Epstein–Barr virus: 40 years on

REVIEWS

Epstein–Barr virus (EBV)1 preferentially infects B lym-phocytes through the binding of the major viral enve-lope glycoprotein gp350 to the CD21 receptor on thesurface of B cells2, and through the binding of a secondglycoprotein, gp42, to human leukocyte antigen (HLA)

CLASS II MOLECULES as a co-receptor3. Infection of other celltypes (principally epithelial cells) is much less efficientand occurs through separate, as yet poorly defined, path-ways3. Cell tropism can be modified to some extent,however, by the cell type from which viral preparationsare made: virions that are produced in HLA-class-II-pos-itive B cells are relatively depleted of gp42, and thereforetarget the HLA-class-II co-receptor less efficiently3.Importantly, EBV has the unique ability to transformresting B cells into permanent, latently infected lym-phoblastoid cell lines (LCLs), an in vitro system that hasprovided an invaluable, albeit incomplete, model of thelymphomagenic potential of the virus. By contrast, infec-tion of epithelial cells in vitro does not activate the fullgrowth-transforming programme of the virus, andrarely — if ever — achieves full LYTIC REPLICATION. B-celltransformation to LCLs therefore remains the dominantin vitro model of infection.

EBV latent genes and transformationIn EBV-transformed LCLs, every cell carries multipleextrachromosomal copies of the viral episome (FIG. 1a)

and constitutively expresses a limited set of viral geneproducts, 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, 2Aand 2B)4 (FIG. 1b). Transcripts from the BamHIA regionof the viral genome (so-called BART transcripts; seelater) are also detected in LCLs. In addition to the latentproteins, LCLs also show abundant expression of thesmall, non-polyadenylated (and therefore non-coding)RNAs, EBER1 and EBER2; the function of these tran-scripts is not clear, but they are consistently expressed inall forms of latent EBV infection4. This pattern of latentEBV gene expression, which appears to be activated onlyin B-cell infections, is referred to as ‘latency III’ (FIG. 1).

LCLs show high levels of expression of the B-cellactivation markers CD23, CD30, CD39 and CD70,and of the cell-adhesion molecules lymphocyte-func-tion-associated antigen 1 (LFA1; also known asCD11a/18), LFA3 (also known as CD58) and intercel-lular cell-adhesion molecule 1 (ICAM1; also known asCD54)5,6. These markers are usually absent orexpressed at low levels on resting B cells, but are transiently induced to high levels when these cells areactivated into short-term growth by antigenic ormitogenic stimulation, indicating that EBV-inducedimmortalization can be elicited through the constitu-tive activation of the same cellular pathways that drivephysiological B-cell proliferation. The ability ofEBNA2, EBNA3C and LMP1 to induce LCL-like phe-notypic changes when expressed individually inhuman B-cell lines indicates that these viral proteins

EPSTEIN–BARR VIRUS: 40 YEARS ONLawrence S. Young and Alan B. Rickinson

Abstract | Epstein–Barr virus (EBV) was discovered 40 years ago from examining electronmicrographs of cells cultured from Burkitt’s lymphoma, a childhood tumour that is common insub-Saharan Africa, where its unusual geographical distribution — which matches that ofholoendemic malaria —indicated a viral aetiology. However, far from showing a restricteddistribution, EBV — a γ-herpesvirus — was found to be widespread in all human populations andto 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’slymphoma proved consistent and became the first of an unexpectedly wide range of associationsdiscovered between this virus and tumours.

HUMAN LEUKOCYTE ANTIGEN

CLASS II MOLECULES

A subset of histocompatabilityantigens that are mainlyexpressed on cells of theimmune system, including Bcells, and are involved in thepresentation of antigens to CD4+

T cells.

LYTIC REPLICATION

The full cycle of virus infection,leading to the production of newvirus progeny and, eventually,lysis of the infected cell.

NATURE REVIEWS | CANCER VOLUME 4 | OCTOBER 2004 | 757

Cancer Research UKInstitute for Cancer Studies,University of Birmingham,Birmingham, B15 2TT, UK.Correspondence to L.S.Y.e-mail:[email protected]:10.1038/nrc1452

HODGKIN’S AND

REED–STERNBERG CELLS

The malignant cells of Hodgkin’slymphoma, named after thepathologists who first identifiedthem as characteristic markers ofthis particular tumour. In EBV-associated Hodgkin’s lymphomalesions, only these malignantcells express EBV latent-cycleantigens.

GERMINAL CENTRES

Structures in peripherallymphoid tissues that arisethrough clonal proliferation ofantigen-stimulated B cells(germinal centroblasts) whoseimmunoglobulin genes undergosomatic hypermutation. A smallfraction of cells expressingimmunoglobulins of higheraffinity for antigen are selectedas memory B cells.

758 | OCTOBER 2004 | VOLUME 4 www.nature.com/reviews/cancer

R E V I E W S

sequence-specific DNA-binding protein, Jκ-recombina-tion-binding protein (RBP-Jκ), to transcriptionally acti-vate cellular genes such as CD23 and the key viral genesLMP1 and LMP2A4,17,18 (FIG. 2). EBNA-LP interacts withEBNA2 and is required for the efficient outgrowth ofvirus-transformed B cells in vitro19,20. The transcrip-tional activation that is mediated by EBNA2 in con-junction with EBNA-LP is modulated by the EBNA3family of proteins, which repress transactivation21,22

(FIG. 2). An essential role for EBNA3A and EBNA3C inB-cell transformation in vitro has been shown usingEBV recombinants23. EBNA3C can cooperate with RASin rodent-fibroblast transformation assays and disruptcell-cycle checkpoints24,25. These effects are partlyexplained by the interaction of EBNA3C with factorsthat modulate transcription (for example, histonedeacetylase 1, nonmetastatic protein 23-homologue 1and C-terminal binding protein) or influence cell-cycleprogression (for example, cyclin A)26.

The EBV-encoded latent membrane proteins LMP1 is the main transforming protein of EBV; it func-tions as a classic oncogene in rodent-fibroblast transfor-mation assays and is essential for EBV-induced B-celltransformation in vitro27,28. LMP1 has pleiotropic effectswhen it is expressed in cells, resulting in the induction ofcell-surface adhesion molecules and activationantigens7, and upregulation of anti-apoptotic proteins(for example, BCL2 and A20)31,32. LMP1 functions as aconstitutively activated member of the tumour necrosisfactor receptor (TNFR) superfamily, and activates sev-eral signalling pathways in a ligand-independent man-ner33–35 (FIG. 3). Functionally, LMP1 resembles CD40 —another member of the TNFR superfamily — and canpartially substitute for CD40 in vivo, providing bothgrowth and differentiation signals to B cells36. TheLMP1 protein activates several downstream signallingpathways that contribute to the many phenotypic con-sequences of LMP1 expression, including the inductionof various genes that encode anti-apoptotic proteinsand cytokines37 (see the legend for FIG. 3 for details).

