobic transmembrane loops, which are involved in self aggregation and oligomerization (amino acids ); third, a long carboxyterminal cytoplasmic region (amino acids ), which possesses most of the signalling activity of the molecule. Two distinct functional domains referred to as C-terminal activation regions 1 and 2 (CTAR1 and CTAR2) have been identified on the basis of their ability to activate the nuclear factor-κb (NF-κB) transcription-factor pathway 133. The signalling effects of LMP1 result from the ability of tumour necrosis factor receptor (TNFR)-associated factors (TRAFs) to interact either directly with CTAR1 or indirectly by interacting with the death-domain-containing protein TRADD, which binds to CTAR2 (REF. 37). These adaptor proteins subsequently recruit a multiprotein catalytic complex containing the NF-κB-inducing kinase (NIK) and the IκB kinases (IKKs). This results in the activation of both the classic IκBα-dependent NF-κB pathway (involving p50 p65 heterodimers) and the processing of p100 NF-κB2 to generate p52 p65 heterodimers 134. Other kinases are recruited to LMP1 through interactions with TRAF molecules including the mitogen-activated protein kinase kinase kinases (MAPKKKs) TPL2 and TAK1, and these contribute to the activation of the NF-κB, MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. ERK, extracellular signal-regulated kinase; JNK, c-jun amino-terminal kinase. late-stage AIDS patients the proportion of EBVassociated cases can fall below 50%. EBV-positive lateonset PTLs are typically monoclonal tumours, some of which mimic the latency III phenotype of classic early-onset disease, whereas others are EBNA2- and LMP1-negative or, occasionally, EBNA2-negative but LMP1-positive for a proportion of cells 62,63.Such tumours might therefore have evolved from EBVtransformed LCL-like lesions through the acquisition of additional cellular genetic changes that render certain viral functions redundant. However, with the exception of BCL6 mutations 63 (which might be a coincidental consequence of germinal-centre transit), no specific change occurs consistently or can be linked to tumours of a particular phenotype. As described in BOX 1, Ig-gene sequencing has shown that PTLs can arise from a range of positions on the B-cell differentiation pathway, including some that indicate pathogenetic similarities with other EBVassociated B-lymphomas. Hodgkin s lymphoma. In this unusual tumour, the clone of malignant HRS cells is vastly outnumbered by a nonmalignant infiltrate, the appearance of which distinguishes the nodular sclerosing (NS), mixed cellularity (MC) and rarer lymphocyte-depleted (LD) subtypes. Approximately 40% of cases of classic Hodgkin s lymphoma in the developed world are associated with EBV; this figure includes most MC and LD cases but, ironically, only a minority of the NS tumours that make up the marked peak of Hodgkin s lymphoma incidence that is seen in the third decade of life in the developed world, and that first raised the issue of an infectious aetiology. Hodgkin s lymphoma in the developing world seems to show an even higher overall association with EBV, reflecting at least in part the absence of this peak in young adults 64. The existence of EBV-negative disease raises the question of whether EBV, when present, is simply a passenger. Although this remains formally possible, the likelihood of Hodgkin s lymphoma having arisen in an EBV-positive target cell by chance seems remote, given that infected cells normally make up a tiny fraction (1 100 cells per million) of the total B- cell pool 59.Furthermore, in EBV-positive tumours, the viral genome is present in every HRS cell and expresses a particular subset of latent-cycle proteins EBNA1, LMP1 and LMP2, the so-called latency II programme 47.The identification of HRS cells as failed products of germinal-centre reactions by Ig genotyping 65,66 (BOX 1) does indeed indicate a plausible pathogenetic role for the virus, based on rescuing such tumour progenitors from apoptosis. LMP1 is therefore capable of constitutively activating the CD40 pathway, thereby replacing a signal that is normally provided by cognate T cells during memorycell selection, and LMP2A can mimic signalling from surface Ig, replacing the usual requirement for highaffinity binding to cognate antigen. Whether EBV still contributes to the malignant phenotype at the time of tumour presentation is more difficult to determine, particularly as there are no EBV-positive HRS cell lines available as in vitro models that retain the classic latency II form of infection. Because the downstream components of surface Ig signalling have been eliminated in HRS cells 67, LMP2A might no longer be operational, at least in the context of that pathway. By contrast, HRS cells continue to show many characteristics of LMP1-induced phenotypic changes, including the strong activation of nuclear factor-κb (NF-κB) and its associated downstream effects. Interestingly, the HRS cells of EBV-negative Hodgkin s lymphoma show a very similar phenotype, which, in at least some cases, seems to have been caused by inactivation of inhibitor of κbα (IκBα), the physiological regulator of NF-κB activity, or by amplification of the REL gene, which encodes an NF-κB family member 66.This parallel indicates that NF-κB deregulation is an important feature of the pathogenesis of Hodgkin s lymphoma, and that EBVpositive and EBV-negative tumours have achieved the same end point by different routes. NATURE REVIEWS CANCER VOLUME 4 OCTOBER

