EBV the prototypical human tumor virus—just how bad is it?
2005; Elsevier BV; Volume: 116; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2005.05.038
ISSN1097-6825
Autores Tópico(s)Herpesvirus Infections and Treatments
ResumoEBV was the first candidate human tumor virus. It is found in several human cancers, particularly lymphomas and carcinomas, and has potent transforming activity in vitro. Yet the virus persists benignly for the lifetime of more than 90% of the human population. Thus it seems that EBV has the potential to be highly pathogenic yet rarely manifests this potential. Studies over the last several years show this is because the virus actually persists in resting memory B cells and not proliferating cells. EBV needs its growth-promoting ability to gain access to the memory compartment but has evolved to minimize its oncogenic potential. These studies also reveal that the different EBV-associated tumors apparently arise from different and discrete stages in the life cycle of B cells latently infected with EBV. This raises the question of how actively EBV participates in the development of human tumors. Does the virus cause the disease, or is it simply a passenger? In the case of immunoblastic lymphoma in the immunosuppressed patient, the virus almost certainly plays a causative role, but in other cases, such as Burkitt's lymphoma, the contribution of EBV remains less clear. EBV was the first candidate human tumor virus. It is found in several human cancers, particularly lymphomas and carcinomas, and has potent transforming activity in vitro. Yet the virus persists benignly for the lifetime of more than 90% of the human population. Thus it seems that EBV has the potential to be highly pathogenic yet rarely manifests this potential. Studies over the last several years show this is because the virus actually persists in resting memory B cells and not proliferating cells. EBV needs its growth-promoting ability to gain access to the memory compartment but has evolved to minimize its oncogenic potential. These studies also reveal that the different EBV-associated tumors apparently arise from different and discrete stages in the life cycle of B cells latently infected with EBV. This raises the question of how actively EBV participates in the development of human tumors. Does the virus cause the disease, or is it simply a passenger? In the case of immunoblastic lymphoma in the immunosuppressed patient, the virus almost certainly plays a causative role, but in other cases, such as Burkitt's lymphoma, the contribution of EBV remains less clear. EBV is well known because of its characteristic biology.1Rickinson A.B. Kieff E. Epstein-Barr virus.in: Knipe D.M. Howley P.M. Virology. 4th ed. Lippincott Williams and Wilkins, New York2001: 2575-2628Google Scholar, 2Kieff E. Rickinson A.B. Epstein-Barr virus and its replication.in: Knipe D.M. Howley P.M. Virology. 4th ed. Lippincott Williams and Wilkins, New York2001: 2511-2574Google Scholar, 3Thorley-Lawson D.A. Epstein-Barr virus.in: Cantor H. Sampter's immunologic diseases. Williams and Wilkins, New York2001: 970-985Google Scholar If you define the success of a pathogen by the number and extent of hosts it infects, EBV is the most successful human pathogen because it latently infects virtually the whole human population and persists for life.4Henle W. Henle G. Seroepidemiology of the virus.in: Epstein M.A. Achong B.G. The Epstein-Barr virus. Springer-Verlag, Berlin1979: 61-78Crossref Google Scholar In tissue culture EBV is one of the most potent transforming viruses,5Pope J.H. Horne M.K. Scott W. Transformation of foetal human leukocytes in vitro by filtrates of a human leukaemic cell line containing herpes-like virus.Int J Cancer. 1968; 3: 857-866Crossref PubMed Scopus (359) Google Scholar, 6Henle W. Diehl V. Kohn G. Zur Hausen H. Henle G. Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells.Science. 1967; 157: 1064-1065Crossref PubMed Scopus (359) Google Scholar and it is found in several human cancers,1Rickinson A.B. Kieff E. Epstein-Barr virus.in: Knipe D.M. Howley P.M. Virology. 