The LMP2 proteins, LMP2A and LMP2B, are notessential for EBV-induced B-cell transformation in vitro38

(FIG. 4). However, expression of LMP2A in B cells in trans-genic mice abrogates normal B-cell development, allow-ing immunoglobulin (Ig)-negative cells to colonizeperipheral lymphoid organs39. This indicates that LMP2Acan drive the proliferation and survival of B cells in theabsence of signalling through the B-cell receptor (BCR).LMP2A can transform epithelial cells and enhance theiradhesion and motility, effects that might be mediated bythe activation of the phosphatidylinositol-3-kinase–AKTpathway40. Repressive effects of LMP2A expression haverecently been reported in human and murine B cells, andmany of these target B-cell-specific factors, resulting in aphenotype that is similar to those of malignant HODGKIN’S

AND REED STERNBERG (HRS) CELLS in Hodgkin’s lymphoma andGERMINAL-CENTRE B cells41,42. In addition to these effects,LMP2A was found to induce expression of a range ofgenes that are involved in cell-cycle induction, inhibitionof apoptosis and suppression of cell-mediated immunity.

are key effectors of the immortalization process7. Therole of EBV latent genes in the in vitro transformationof B cells has been confirmed more recently by thegeneration of recombinant forms of EBV that lackindividual latent genes. Studies using these viruseshave confirmed the absolute requirement for EBNA2and LMP1 in the transformation process, and havehighlighted a crucial role for EBNA1, EBNA-LP,EBNA3A and EBNA3C4.

The EBV-encoded nuclear antigensEBV-infected cells express a group of nuclear proteinsthat influence both viral and cellular transcription.EBNA1 is expressed in all virus-infected cells, inwhich its role in the maintenance and replication ofthe episomal EBV genome is achieved throughsequence-specific binding to the plasmid origin ofviral replication, OriP4 (FIG. 1b,c). EBNA1 can alsointeract with certain viral promoters, thereby con-tributing to the transcriptional regulation of theEBNAs (including EBNA1 itself ) and of LMP1.EBNA1 is separated into amino- and carboxy-termi-nal domains by a Gly-Ala repeat sequence, the mainfunction of which seems to be to stabilize the matureprotein — preventing its proteasomal breakdown8 —rather than functioning in its originally suggested roleas an immune-evasion domain9–11. Gene-knockoutstudies indicate that EBNA1 does not have a crucialfunction in in vitro B-cell transformation beyond themaintenance of the viral genome12; on the other hand,a more direct involvement in oncogenesis is indicatedby the ability of B-cell-directed EBNA1 expression toproduce B-cell lymphomas in transgenic mice13, andby its possible contribution to the survival of Burkitt’slymphoma cells in vitro14.

The inability of one EBV strain — P3HR-1, whichcarries a deletion of the gene that encodes EBNA2 andthe last two exons of that for EBNA-LP — to transformB cells in vitro was the first indication of the crucial roleof EBNA2 in the transformation process4. Restorationof the EBNA2 gene in P3HR-1 has unequivocally con-firmed the importance of EBNA2 in B-cell transforma-tion and has allowed the functionally relevant domainsof EBNA2 to be identified15,16. EBNA2 interacts with a

Summary

• Epstein–Barr virus (EBV) infection is implicated in the aetiology of several differentlymphoid and epithelial malignancies.

• EBV-encoded latent genes induce B-cell transformation in vitro by altering cellulargene transcription and constitutively activating key cell-signalling pathways.

• EBV exploits the physiology of normal B-cell differentiation to persist within thememory-B-cell pool of the immunocompetent host.

• Immunosuppressed transplant patients are at risk of developing fatal EBV-transformed 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 orprevent EBV-associated tumours.


R E V I E W S

protein L22, and bind the interferon-inducible, double-stranded-RNA-activated protein kinase PKR43. PKRhas a role in mediating the antiviral effects of the inter-ferons, and it has been suggested that EBER-mediatedinhibition of PKR function might be important forviral persistence44. Expression of the EBERs in Burkitt’slymphoma cell lines has been found to increasetumorigenicity, promote cell survival and induce inter-leukin-10 (IL-10) expression43,45. Such studies indicatethat EBV genes that were previously shown to be dis-pensable for transformation in B-cell systems mightmake more important contributions to the pathogene-sis of some EBV-associated malignancies, and to EBVpersistence, than was previously appreciated.

A group of abundantly expressed RNAs that areencoded by the BamHIA region of the EBV genomewere originally identified in nasopharyngeal carci-noma (NPC), but were subsequently found to beexpressed in other EBV-associated malignancies, suchas Burkitt’s lymphoma, Hodgkin’s lymphoma andnasal T-cell lymphoma, as well as in the peripheralblood of healthy individuals46–48. These highly splicedtranscripts are commonly referred to as eitherBamHIA rightward transcripts (BARTs) or comple-mentary-strand transcripts (CSTs)49,50. The proteinproducts of these open reading frames remain to beconclusively identified. Another transcript that is gen-erated from the BamHIA region is BARF1, whichencodes a 31-kDa protein that was originally identi-fied as an early antigen expressed on induction of theEBV lytic cycle. Recent studies have shown thatBARF1 is a secreted protein that is expressed as alatent protein in EBV-associated NPC and gastric car-cinoma51,52. BARF1 shares limited homology with thehuman colony-stimulating factor 1 receptor (the FMSoncogene) and displays oncogenic activity when it isexpressed in rodent fibroblasts and simian primaryepithelial cells53.