6 EBV LMP2A Tyr112 Tyr85 Tyr74 PY PY LYN SRC/SYC NEDD4 Lipid raft NH 2 Figure 4 Structure and function of LMP2. The structures of the Epstein Barr virus (EBV) latent membrane proteins LMP2A and LMP2B are similar; both have 12 transmembrane domains and a 27-amino-acid cytoplasmic carboxyl terminus. In addition, LMP2A has a 119-amino-acid cytoplasmic amino-terminal domain that contains eight tyrosine residues, two of which (Tyr74 and Tyr85) form an immunoreceptor tyrosine-based activation motif (ITAM) 38. The phosphorylated ITAM recruits members of the SRC family of protein tyrosine kinases and the SYK tyrosine kinase and negatively regulates their activities. A membrane-proximal tyrosine residue (Tyr112) binds the LYN tyrosine kinase and mediates the constitutive phosphorylation of the other tyrosine residues in LMP2A 38. The LMP2A ITAM blocks signalling from the B-cell receptor (BCR) by sequestering these tyrosine kinases and by blocking the translocation of the BCR into lipid rafts 135. LMP2A also recruits NEDD4-like ubiquitin protein ligases through phosphotyrosine (PY) motifs, and these promote the degradation of LYN and LMP2A by a ubiquitin-dependent mechanism 136. LMP2A interacts with the extracellular signal-regulated kinase 1 (ERK1) mitogen-activated protein kinase (MAPK), and this results in the phosphorylation of two serine residues (Ser15 and Ser102) in LMP2A, and might contribute to LMP2A-induced activation of JUN 137. MAPKKK, MAPK kinase kinase; PI3K, phosphatidylinositol 3-kinase. SOMATIC HYPERMUTATION Point mutations that occur in the immunoglobulin-gene variable regions (and some other genes) during B-cell differentiation. LYMPHADENOPATHY A marked swelling of peripheral lymphoid tissues in situations arising from chronic antigenic stimulation by an infectious agent. PI3K MAPKKK AKT RHO GTPase ERK Ubiquitylation of LMP2A and LMP2A-associated proteins Cell survival and motility JUN? Burkitt s lymphoma. EBV is present in all cases of endemic Burkitt s lymphoma the high-incidence form of the tumour that affects children in areas of Africa and New Guinea in which malaria is holoendemic and in up to 85% of cases in areas of intermediate incidence such as Brazil and North Africa, but in only 15% of the low-incidence sporadic tumours that are seen in children in the developed world. Remarkably, Burkitt s lymphoma is also common among adult human immunodeficiency virus (HIV) carriers in the developed world and often arises as the first AIDS-defining illness in relatively immunocompetent patients; some 30 40% of these tumours are EBV-associated 5.All Burkitt s lymphomas carry one of three characteristic chromosomal translocations that place the MYC oncogene under the control of the Ig heavy chain or one of the light-chain loci. The primacy of MYC deregulation as the key factor in the pathogenesis of Burkitt s lymphoma is clear from a range of experimental systems In addition, many tumours have TP53 mutations or other defects in the p53 ARF pathway, as well as mutations in the putative tumour-suppressor gene retinoblastoma-like (REF. 71). Irrespective of their EBV status, the phenotype of Burkitt s lymphoma cells (CD10 +, CD77 +, BCL6 + ) is remarkably similar to that of germinal centroblasts, and the detection of ongoing Ig-gene mutation in tumour cells (BOX 1) supports the suggestion that they originate in germinal centres. Indeed, the MYC translocation itself is likely to have occurred as an error of the SOMATIC HYPERMUTATION process. At presentation, the vast majority of EBV-positive tumours show a highly restricted latency I form of infection, with viral antigen expression limited to that of EBNA1 (REFS 6,75).So,how EBV contributes to the pathogenesis of Burkitt s lymphoma remains a matter of speculation. The virus might have an initiating role in which growth-transforming B-cell infections establish a pool of target cells that are at risk of a subsequent MYC translocation, a process that has been successfully modelled in vitro 68.This latter study highlighted the apparent incompatibility of the EBV latency-iii-driven and MYC-driven growth programmes in B cells 76, indicating that the evolution to Burkitt s lymphoma can only occur if the EBV programme is suppressed. Indeed, the strength of selection against full expression of viral latent genes is illustrated by a subset of Burkitt s lymphomas in which the transcriptional features of latency III are retained, but the resident viral genome has deleted the EBNA2 gene and therefore abrogated