4th ed. Lippincott Williams and Wilkins, New York2001: 2575-2628Google Scholar, 3Thorley-Lawson D.A. Epstein-Barr virus.in: Cantor H. Sampter's immunologic diseases. Williams and Wilkins, New York2001: 970-985Google Scholar yet for most of the population, it remains benign. The collection of viral latent proteins expressed is different in each tumor type (Table I). Sometimes all of the known latent proteins are expressed, sometimes a limited subset, and sometimes only one.Table IThe EBV transcription programs in normal B cells and tumorsTranscription programGenes expressed∗Does not include the noncoding EBER and BART RNAs that are assumed to be ubiquitous but have not been rigorously identified in all of the infected subtypes.Infected normal B-cell type†Except where indicated, the cell types are primarily restricted to the lymphoid tissue of the Waldeyer ring.FunctionInfected tumor typeGrowthEBNA1, 2, 3a, 3b, 3c, LP, LMP1, LMP2a, and LMP2bNaiveActivate B cellImmunoblastic lymphomaDefaultEBNA1, LMP1, and LMP2aGerminal centerDifferentiate activated B cell into memoryHDLatencyNonePeripheral memoryAllow lifetime persistenceEBNA1 onlyEBNA1Dividing peripheral memoryAllow virus in latency program cell to divideBurkitt's lymphomaLyticAll lytic genesPlasma cellReplicate the virus in plasma cell∗ Does not include the noncoding EBER and BART RNAs that are assumed to be ubiquitous but have not been rigorously identified in all of the infected subtypes.† Except where indicated, the cell types are primarily restricted to the lymphoid tissue of the Waldeyer ring. Open table in a new tab Despite the apparent robustness with which the human population deals with EBV (>95% of all adults carry the virus), the diseases caused by EBV indicate that the situation is finely balanced. The first indication comes from X-linked lymphoproliferative disease.7Seemayer T.A. Gross T.G. Egeler R.M. Pirruccello S.J. Davis J.R. Kelly C.M. et al.X-linked lymphoproliferative disease: twenty-five years after the discovery.Pediatr Res. 1995; 38: 471-478Crossref PubMed Scopus (269) Google Scholar In this disease persistent infection is not established because mutations in the SH2D1A gene8Coffey A.J. Brooksbank R.A. Brandau O. Oohashi T. Howell G.R. Bye J.M. et al.Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.Nat Genet. 1998; 20: 129-135Crossref PubMed Scopus (656) Google Scholar, 9Sayos J. Wu C. Morra M. Wang N. Zhang X. Allen D. et al.The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM.Nature. 1998; 395: 462-469Crossref PubMed Scopus (820) Google Scholar cause acute EBV infection to become a fatal disease. Put melodramatically, a single nucleotide change in the SH2D1A gene is all that prevents the vast majority of the human race from dying of acute EBV infection. The second indication comes from the observation that immunologic disturbance, as a predisposing factor, is a unifying theme for all of the EBV B-cell lymphomas. This also suggests that the regulation of EBV infection in B cells is finely balanced. Disruptions can lead to deregulation and EBV-driven tumor development, even in otherwise healthy carriers of the virus. The clearest example of this is individuals who are immunosuppressed, such as patients undergoing organ transplantation, who are iatrogenically immunosuppressed, or patients with AIDS, who are immunosuppressed by HIV. These individuals are at risk for EBV lymphomas that are aggressive and often fatal.10Hopwood P. Crawford D.H. The role of EBV in post-transplant malignancies: a review.J Clin Pathol. 2000; 53: 248-254Crossref PubMed Scopus (110) Google Scholar This means that it is only courtesy of an active immune response that we are protected from fatal EBV-driven lymphoma. Yet there are some curious properties of these tumors that suggest the risk is not as high as might be expected. For example, not every immunosuppressed patient has the tumors, and the tumors are frequently oligoclonal. This is not the expected outcome. If it were simply a case of the immune system failing to control the EBV-infected cells, every immune-suppressed person should fill up with multiple tumors because everybody carries approximately 5 × 105 infected cells,11Khan G. Miyashita E.M. Yang B. Babcock G.J. Thorley-Lawson D.A. Is EBV persistence in vivo a model for B cell homeostasis?.Immunity. 1996; 5: 173-179Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar and immunosuppressed individuals carry perhaps 50 times more.12Babcock G.J. Decker L.L. Freeman R.B. Thorley-Lawson D.A. Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients.J Exp Med. 1999; 190: 567-576Crossref PubMed Scopus (273) Google Scholar Taken together, these observations raise several questions. How does EBV persist benignly for the lifetime of a human despite its pathogenic potential? Why does EBV have such potent and pathogenic properties if it has evolved to persist for the lifetime of the human host it puts at risk by manifesting those properties? Where do the EBV-associated tumors come from, why do they have different patterns of latent gene expression, and why does disruption of the immune system predispose to EBV lymphoma development? Lastly, what goes wrong in the maintenance of persistence that leads to EBV-associated diseases? The key to answering these questions comes from a model of EBV persistence13Thorley-Lawson D.A. Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas.N Engl J Med. 2004; 350: 1328-1337Crossref PubMed Scopus (796) Google Scholar, 14Thorley-Lawson D.A. Epstein-Barr virus: exploiting the immune system.Nat Rev Immunol. 2001; 1: 75-82Crossref PubMed Scopus (760) Google Scholar developed from the observation that despite EBV's transforming ability, it persists in vivo in resting15Miyashita E.M. Yang B. Babcock G.J. Thorley-Lawson D.A. Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell.J Virol. 1997; 71: 4882-4891Crossref PubMed Google Scholar memory16Babcock G.J. Decker L.L. Volk M. Thorley-Lawson D.A. EBV persistence in memory B cells in vivo.Immunity. 1998; 9: 395-404Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar B cells that do not express any viral proteins.17Hochberg D. Middeldorp J.M. Catalina M. Sullivan J.L. Luzuriaga K. Thorley-Lawson D.A. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.Proc Natl Acad Sci U S A. 2003; 101: 239-244Crossref PubMed Scopus (227) Google Scholar This article will first briefly review the complete life cycle of EBV infection and then discuss how the origins of EBV-associated tumors can be explained in the context of this model, with special emphasis on the role of an impaired immune response. Finally, the model will be used to attempt to answer the questions posed above. The essence of EBV's behavior is that under normal conditions, it does not aberrantly deregulate the behavior of infected B cells in vivo. It initiates, establishes, and maintains persistent infection by subtly using virtually every aspect of normal B-cell biology. Ultimately, this allows the virus to persist within memory B cells for the lifetime of the host in a fashion that is nonpathogenic. The thesis of this review is that EBV is not a natural tumor virus and that it has developed strategies to minimize its pathogenic potential to the host. To understand EBV biology, it is first necessary to understand the biology of the B lymphocyte in the mucosal lymphoepithelium of the tonsil (Fig 1). A summary of normal mature B-cell biology and the proposed parallels with EBV is given in Figs 2 and 3, and a summary of information on the different viral latency programs is presented in Table I. The model has been described in detail elsewhere.13Thorley-Lawson D.A. Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas.N Engl J Med. 2004; 350: 1328-1337Crossref PubMed Scopus (796) Google Scholar, 14Thorley-Lawson D.A. Epstein-Barr virus: exploiting the immune system.Nat Rev Immunol. 2001; 1: 75-82Crossref PubMed Scopus (760) Google ScholarFig 2A model of how EBV uses normal B-cell biology to establish and maintain persistent infection in memory B cells. The response of a normal B cell to antigen, leading to the production of antigen-specific memory cells in the peripheral circulation, is diagrammed to the left, and the parallel series of steps by which EBV establishes latent infection in peripheral memory B cells is shown to the right. The specific viral transcription programs are labeled in blue to the right. For details, see the text and Table I.View Large Image Figure ViewerDownload (PPT)Fig 3A model of how EBV uses normal B-cell biology to replicate and be shed into saliva. The pathway by which antigen-specific B cells become activated and differentiate into antibody-producing plasma cells is shown to the left, and the parallels that lead to shedding of EBV are shown to the right. The EBV transcription program is indicated in blue to the right. For details, see text and Table I.View Large Image Figure ViewerDownload (PPT) Environmental antigens entering the mouth are continuously sampled by the epithelium of the tonsil. Underneath the epithelium is a bed of lymphoid tissue including large numbers of naive lymphocytes.18Brandtzaeg P. Farstad I.N. Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track.Immunol Today. 1999; 20: 267-277Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19Perry M. Whyte A. Immunology of the tonsils.Immunol Today. 