EBV infections in immunocompetent hostsIn contrast to in vitro studies of EBV infection andlatent-gene function, our understanding of the biologyof EBV infection in vivo (FIG. 5) is still rudimentary.Primary infection (by oral transmission) is usuallyasymptomatic, but if it is delayed until adolescence itoccasionally presents as INFECTIOUS MONONUCLEOSIS (IM).Patients with acute IM shed high titres of infectiousvirus in the throat from lytic infection at oropharyn-geal sites. It is possible that this occurs in local mucosalB cells but, from evidence of virus replicative lesionsthat are seen in the oral mucosa of immunocompro-mised patients, it is likely that this also involves theoropharyngeal epithelium. At the same time, largenumbers of latently infected B cells — at least some ofwhich represent transformed EBNA2+, LMP1+ lym-phoblasts54,55 — appear in tonsillar (and possiblyother) lymphoid tissues. In vitro, both naive and MEM-

ORY B CELLS seem equally susceptible to EBV infection.However, although some of the infected tonsillar cellsin IM tonsils have a naive Ig genotype, the expandingclones preferentially involve cells with mutated Ig

Other EBV latent transcriptsIn addition to the latent proteins, the two small non-polyadenylated (non-coding) RNAs — EBER1 andEBER2 — are expressed in all forms of latency.However, the EBERs are not essential for the EBV-induced transformation of primary B lymphocytes4.The EBERs assemble into stable ribonucleoprotein particles with the autoantigen La and ribosomal

BamHIA region

BARF0

BARF1

LMP2A

LMP2BCp or Wp

OriPLMP1

EBER1 EBER2

EBNA-LP

EBNA2

EBNA3CEBNA3B

EBNA3A

Double-stranded DNA episome

EBNA1

Qp

TR

CW W W W W W Y H F Q U P O M L E K B G D X V I A

W W W W W W

OriPNhet Nhet

TR TR

EBNA-LP EBNA2 EBNA3A EBNA3B EBNA3C EBNA1 LMP1

Sa Re1–e3 dZ c b T

c Open reading frames for the EBV latent proteins

a EBV electron micrograph b EBV genome: latent genes

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 thedouble-stranded viral DNA episome. The origin of plasmid replication (OriP) is shown in orange. Thelarge green solid arrows represent exons encoding each of the latent proteins, and the arrowsindicate the direction in which the genes encoding these proteins are transcribed. The latentproteins include the six nuclear antigens (EBNAs 1, 2, 3A, 3B and 3C, and EBNA-LP) and the threelatent membrane proteins (LMPs 1, 2A and 2B). EBNA-LP is transcribed from a variable number ofrepetitive exons. LMP2A and LMP2B are composed of multiple exons, which are located on eitherside of the terminal repeat (TR) region, which is formed during the circularization of the linear DNA toproduce the viral episome. The blue arrows at the top represent the highly transcribed non-polyadenylated RNAs EBER1 and EBER2; their transcription is a consistent feature of latent EBVinfection. The long outer green arrow represents EBV transcription during a form of latency knownas latency III (Lat III), in which all the EBNAs are transcribed from either the Cp or Wp promoter; thedifferent EBNAs are encoded by individual mRNAs that are generated by differential splicing of thesame long primary transcript. The inner, shorter red arrow represents the EBNA1 transcript, whichoriginates from the Qp promoter during Lat I and Lat II. Transcripts from the BamHIA region can bedetected 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 openreading frames for the EBV latent proteins on the BamHI restriction-endonuclease map of theprototype B95.8 genome. The BamHI fragments are named according to size, with A being thelargest. Lowercase letters indicate the smallest fragments. Note that the LMP2 proteins areproduced from mRNAs that splice across the terminal repeats (TRs) in the circularized EBVgenome. This region is referred to as Nhet, to denote the heterogeneity in this region due to thevariable number of TRs in different virus isolates and in different clones of EBV-infected cells. b and cmodified with permission from REF. 138 © (2003) Nature Publishing Group.

INFECTIOUS MONONUCLEOSIS

A transient illness, associatedwith hyperactivation of theCD8+ T-cell response, thatoccurs in some individualswhose primary EBV infection isdelayed until the second or thirddecade of life. Primary infectionduring childhood is almostalways asymptomatic.


R E V I E W S

infected cells into germinal centres, leading to progenythat either re-enter the circulating memory pool or dif-ferentiate to become plasma cells that might migrate tomucosal sites. The different forms of latency that areseen in virus-associated malignancies might representlatency programmes that have evolved to accommodatesuch changes in host-cell physiology. Germinal-centretransit therefore seems to activate a latency programmein which only the genome-maintenance protein EBNA1is expressed, whereas exit from germinal centres is possi-bly linked to the transient activation of LMP1 andLMP2 expression58. Similarly, a commitment to plasma-cytoid differentiation is thought to trigger these cells toundergo lytic viral replication, providing a source oflow-level virus shedding into the oropharynx. Theremight also be circumstances in which infected cells inthe reservoir can become reactivated to produce furtherproliferative latency III infections.

Primary EBV infection elicits strong cellularimmune responses that then bring the infectionunder control. The LYMPHOCYTOSIS that typifies acuteIM therefore reflects the hyperexpansion of cytotoxicCD8+ T cells that are reactive to both lytic- andlatent-cycle viral antigens, reactivities that are subse-quently maintained in the CD8+ T-cell memory atlevels that, collectively, might constitute up to 5% ofthe total circulating CD8+ T-cell pool60. This level ofcommitment to a single virus, which is apparent evenin EBV carriers who have no prior history of IM,implies a crucial role for immune T-cell surveillancein controlling persistent EBV infection.

Virus-associated B-cell lymphomasThere are three histologically and clinically distincttypes of EBV-associated B-cell lymphoma that show dif-ferent patterns of latent gene expression and seem, fromIg-gene sequencing, to derive from cells at differentpositions in the B-cell differentiation pathway. Here, wedescribe the main features of these tumours and discussthe role of EBV in their pathogenesis.

Lymphomas in immunosuppressed individuals. T-CELL-

IMMUNOCOMPROMISED patients are at high risk of devel-oping B-cell lymphomas, and those that arise intransplant patients are the best studied of these lym-phomas. Most ‘post-transplant lymphomas’ (PTLs)arise as polyclonal or monoclonal lesions within thefirst year of allografting, when immunosuppression ismost severe. Almost all of these early-onset tumoursare EBV-positive and (on the basis of positive EBNA2and LMP1 staining) express the latency III pro-gramme, which identifies them as virus-transformedB cells that grow out in the absence of effective T-cellsurveillance61. Some of the lymphomas that are seenin highly immunocompromised AIDS patients, par-ticularly central nervous system (CNS) lesions, showessentially the same phenotype. Transplant cohortscontinue to show a significant, albeit lower, risk ofdeveloping lymphomas well beyond the first year, butthese late-onset tumours are a more heterogeneousgroup and — as with the non-CNS lymphomas of

sequences that are typical of antigen-selected memorycells. This is consistent with the important findingthat, in the blood of patients with acute IM and subse-quently of long-term virus carriers, EBV-infected cellsare concentrated in the IgD–CD27+ memory-B-cellsubset56,57. Furthermore these cells have by that timereturned to a resting state and have downregulated theexpression of most, and possibly all, viral proteins58.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 physi-ological controls as the general mucosa-associatedmemory-B-cell pool59. Such a strategy brings with it thepossibility of fortuitous, antigen-driven recruitment of

MEMORY B CELLS

B cells that have experiencedantigen stimulation and, usually,somatic hypermutation andgerminal-centre transit, beforesubsequent selection into a poolof long-lived recirculating cells.These cells rapidly respond to alater re-challenge with theirspecific antigen, mounting anefficient secondary antibodyresponse.