the conventional B-cell transforming function 75. Alternatively, EBV might contribute to the Burkitt s lymphoma phenotype through the latency- I-active genes themselves. EBNA1 is an obvious candidate, but its reported oncogenicity in mouse transgene assays 13 remains controversial, and its contribution to virus-induced B-cell transformation in vitro seems to be limited to maintenance of the viral genome 12,77 ;however, recent experiments in which EBNA1 function was blocked in EBV-positive Burkitt s lymphoma cell lines have indicated that the protein does promote cell survival 14.Other studies, based on infection or transfection of spontaneous EBV genome-loss derivatives of the Akata Burkitt s lymphoma cell line, have also shown increased survival mediated either by virus-induced upregulation of the TCL1 oncogene 78 or through induction of the expression of the IL-10 cytokine by the non-coding EBER RNAs 43.The wider relevance of these effects beyond the Akata Burkitt s lymphoma model system remains to be determined. Any comprehensive view of Burkitt s lymphoma pathogenesis must take into account the marked increases in tumour incidence that are associated with holoendemic malaria and HIV infections. In this regard, both agents can act as chronic stimuli of the B-cell system and, at least for HIV carriage, the persistent generalized LYMPHADENOPATHY that results from such stimulation is characterized by exaggerated germinal-centre activity 79.We suggest that this greatly increases the chances of productive MYC translocations occurring. Furthermore, both agents disturb the EBV host balance and probably increase the numbers of EBV-infected B cells that are at risk of being recruited into germinal-centre reactions 80.There are differences between the two situations, however, and it will be interesting to see whether malaria has its own specific effects on the EBV host balance as, unlike HIV, the 100-fold increase in the incidence of Burkitt s lymphoma seen in patients with malaria consists entirely of EBV-positive disease OCTOBER 2004 VOLUME 4

7 a Primary infection Lytic replication Epithelium b Persistent infection Latency 0 Memory- B-cell reservoir Naive B cell Memory B cell Latency I/II Germinal centre Latency III Latency I/II Latency 0 Primary- T-cell response Memory- T-cell response Latency III Naive B cell Memory B cell Germinal centre Plasma cell Memory B-cell reservoir Lytic replication Epithelium Figure 5 Putative in vivo interactions between Epstein Barr virus and host cells. a Primary infection. Incoming virus establishes a primary focus of lytic replication in the oropharynx (possibly in the mucosal epithelium), after which the virus spreads throughout the lymphoid tissues as a latent (latency III) growth-transforming infection of B cells. Many of these proliferating cells are removed by the emerging latent-antigen-specific primary-t-cell response, but some escape by downregulating antigen expression and establishing a stable reservoir of resting viral-genomepositive memory B cells, in which viral antigen expression is mostly suppressed (latency 0). Different views of these events are shown. One view is that naive B cells are the main targets of new EBV infections in vivo. In this scenario, viral transformation drives naive cells into memory by mimicking the physiological process of antigen-driven memory-cell development in lymphoid tissues, a process involving somatic immunoglobulin-gene hypermutation during transit through a germinal centre. However, this is difficult to reconcile with the finding that EBV-infected B cells in tonsils from patients with infectious mononucleosis (IM) localize to extrafollicular areas not to germinal centres and show no evidence of ongoing hypermutation within expanding clones. An alternative view therefore envisages infection of pre-existing memory cells as a direct route into memory; this is consistent with the above observations on IM tonsils, but still leaves unexplained the apparent disappearance of the infected naive cell population. b Persistent infection. The reservoir of EBVinfected memory B cells becomes subject to the physiological controls governing memory-b-cell migration and differentiation as a whole. Occasionally, these EBV-infected cells might be recruited into germinal-centre reactions, entailing the activation of different latency programmes, after which they might either re-enter the reservoir as memory cells or commit to plasma-cell differentiation possibly moving to mucosal sites in the oropharynx and, in the process, activating the viral lytic cycle. Virions produced at these sites might initiate foci of lytic replication in permissive epithelial cells, allowing low-level shedding of infectious virus in the oropharynx, and might also initiate new growthtransforming latency III infections of naive and/or memory B cells; these new infections might possibly replenish the B-cell reservoir, but are more likely to be efficiently removed by the now wellestablished memory-t-cell response. Virus-associated T-cell and NK-cell lymphomas EBV is so markedly B-lymphotropic when it is exposed to human lymphocyte preparations in vitro that the association of the virus with rare but specific types of T-cell and natural killer (NK)-cell lymphomas 81,82 was completely unexpected. How the virus accesses these cell lineages in vivo is still uncertain, but most evidence indicates that such infections are rare and, when they do occur, confer a high risk of lymphoma development. One of the best examples is an EBV-genome-positive T-cell lymphoma, which is seen worldwide but is most common in southeast Asian populations. This type of lymphoma arises either after acute primary infection, manifesting as virus-associated haemophagocytic syndrome (VAHS), or in the setting of a chronic active EBV infection with VAHS-like symptoms 81,83.These are monoclonal tumours of CD4 + or CD8 + T-cell origin, in which viral gene expression is restricted to the production of EBNA1 and LMP2, with variable levels of LMP1 being detectable in only a fraction of cells (designated latency I/II). The tumours seem to arise rapidly from a pre-malignant pool of EBV-infected T cells that, uniquely, are present in the blood of patients who have VAHS-like disease 84 and might, through cytokine release, drive macrophage activation and haemophagocytosis. A second example, which is again most common in south-east Asia, is an extranodal lymphoma that usually presents as an erosive lesion ( lethal midline granuloma ) in the nasal cavity 82.Some of these are CD3 + CD56 T-cell lymphomas, but most are CD3 CD56 + tumours of NK-cell origin, again with latency I/II gene expression 85. Carcinomas associated with the virus Nasopharyngeal carcinoma. An important consequence of epithelial infection with EBV is malignant transformation, resulting in the development of NPC, a subset of gastric adenocarcinomas and certain salivarygland carcinomas 5,86.The EBV-associated, undifferentiated form of NPC World Health Organization (WHO) type III shows the most consistent worldwide association with EBV and is particularly common in areas of China and south-east Asia, reaching a peak incidence of around cases per 100,000 (REF. 87). Incidence rates are high in individuals of Chinese descent, irrespective of where they live, and particularly in Cantonese males. In addition to this genetic predisposition, environmental cofactors such as dietary components (for example, salted fish) are thought to be important in the aetiology of NPC 88. NPC tumours are characterized by the presence of undifferentiated carcinoma cells and a prominent lymphocytic infiltrate, and this interaction between tumour cells and lymphocytes seems to be crucial for the continued propagation of the malignant component. EBV latent-gene expression in NPC is predominantly restricted to the EBNA1 nuclear antigen, the latent membrane proteins (LMP2A and LMP2B) and the BamHIA transcripts, with ~20% of tumours also expressing the oncogenic LMP1 protein 86.Southern-blot hybridization of DNA from NPC tissues demonstrates the monoclonality of NATURE REVIEWS CANCER VOLUME 4 OCTOBER

8 Box 1 Cellular origins of EBV-positive B-cell lymphomas Post-transplantation lymphomas From immunoglobulin variable (IgV) gene sequencing, post-transplantation lymphomas (PTLs) seem able to arise from naive cells, memory cells or more commonly in late onset cases cells that have atypical or non-functional mutations that are normally inconsistent with cell survival; a minority of these latter cases even show ongoing hypermutation. Epstein Barr virus (EBV) might create a pool of nonmalignant, but atypical, EBV-carrying B cells in vivo,either by directly rescuing cells from within germinal centres or possibly by activating somatic hypermutation in cells outside the germinal-centre environment. The latter scenario is supported by evidence from recent studies of EBV-infected non-malignant B-cell clones, both in a posttransplantation Hodgkin s lymphoma biopsy 125 and in infiltrating an EBV-negative T-cell lymphoma of the angioimmunoblastic lymphadenopathy type 126. Hodgkin s lymphoma All Hodgkin s and Reed-Sternberg cells within a tumour are part of the same clone and almost always carry a mutated IgV sequence that lacks intraclonal diversity. Sequences that contain nonsense mutations or deletions are seen in 25% of cases. As this almost certainly underestimates the true incidence of gene inactivation, most cases of Hodgkin s lymphoma probably derive from crippled germinal-centre cells that have been rescued from the germinal-centre reaction. There are clear analogies here with EBV-positive PTLs that carry inactivated Ig genes or other apparently non-antigenselected mutations. Burkitt s lymphoma IgV sequencing of Burkitt s lymphoma biopsy cells and derived cell lines (from both EBV-positive and EBV-negative cases) has identified multiple mutations and, in many cases, ongoing sequence diversification within the malignant clone. This confirmed Burkitt s lymphoma as a tumour