LYMPHOCYTOSIS

A marked expansion oflymphocyte numbers in theblood, caused by proliferation ofEBV-specific CD8+ T cells in theblood of patients with infectiousmononucleosis.

T-CELL IMMUNOCOMPROMISED

A state of immune T-cellimpairment seen, for instance,in transplant patients receivinghigh doses of T-cell-suppressivedrugs to prevent rejection of thetransplant and in late-stageAIDS patients; in bothsituations, immune control overpersistent viral infections, suchas EBV, is impaired.

SMRT

SKIP

SKIP

HDAC1/2

SAP30

CIR

RBP-Jκ

RBP-Jκ

GTGGGAA

GTGGGAA

Off

On

SIN3A

SMRT HDAC1/2

SAP30

CIR

SIN3AEBNA2

EBNA2 EBNA-LP

BTM

EBNA3A

EBNA3B

EBNA3C

a Repression

b Activation

Figure 2 | The EBV-encoded nuclear antigens.a | Epstein–Barr virus (EBV)-encoded nuclear antigen 2(EBNA2) functions as a transcriptional activator by interactingwith the DNA-binding Jκ-recombination-binding protein (RBP-Jκ) and relieving the transcriptional repression that ismediated 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-Jκ-interacting protein that also interacts with the SMRT–HDACcorepressor complex. EBNA2 abolishes RBP-Jκ mediatedrepression by competing for the SMRT–HDAC corepressorcomplex through binding to both RBP-Jκ and SKIP130. b | The acidic domain of EBNA2 then recruits the basaltranscription machinery (TFIIB, TFIIH and p300; not shown) to activate transcription. EBNA-LP cooperates with EBNA2 inRBP-Jκ-mediated transcriptional activation by interacting withthe acidic activation domain of EBNA2 (REF. 4). The EBNA3family of proteins modulate EBNA2-mediated RBP-Jκactivation by interacting with RBP-Jκ and competing forbinding and activation by EBNA2. The RBP-Jκ homologue inDrosophila is involved in signal transduction from the Notchreceptor, a pathway that is important in cell-fate determinationin Drosophila and has also been implicated in the developmentof T-cell tumours in humans131. EBNA2 can functionally replacethe intracellular region of Notch132. BTM, basal transcriptionmachinery; CIR, CBFI (RBP-Jκ)-interacting corepressor;SAP30, SIN3-associated protein 30.


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Hodgkin’s lymphoma. In this unusual tumour, the cloneof malignant HRS cells is vastly outnumbered by a non-malignant infiltrate, the appearance of which distin-guishes the nodular sclerosing (NS), mixed cellularity(MC) and rarer lymphocyte-depleted (LD) subtypes.Approximately 40% of cases of classic Hodgkin’s lym-phoma in the developed world are associated with EBV;this figure includes most MC and LD cases but, ironi-cally, only a minority of the NS tumours that make upthe marked peak of Hodgkin’s lymphoma incidencethat is seen in the third decade of life in the developedworld, and that first raised the issue of an infectious aeti-ology. Hodgkin’s lymphoma in the developing worldseems to show an even higher overall association withEBV, reflecting at least in part the absence of this peak inyoung adults64.

The existence of EBV-negative disease raises thequestion of whether EBV, when present, is simply a‘passenger’. Although this remains formally possible,the likelihood of Hodgkin’s lymphoma having arisenin an EBV-positive target cell by chance seemsremote, given that infected cells normally make up atiny fraction (1–100 cells per million) of the total B-cell pool59. Furthermore, in EBV-positive tumours,the viral genome is present in every HRS cell andexpresses a particular subset of latent-cycle proteins— EBNA1, LMP1 and LMP2, the so-called latency IIprogramme47. The identification of HRS cells asfailed products of germinal-centre reactions by Iggenotyping65,66 (BOX 1) does indeed indicate a plausi-ble pathogenetic role for the virus, based on rescuingsuch tumour progenitors from apoptosis. LMP1 istherefore capable of constitutively activating theCD40 pathway, thereby replacing a signal that is nor-mally provided by cognate T cells during memory-cell selection, and LMP2A can mimic signalling fromsurface Ig, replacing the usual requirement for high-affinity binding to cognate antigen. Whether EBV stillcontributes to the malignant phenotype at the timeof tumour presentation is more difficult to deter-mine, particularly as there are no EBV-positive HRScell lines available as in vitro models that retain theclassic latency II form of infection. Because thedownstream components of surface Ig signallinghave been eliminated in HRS cells67, LMP2A mightno longer be operational, at least in the context ofthat pathway. By contrast, HRS cells continue to showmany characteristics of LMP1-induced phenotypicchanges, including the strong activation of nuclearfactor-κB (NF-κB) and its associated downstreameffects. Interestingly, the HRS cells of EBV-negativeHodgkin’s lymphoma show a very similar phenotype,which, in at least some cases, seems to have beencaused by inactivation of inhibitor of κBα (IκBα),the physiological regulator of NF-κB activity, or byamplification of the REL gene, which encodes an NF-κB family member66. This parallel indicates thatNF-κB deregulation is an important feature of thepathogenesis of Hodgkin’s lymphoma, and that EBV-positive and EBV-negative tumours have achieved thesame end point by different routes.

late-stage AIDS patients — the proportion of EBV-associated cases can fall below 50%. EBV-positive late-onset PTLs are typically monoclonal tumours, someof which mimic the latency III phenotype of classicearly-onset disease, whereas others are EBNA2- andLMP1-negative or, occasionally, EBNA2-negative butLMP1-positive for a proportion of cells62,63. Suchtumours might therefore have evolved from EBV-transformed LCL-like lesions through the acquisitionof additional cellular genetic changes that render cer-tain viral functions redundant. However, with theexception of BCL6 mutations63 (which might be acoincidental consequence of germinal-centre transit),no specific change occurs consistently or can belinked to tumours of a particular phenotype. Asdescribed in BOX 1, Ig-gene sequencing has shownthat PTLs can arise from a range of positions on theB-cell differentiation pathway, including some thatindicate pathogenetic similarities with other EBV-associated B-lymphomas.