of germinal-centroblast origin. Burkitt s lymphoma cell lines that had switched to a latency III infection and an LCL-like surface phenotype continued to show Ig-gene diversification. EBV therefore does not suppress ongoing hypermutation, even when the activation of the viral growth-transforming programme eliminates other markers of germinal centroblast origin. the resident viral genomes, indicating that EBV infection takes place before the clonal expansion of the population of malignant cells 89.Studies of normal nasopharyngeal tissue and pre-malignant biopsies indicate that genetic events occur early in the pathogenesis of NPC, and that these might predispose to subsequent EBV infection (BOX 2).Extensive serological screening has identified increased EBV-specific antibody titres in high-incidence areas; in particular, IgA antibodies to the EBV capsid antigen and early antigens have proved useful in diagnosis and in monitoring the effectiveness of therapy 90.More recent studies using real-time quantitative PCR to measure circulating tumour-derived EBV DNA in the blood of patients with NPC have shown that the level of pre-treatment EBV DNA is strongly associated with overall survival, and that posttreatment EBV DNA levels predict progression-free and overall survival 91.Association of EBV with the other more differentiated forms of NPC (WHO types I and II) has been shown, particularly in those geographical regions with a high incidence of undifferentiated NPC 92.Carcinomas that have similar features to undifferentiated NPC have been described at other sites, including the thymus, tonsils, lungs, stomach, skin and uterine cervix, and are often referred to as undifferentiated carcinomas of nasopharyngeal type (UCNT) or lymphoepitheliomas. There is geographical variation in the extent of the association of EBV with UCNTs. Gastric carcinoma. EBV is also found in ~10% of more typical gastric adenocarcinomas, accounting for up to 75,000 new cases per year 93,94.These tumours display a restricted pattern of EBV latent-gene expression (resulting expression of EBERs, EBNA1, LMP2A, BARTs and BARF1), similar to that seen in NPC 95.There is significant geographical variation in the association of EBV with gastric carcinoma, which might be due to ethnic and genetic differences. EBV-positive gastric carcinomas have distinct phenotypic and clinical characteristics compared with EBV-negative tumours, including loss of expression of INK4A (also known as p16) and improved patient survival 96,97.As in NPC, the precise role of EBV in the pathogenesis of gastric carcinoma remains to be determined, but the absence of EBV infection in pre-malignant gastric lesions supports the suggestion that viral infection is a relatively late event in gastric carcinogenesis 98. Is EBV associated with other common epithelial malignancies? Detection of the EBERs by in situ hybridization has become the standard method of detecting EBV infection in the routine processing of tumour tissues. Although the EBERs were previously considered to be expressed in all forms of EBV latency, two studies have raised the possibility that EBER-negative forms of latency might exist in previously unrecognized EBV-associated malignancies, such as carcinomas of the breast and liver 99,100.Difficulties in confirming these associations have raised concerns about the use of PCR analysis to detect EBV infection, and have also questioned the specificity of monoclonal-antibody reagents 101.It is our contention that the definitive designation of a tumour as EBV-associated should require unequivocal demonstration of the presence of the EBV genome or viral gene products within most of the tumour-cell population. Novel therapeutic approaches Given the significant burden of EBV-associated tumours worldwide, an important priority is to design novel therapies that specifically target viral proteins or otherwise exploit the presence of the virus in malignant cells. Pharmacological approaches. One potential approach is the use of gene-therapy constructs to express either cytotoxic or inhibitory proteins selectively in tumour cells. For example, OriP-based constructs that are responsive to endogenous EBNA1 in infected cells have been used to express cytotoxic proteins (for example, FAS ligand) or wild-type p53 in in vitro and in vivo models of NPC 102,103. Other approaches are based on the induction of the EBV lytic cycle, either by pharmacological agents or by delivery of EBV immediate-early genes, thereby inducing virusencoded kinases (EBV thymidine kinase and BGLF4, a protein kinase) that phosphorylate the nucleoside analogue gancyclovir to produce its active cytotoxic form 104,105.Demethylating agents such as 5-azacytidine are able to de-repress lytic, as well as potentially immunogenic, latent genes 106 and are now in early-stage clinical trials in patients with NPC, Hodgkin s lymphoma and AIDS-associated lymphoma 107. Another common chemotherapeutic agent, hydroxyurea, is able to induce 764 OCTOBER 2004 VOLUME 4