EBVLMP1EBVLMP1

CTAR1

CTAR2

NH2TRAFs

TRAF2TRADD

Lipid raft

PI3K

MAPKKK

MAPKKK

AKT

RHO GTPase

p38, ERK?

IKKαIKKαNIK

IKKγIKKβ

p100

p65 p50

p65 p50p65 p52

IκB

NF-κB2 NF-κB1

JNK, ERK?

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 plasmamembrane and orientates the protein; second, six hydrophobic transmembrane loops, which areinvolved in self aggregation and oligomerization (amino acids 24–186); third, a long carboxy-terminal cytoplasmic region (amino acids 187–386), which possesses most of the signallingactivity of the molecule. Two distinct functional domains referred to as C-terminal activationregions 1 and 2 (CTAR1 and CTAR2) have been identified on the basis of their ability to activatethe nuclear factor-κB (NF-κB) transcription-factor pathway133. The signalling effects of LMP1result from the ability of tumour necrosis factor receptor (TNFR)-associated factors (TRAFs) tointeract either directly with CTAR1 or indirectly by interacting with the death-domain-containingprotein TRADD, which binds to CTAR2 (REF. 37). These adaptor proteins subsequently recruit amultiprotein 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 (involvingp50–p65 heterodimers) and the processing of p100 NF-κB2 to generate p52–p65heterodimers134. Other kinases are recruited to LMP1 through interactions with TRAF moleculesincluding the mitogen-activated protein kinase kinase kinases (MAPKKKs) TPL2 and TAK1, andthese 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.


R E V I E W S

At presentation, the vast majority of EBV-positivetumours show a highly restricted latency I form ofinfection, with viral antigen expression limited to thatof EBNA1 (REFS 6,75). So, how EBV contributes to thepathogenesis of Burkitt’s lymphoma remains a matterof speculation. The virus might have an initiating rolein which growth-transforming B-cell infections estab-lish a pool of target cells that are at risk of a subse-quent MYC translocation, a process that has been successfully modelled in vitro68. This latter study high-lighted the apparent incompatibility of the EBVlatency-III-driven and MYC-driven growth pro-grammes in B cells76, indicating that the evolution toBurkitt’s lymphoma can only occur if the EBV pro-gramme is suppressed. Indeed, the strength of selec-tion against full expression of viral latent genes isillustrated by a subset of Burkitt’s lymphomas inwhich the transcriptional features of latency III areretained, but the resident viral genome has deleted theEBNA2 gene and therefore abrogated the conventionalB-cell transforming function75.

Alternatively, EBV might contribute to theBurkitt’s lymphoma phenotype through the latency-I-active genes themselves. EBNA1 is an obvious candidate, but its reported oncogenicity in mousetransgene assays13 remains controversial, and its con-tribution to virus-induced B-cell transformation in vitro seems to be limited to maintenance of theviral genome12,77; however, recent experiments inwhich EBNA1 function was blocked in EBV-positiveBurkitt’s lymphoma cell lines have indicated that theprotein does promote cell survival14. Other studies,based on infection or transfection of spontaneousEBV genome-loss derivatives of the Akata Burkitt’slymphoma cell line, have also shown increased sur-vival mediated either by virus-induced upregulationof the TCL1 oncogene78 or through induction of theexpression of the IL-10 cytokine by the non-codingEBER RNAs43. The wider relevance of these effectsbeyond the Akata Burkitt’s lymphoma model systemremains to be determined.

Any comprehensive view of Burkitt’s lymphomapathogenesis must take into account the markedincreases in tumour incidence that are associated withholoendemic malaria and HIV infections. In thisregard, both agents can act as chronic stimuli of the B-cell system and, at least for HIV carriage, the persis-tent generalized LYMPHADENOPATHY that results fromsuch stimulation is characterized by exaggerated ger-minal-centre activity79. We suggest that this greatlyincreases the chances of productive MYC transloca-tions occurring. Furthermore, both agents disturb theEBV–host balance and probably increase the numbersof EBV-infected B cells that are at risk of beingrecruited into germinal-centre reactions80. There aredifferences between the two situations, however, and itwill be interesting to see whether malaria has its ownspecific effects on the EBV–host balance as, unlikeHIV, the 100-fold increase in the incidence of Burkitt’slymphoma seen in patients with malaria consistsentirely of EBV-positive disease5.

Burkitt’s lymphoma. EBV is present in all cases of‘endemic’ Burkitt’s lymphoma — the high-incidenceform of the tumour that affects children in areas of Africaand New Guinea in which malaria is holoendemic — andin up to 85% of cases in areas of intermediate incidencesuch as Brazil and North Africa, but in only 15% of thelow-incidence ‘sporadic’ tumours that are seen in children in the developed world. Remarkably, Burkitt’slymphoma is also common among adult humanimmunodeficiency virus (HIV) carriers in the developedworld and often arises as the first AIDS-defining illness inrelatively immunocompetent patients; some 30–40% ofthese tumours are EBV-associated5. All Burkitt’s lym-phomas carry one of three characteristic chromosomaltranslocations that place the MYC oncogene under thecontrol of the Ig heavy chain or one of the light-chainloci. The primacy of MYC deregulation as the key factorin the pathogenesis of Burkitt’s lymphoma is clear from arange of experimental systems68–70. In addition, manytumours have TP53 mutations or other defects in thep53–ARF pathway, as well as mutations in the putativetumour-suppressor gene retinoblastoma-like (REF. 71).Irrespective of their EBV status, the phenotype ofBurkitt’s lymphoma cells (CD10+, CD77+, BCL6+) isremarkably similar to that of germinal centroblasts, andthe detection of ongoing Ig-gene mutation in tumourcells72–74 (BOX 1) supports the suggestion that they origi-nate in germinal centres. Indeed, the MYC translocationitself is likely to have occurred as an error of the SOMATIC

HYPERMUTATION process.

Tyr112

Tyr85

Tyr74

PYPY

NH2

EBVLMP2AEBVLMP2A

Lipid raft

SRC/SYC

LYN

NEDD4

PI3K

MAPKKK

Ubiquitylation of LMP2A andLMP2A-associated proteins

AKT

RHO GTPase

Cell survivaland motility

ERK JUN?

Figure 4 | Structure and function of LMP2. The structures of the Epstein–Barr virus (EBV) latentmembrane proteins LMP2A and LMP2B are similar; both have 12 transmembrane domains and a27-amino-acid cytoplasmic carboxyl terminus. In addition, LMP2A has a 119-amino-acidcytoplasmic amino-terminal domain that contains eight tyrosine residues, two of which (Tyr74 andTyr85) form an immunoreceptor tyrosine-based activation motif (ITAM)38. The phosphorylated ITAMrecruits members of the SRC family of protein tyrosine kinases and the SYK tyrosine kinase andnegatively regulates their activities. A membrane-proximal tyrosine residue (Tyr112) binds the LYNtyrosine kinase and mediates the constitutive phosphorylation of the other tyrosine residues inLMP2A38. The LMP2A ITAM blocks signalling from the B-cell receptor (BCR) by sequestering thesetyrosine kinases and by blocking the translocation of the BCR into lipid rafts135. LMP2A also recruitsNEDD4-like ubiquitin protein ligases through phosphotyrosine (PY) motifs, and these promote thedegradation of LYN and LMP2A by a ubiquitin-dependent mechanism136. LMP2A interacts with theextracellular signal-regulated kinase 1 (ERK1) mitogen-activated protein kinase (MAPK), and thisresults in the phosphorylation of two serine residues (Ser15 and Ser102) in LMP2A, and mightcontribute to LMP2A-induced activation of JUN137. MAPKKK, MAPK kinase kinase; PI3K,phosphatidylinositol 3-kinase.

SOMATIC HYPERMUTATION

Point mutations that occur inthe immunoglobulin-genevariable regions (and some othergenes) during B-celldifferentiation.

LYMPHADENOPATHY

A marked swelling of peripherallymphoid tissues in situationsarising from chronic antigenicstimulation by an infectiousagent.


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Virus-associated T-cell and NK-cell lymphomasEBV is so markedly B-lymphotropic when it is exposedto human lymphocyte preparations in vitro that theassociation of the virus with rare but specific types ofT-cell and natural killer (NK)-cell lymphomas81,82 wascompletely unexpected. How the virus accesses these celllineages in vivo is still uncertain, but most evidence indi-cates that such infections are rare and, when they dooccur, 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 mostcommon in southeast Asian populations. This type oflymphoma arises either after acute primary infection,manifesting as virus-associated haemophagocytic syn-drome (VAHS), or in the setting of a chronic active EBVinfection with VAHS-like symptoms81,83. These aremonoclonal tumours of CD4+ or CD8+ T-cell origin, inwhich viral gene expression is restricted to the produc-tion of EBNA1 and LMP2, with variable levels of LMP1being detectable in only a fraction of cells (designatedlatency 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 haveVAHS-like disease84 and might, through cytokinerelease, drive macrophage activation and haemophago-cytosis. A second example, which is again most com-mon in south-east Asia, is an extranodal lymphoma that usually presents as an erosive lesion (‘lethal mid-line granuloma’) in the nasal cavity 82. Some of these are CD3+CD56– T-cell lymphomas, but most areCD3–CD56+ tumours of NK-cell origin, again withlatency I/II gene expression85.

Carcinomas associated with the virusNasopharyngeal carcinoma. An important conse-quence of epithelial infection with EBV is malignanttransformation, resulting in the development of NPC, asubset of gastric adenocarcinomas and certain salivary-gland carcinomas5,86. The EBV-associated, undifferenti-ated form of NPC — World Health Organization(WHO) type III — shows the most consistent world-wide association with EBV and is particularly commonin areas of China and south-east Asia, reaching a peakincidence of around 20–30 cases per 100,000 (REF. 87).Incidence rates are high in individuals of Chinesedescent, irrespective of where they live, and particularlyin Cantonese males. In addition to this genetic predis-position, environmental cofactors such as dietary com-ponents (for example, salted fish) are thought to beimportant in the aetiology of NPC88. NPC tumours arecharacterized by the presence of undifferentiated carci-noma cells and a prominent lymphocytic infiltrate, andthis interaction between tumour cells and lymphocytesseems to be crucial for the continued propagation ofthe malignant component. EBV latent-gene expressionin NPC is predominantly restricted to the EBNA1nuclear antigen, the latent membrane proteins(LMP2A and LMP2B) and the BamHIA transcripts,with ~20% of tumours also expressing the oncogenicLMP1 protein86. Southern-blot hybridization of DNAfrom NPC tissues demonstrates the monoclonality of

a Primary infection

Epithelium

Naive B cell

Lytic replication

MemoryB-cellreservoir

Latency I/IILatency III Latency 0

Germinal centre

Germinal centre

MemoryB cell

b Persistent infection

Plasma cell

Primary- T-cell response

Epithelium

Naive B cell

Lytic replication

Memory-B-cellreservoir

Latency I/II Latency IIILatency 0

MemoryB cell

Memory-T-cell response

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 tissuesas a latent (latency III) growth-transforming infection of B cells. Many of these proliferating cells areremoved by the emerging latent-antigen-specific primary-T-cell response, but some escape bydownregulating antigen expression and establishing a stable reservoir of resting viral-genome-positive memory B cells, in which viral antigen expression is mostly suppressed (latency 0). Differentviews of these events are shown. One view is that naive B cells are the main targets of new EBVinfections in vivo. In this scenario, viral transformation drives naive cells into memory by mimickingthe physiological process of antigen-driven memory-cell development in lymphoid tissues, aprocess involving somatic immunoglobulin-gene hypermutation during transit through a germinalcentre. However, this is difficult to reconcile with the finding that EBV-infected B cells in tonsils frompatients with infectious mononucleosis (IM) localize to extrafollicular areas — not to germinal centres— and show no evidence of ongoing hypermutation within expanding clones. An alternative viewtherefore envisages infection of pre-existing memory cells as a direct route into memory; this isconsistent with the above observations on IM tonsils, but still leaves unexplained the apparentdisappearance of the infected naive cell population. b | Persistent infection. The reservoir of EBV-infected memory B cells becomes subject to the physiological controls governing memory-B-cellmigration and differentiation as a whole. Occasionally, these EBV-infected cells might be recruitedinto germinal-centre reactions, entailing the activation of different latency programmes, after whichthey 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 lyticcycle. 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 growth-transforming latency III infections of naive and/or memory B cells; these new infections mightpossibly replenish the B-cell reservoir, but are more likely to be efficiently removed by the now well-established memory-T-cell response.


R E V I E W S

Gastric carcinoma. EBV is also found in ~10% of moretypical gastric adenocarcinomas, accounting for up to75,000 new cases per year93,94. These tumours display arestricted pattern of EBV latent-gene expression (result-ing expression of EBERs, EBNA1, LMP2A, BARTs andBARF1), similar to that seen in NPC95. There is significantgeographical variation in the association of EBV with gas-tric carcinoma, which might be due to ethnic and geneticdifferences. EBV-positive gastric carcinomas have distinctphenotypic and clinical characteristics compared withEBV-negative tumours, including loss of expression ofINK4A (also known as p16) and improved patient sur-vival96,97.As in NPC, the precise role of EBV in the patho-genesis of gastric carcinoma remains to be determined,but the absence of EBV infection in pre-malignant gastriclesions supports the suggestion that viral infection is a relatively late event in gastric carcinogenesis98.

Is EBV associated with other common epithelial malig-nancies? Detection of the EBERs by in situ hybridizationhas become the standard method of detecting EBV infec-tion in the routine processing of tumour tissues.Although the EBERs were previously considered to beexpressed in all forms of EBV latency, two studies haveraised the possibility that EBER-negative forms of latencymight exist in previously unrecognized EBV-associatedmalignancies, such as carcinomas of the breast andliver99,100. Difficulties in confirming these associationshave raised concerns about the use of PCR analysis todetect EBV infection, and have also questioned the speci-ficity of monoclonal-antibody reagents101. It is our con-tention that the definitive designation of a tumour as‘EBV-associated’ should require unequivocal demonstra-tion of the presence of the EBV genome or viral geneproducts within most of the tumour-cell population.

Novel therapeutic approaches Given the significant burden of EBV-associated tumoursworldwide, an important priority is to design novel ther-apies that specifically target viral proteins or otherwiseexploit the presence of the virus in malignant cells.

Pharmacological approaches. One potential approach isthe use of gene-therapy constructs to express either cyto-toxic or inhibitory proteins selectively in tumour cells. Forexample, OriP-based constructs that are responsive toendogenous EBNA1 in infected cells have been used toexpress cytotoxic proteins (for example, FAS ligand) orwild-type p53 in in vitro and in vivo models of NPC102,103.Other approaches are based on the induction of the EBVlytic cycle, either by pharmacological agents or by deliveryof EBV immediate-early genes, thereby inducing virus-encoded kinases (EBV thymidine kinase and BGLF4, aprotein kinase) that phosphorylate the nucleoside ana-logue gancyclovir to produce its active cytotoxicform104,105. Demethylating agents such as 5-azacytidineare able to de-repress lytic, as well as potentially immuno-genic, latent genes106 and are now in early-stage clinicaltrials in patients with NPC, Hodgkin’s lymphoma andAIDS-associated lymphoma107. Another commonchemotherapeutic agent, hydroxyurea, is able to induce

the resident viral genomes, indicating that EBV infec-tion takes place before the clonal expansion of the population of malignant cells89. Studies of normalnasopharyngeal tissue and pre-malignant biopsies indi-cate that genetic events occur early in the pathogenesisof NPC, and that these might predispose to subsequentEBV infection (BOX 2). Extensive serological screeninghas identified increased EBV-specific antibody titres inhigh-incidence areas; in particular, IgA antibodies tothe EBV capsid antigen and early antigens have proveduseful in diagnosis and in monitoring the effectivenessof therapy90. More recent studies using real-time quan-titative PCR to measure circulating tumour-derivedEBV DNA in the blood of patients with NPC haveshown that the level of pre-treatment EBV DNA isstrongly associated with overall survival, and that post-treatment EBV DNA levels predict progression-freeand overall survival91. Association of EBV with theother more differentiated forms of NPC (WHO types Iand II) has been shown, particularly in those geograph-ical regions with a high incidence of undifferentiatedNPC92. Carcinomas that have similar features to undif-ferentiated NPC have been described at other sites,including the thymus, tonsils, lungs, stomach, skin anduterine cervix, and are often referred to as ‘undifferenti-ated carcinomas of nasopharyngeal type’ (UCNT) or‘lymphoepitheliomas’. There is geographical variationin the extent of the association of EBV with UCNTs.

Box 1 | Cellular origins of EBV-positive B-cell lymphomas

Post-transplantation lymphomasFrom immunoglobulin variable (IgV) gene sequencing, post-transplantationlymphomas (PTLs) seem able to arise from naive cells, memory cells or — morecommonly in late onset cases — cells that have atypical or non-functional mutationsthat are normally inconsistent with cell survival; a minority of these latter cases evenshow ongoing hypermutation. Epstein–Barr virus (EBV) might create a pool of non-malignant, but atypical, EBV-carrying B cells in vivo, either by directly rescuing cellsfrom within germinal centres or possibly by activating somatic hypermutation in cellsoutside the germinal-centre environment. The latter scenario is supported by evidencefrom recent studies of EBV-infected non-malignant B-cell clones, both in a post-transplantation Hodgkin’s lymphoma biopsy125 and in infiltrating an EBV-negative T-cell lymphoma of the angioimmunoblastic lymphadenopathy type126.

Hodgkin’s lymphomaAll Hodgkin’s and Reed-Sternberg cells within a tumour are part of the same clone andalmost always carry a mutated IgV sequence that lacks intraclonal diversity. Sequencesthat contain nonsense mutations or deletions are seen in 25% of cases. As this almostcertainly underestimates the true incidence of gene inactivation, most cases ofHodgkin’s lymphoma probably derive from crippled germinal-centre cells that havebeen rescued from the germinal-centre reaction. There are clear analogies here withEBV-positive PTLs that carry inactivated Ig genes or other apparently non-antigen-selected mutations.

Burkitt’s lymphomaIgV sequencing of Burkitt’s lymphoma biopsy cells and derived cell lines (from bothEBV-positive and EBV-negative cases) has identified multiple mutations and, in manycases, ongoing sequence diversification within the malignant clone. This confirmedBurkitt’s lymphoma as a tumour of germinal-centroblast origin. Burkitt’s lymphomacell lines that had switched to a latency III infection and an LCL-like surface phenotypecontinued to show Ig-gene diversification. EBV therefore does not suppress ongoinghypermutation, even when the activation of the viral growth-transforming programmeeliminates other markers of germinal centroblast origin.

on NF-κB112. More recently, LCL growth in vitro hasbeen impaired by blocking the transactivating functionof EBNA2 using a short-peptide mimic of the RBP-Jκ-interaction domain of the viral protein113 (FIG. 2).EBNA1 — the one viral protein that is expressed in allEBV-positive tumours — is a particularly attractive tar-get, and a dominant-negative form of the protein thatblocks its genome-maintenance function might havetherapeutic potential114.

the loss of EBV episomes in in vitro models and hasshown some limited clinical efficacy in patients withEBV-positive AIDS-related CNS lymphoma108,109.

More focused pharmacological approaches aim toabrogate the functions of individual EBV proteins. Inmodel systems, LMP1 effector function has been tar-geted directly, using single-chain antibodies or antisenseRNA approaches110,111, and indirectly, by the genetic orpharmacological interception of its downstream effects

Box 2 | Role of Epstein–Barr virus in the pathogenesis of nasopharyngeal carcinoma

In both nasopharyngeal carcinoma (NPC) and Epstein–Barr virus (EBV)-positive gastric carcinoma, the tumour cellscarry monoclonal viral genomes, which indicates that EBV infection must have occurred prior to expansion of themalignant cell clone89. However, the difficulty of detecting EBV-infected epithelial cells in normal nasopharyngealbiopsies from individuals who are at high risk of developing NPC argues against a pre-existing normal reservoir ofepithelial cell infection from which virus-positive carcinomas arise. Indeed, EBV infection has been detected both by in situ hybridization to the EBV-encoded RNAs (EBERs) and by the presence of monoclonal EBV genomes in high-gradepre-invasive lesions (severe dysplasia and carcinoma in situ) in the nasopharynx, but not in low-grade disease127. Similarresults have been obtained from EBV-positive gastric carcinoma, in which associated normal gastric mucosa, inflamedmucosa and pre-malignant lesions are EBV-negative98. Multiple genetic changes have been found in NPC, with frequentdeletion of regions on chromosomes 3p, 9p, 11q, 13q and 14q and promoter hypermethylation of specific genes onchromosomes 3p (RASSF1A and retinoic-acid receptor β2) and 9p (genes that encode INK4A, INK4B, ARF and death-associated protein kinase)128. Deletions in both 3p and 9p have been identified in low-grade dysplastic lesions and innormal nasopharyngeal epithelium from individuals who are at high risk of developing NPC in the absence of EBVinfection, indicating that genetic events occur early in the pathogenesis of NPC and that these might cause predispositionto subsequent EBV infection128. This possibility is supported by in vitro data showing that the stable infection of epithelialcells by EBV requires an altered, undifferentiated cellular environment129.

The above scheme (see figure; images show stained epithelial sections) has been proposed, in which loss ofheterozygosity (LOH) occurs early in the pathogenesis of NPC, possibly as a result of exposure to environmentalcofactors such as dietary components (such as salted fish). This results in low-grade pre-invasive lesions that, afteradditional genetic and epigenetic events, become susceptible to EBV infection. Once cells have become infected, EBVlatent genes provide growth and survival benefits, resulting in the development of NPC. Additional genetic andepigenetic changes occur after EBV infection. CIS, carcinoma in situ; EDNRB, endothelin receptor B; H/E, staining withhaematoxylin and eosin; TSLC1, tumour suppressor in lung cancer 1.


R E V I E W S

H/E

EBER

Normal epithelium

Low-grade pre-invasive lesion

High-grade pre-invasive lesion

Nasopharyngealcarcinoma

MetastasisCIS?

LOH on chromosome 3p and 9p

Inactivation of RASSF1A and CDKN2A

EBV latent infection

Telomerase dysregulation

LOH on 14q, 11q, 13q and 16qInactivation of EDNRB and TSLC1?

Other genetic changes(for example, in TP53 and E-cadherin)

BCL2 overexpression


R E V I E W S

These tumours might also be capable of evading or suppressing T-cell immune attack — in the case ofHodgkin’s lymphoma, possibly through immunosup-pressive cytokines that are produced by the HRS cellsthemselves122. More work is needed to determine theinfluence of the tumour microenvironment on T-cellattack and to explore ways of modifying the cytokinemilieu, such as the use of EBV-specific T cells to deliverimmunostimulatory cytokines123.

ConclusionsSince its discovery in 1964, EBV has moved frombeing a bit-part player in the story of an obscureAfrican tumour to its present leading role as theprime example of a human tumour virus that is aeti-ologically linked to an unexpectedly diverse range ofmalignancies. Much early work concentrated on theepidemiology of infection in human populations andon the extent of the associations of EBV withtumours. The publication of the EBV genomesequence in 1984 (REF. 124) allowed the molecularanalysis of the virus, work that continues to illumi-nate the mechanisms of action of the viral proteinsthat contribute to tumorigenesis. Now, the challengeis to exploit these mechanistic insights both to gain abetter understanding of the biology of EBV infectionin vivo and to develop novel therapies for treatingvirus-associated disease.

Immunotherapy. A large body of work attempting to target EBV-positive malignancies with T cells that arespecific for EBV antigens is important both in its ownright and as proof of principle for tumour immunother-apy in general. The approach was first used to targetEBV-positive PTLs. Bone-marrow-transplant patientswere infused with EBV latent-antigen-specific effector Tcells that were prepared from the bone marrow donor byautologous LCL stimulation and expansion in vitro. Thisstrategy was highly effective, both as a therapy for thetreatment of existing disease, and in prophylaxis115.Similar adoptive-transfer approaches have now beenused to treat PTLs in solid-organ transplant settingsusing T cells that are expanded in vitro and are preparedeither from the patient116 or, where necessary, from a par-tially HLA-matched donor117. However, these LCL-stim-ulated effector preparations tend to be dominated byCD8+ T cells that are specific for the immunodominantEBNA3A, EBNA3B and EBNA3C proteins — antigensthat are not expressed in all EBV-associated tumours. ForHodgkin’s lymphoma and NPC, therefore, clinical trialswith LCL-stimulated effectors118 represent just a firststep. Strategies are now being developed either to gener-ate T-cell preparations for transfer that are enriched in CD8+ — and possibly CD4+ — reactivities to availablesub-dominant targets (such as LMP2A and EBNA1)119,120,or to immunize the patient with appropriate antigenicconstructs to boost these particular responses in vivo121.

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AcknowledgementsThe authors apologize to colleagues whose primary researchpapers are not cited because of the limited number of references.The authors thank D. Huang, P. Murray and G. Niedobitek for assis-tance with the figures and D. Williams for secretarial support. Theauthors’ studies are supported by Cancer Research UK, theLeukaemia Research Fund and the Medical Research Council, UK.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Cancer.gov: http://www.cancer.govHodgkin’s lymphoma | nasopharyngeal cancerEntrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBCR | CD21 | INK4A | MYC | NF-κB | PKR | TP53Access to this links box is available online.

Epstein–Barr virus: 40 years on

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