1998; 19: 414-421Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar If antigen is recognized by the antibody on the surface of the naive B cell, it will bind and cause the B cell to become an activated blast and migrate into the follicle to form a germinal center (Fig 2).20Liu Y.J. Arpin C. Germinal center development.Immunol Rev. 1997; 156: 111-126Crossref PubMed Scopus (309) Google Scholar Here the cell undergoes rounds of rapid proliferation associated with isotype switching and mutation of the immunoglobulin genes, followed by competitive selection for those with the antibody that binds the antigen best. Those who lose in the competition to bind antigen die by apoptosis. Ultimately, the surviving cells leave the germinal center as memory cells primed to make a rapid response to rechallenge with the antigen. This process requires, in addition to the antigen, a signal to the B cell from an antigen-specific T helper cell. EBV also transits the epithelium and infects naive B cells21Joseph A.M. Babcock G.J. Thorley-Lawson D.A. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils.J Virol. 2000; 74: 9964-9971Crossref PubMed Scopus (96) Google Scholar in the underlying tissue, where it expresses a set of latent genes that cause the cell to become activated and proliferate as though it were responding to antigen. This EBV transcription program (the growth program, Fig 2) involves 9 latent proteins, including nuclear antigens (EBV nuclear antigens [EBNAs]) and membrane proteins (latent membrane proteins [LMPs]).2Kieff E. Rickinson A.B. Epstein-Barr virus and its replication.in: Knipe D.M. Howley P.M. Virology. 4th ed. Lippincott Williams and Wilkins, New York2001: 2511-2574Google Scholar These proteins have all the necessary activities to push the B cell to become an activated blast without any necessity for external signaling. This cell migrates to the follicle, where the viral transcription program changes,22Babcock G.J. Hochberg D. Thorley-Lawson A.D. The expression pattern of Epstein-Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell.Immunity. 2000; 13: 497-506Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar such that only 3 of the latent proteins are expressed: EBNA1 (required to replicate the viral DNA) and 2 membrane proteins, LMP1 and LMP2 (the default program, Fig 2). The functions of LMP1 and LMP2 have evolved to steer the latently infected B cell through the germinal center environment. LMP2 alone will push B cells to form a germinal center in the mucosal follicle23Casola S. Otipoby K.L. Alimzhanov M. Humme S. Uyttersprot N. Kutok J.L. et al.B cell receptor signal strength determines B cell fate.Nat Immunol. 2004; 5: 317-327Crossref PubMed Scopus (471) Google Scholar; LMP1 and LMP2 can drive immunoglobulin gene mutation23Casola S. Otipoby K.L. Alimzhanov M. Humme S. Uyttersprot N. Kutok J.L. et al.B cell receptor signal strength determines B cell fate.Nat Immunol. 2004; 5: 317-327Crossref PubMed Scopus (471) Google Scholar and isotype switching24He B. Raab-Traub N. Casali P. Cerutti A. EBV-encoded latent membrane protein 1 cooperates with BAFF/BLyS and APRIL to induce T cell-independent Ig heavy chain class switching.J Immunol. 2003; 171: 5215-5224PubMed Google Scholar (the defining markers of the germinal center), respectively, and LMP1 downregulates expression of the germinal center regulatory transcription factor bcl-6,25Panagopoulos D. Victoratos P. Alexiou M. Kollias G. Mosialos G. Comparative analysis of signal transduction by CD40 and the Epstein-Barr virus oncoprotein LMP1 in vivo.J Virol. 2004; 78: 13253-13261Crossref PubMed Scopus (50) Google Scholar the signal for a memory cell to exit the germinal center.26Calame K.L. Lin K.I. Tunyaplin C. Regulatory mechanisms that determine the development and function of plasma cells.Annu Rev Immunol. 2003; 21: 205-230Crossref PubMed Scopus (294) Google Scholar This implies coordinated expression of LMP1 and LMP2, where LMP2 is turned on before and LMP1 is turned on during the germinal center reaction. Thus constitutive expression of LMP1 in the absence of LMP2 blocks germinal center formation because the cells can never turn on bcl-6, an essential step in germinal center formation.27Uchida J. Yasui T. Takaoka-Shichijo Y. Muraoka M. Kulwichit W. Raab-Traub N. et al.Mimicry of CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte responses.Science. 1999; 286: 300-303Crossref PubMed Scopus (341) Google Scholar This explains why EBV has the ability to make cells proliferate, despite the fact that this puts the host at risk for neoplastic disease. Essentially it has to because this is the mechanism, activation followed by differentiation, by which a normal B cell enters the B-cell memory pool. Once in the periphery, the latently infected cells shut down all viral protein expression (the latency program) and appear to be maintained as normal memory B cells.17Hochberg D. Middeldorp J.M. Catalina M. Sullivan J.L. Luzuriaga K. Thorley-Lawson D.A. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.Proc Natl Acad Sci U S A. 2003; 101: 239-244Crossref PubMed Scopus (227) Google Scholar In the early stages of acute infectious mononucleosis (IM; primary EBV infection in the adult), the number of such cells in the blood can reach staggering proportions, with 50% or more of all memory cells being infected.28Hochberg D. Souza T. Catalina M. Sullivan J.L. Luzuriaga K. Thorley-Lawson D.A. et al.Acute infection with Epstein-Bar virus targets and overwhelms the peripheral memory B cell compartment with resting, latently infected cells.J Virol. 2004; 78: 5194-5204Crossref PubMed Scopus (138) Google Scholar However, the numbers decrease rapidly (half-life of 7 days; Hadinoto and Thorley-Lawson, unpublished data) for the first 2 months and then more steadily after that, until by 1 year there are typically only about 1 in 105 to 106 infected memory B cells. After this time, the level of infected cells appears to be relatively stable over many years.11Khan G. Miyashita E.M. Yang B. Babcock G.J. Thorley-Lawson D.A. Is EBV persistence in vivo a model for B cell homeostasis?.Immunity. 1996; 5: 173-179Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar This presumably represents a balance between the replenishment of latently infected memory cells through cell division17Hochberg D. Middeldorp J.M. Catalina M. Sullivan J.L. Luzuriaga K. Thorley-Lawson D.A. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.Proc Natl Acad Sci U S A. 2003; 101: 239-244Crossref PubMed Scopus (227) Google Scholar and their loss through viral replication (see below). This cell division must be regulated as part of normal memory B-cell homeostasis because there are no viral proteins expressed that could cause the cell to divide. When they divide, they express EBNA1 (the EBNA1-only program, Fig 2),17Hochberg D. Middeldorp J.M. Catalina M. Sullivan J.L. Luzuriaga K. Thorley-Lawson D.A. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.Proc Natl Acad Sci U S A. 2003; 101: 239-244Crossref PubMed Scopus (227) Google Scholar which is needed to allow the viral DNA to replicate with the cells.29Yates J.L. Warren N. Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.Nature. 1985; 313: 812-815Crossref PubMed Scopus (1054) Google Scholar Perhaps not surprisingly, because EBNA1 represents the only point of immune attack of the memory cells, EBNA1 has evolved to be poorly recognized by the immune system.30Levitskaya J. Coram M. Levitsky V. Imreh S. Steigerwald M.P. Klein G. et al.Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1.Nature. 1995; 375: 685-688Crossref PubMed Scopus (681) Google Scholar By gaining entrance to normal memory B cells and shutting down viral protein expression, the virus is safe from immune surveillance. It is also benign because none of the latent proteins that drive growth are expressed. This explains why EBV is able to persist benignly in the vast majority of human subjects: EBV infection in vivo does not drive limitless proliferation. Rather it drives transient proliferation so that the cells can become resting memory cells. The virus persists in nonpathogenic resting cells not proliferating blasts. This also explains why EBV-associated tumors do not arise in every infected individual, even when they are immunosuppressed; something must go wrong with the normal biology that takes the latently infected cells into a resting state before EBV could be involved in tumor development. By accessing the memory compartment, EBV has a site for long-term persistence. However, it must replicate and be shed to spread to new hosts. The parallels between normal B-cell biology and the mechanism of viral shedding are shown in Fig 3. Signals that cause the B cell to differentiate into an antibody-secreting plasma cell will in turn reactivate the virus.31Laichalk L.L. Thorley-Lawson D.A. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo.J Virol. 2005; 79: 1296-1307Crossref PubMed Scopus (173) Google Scholar Because antibody-secreting plasma cells migrate into the mucosal epithelium,18Brandtzaeg P. Farstad I.N. Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track.Immunol Today. 1999; 20: 267-277Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 32Brandtzaeg P. Baekkevold E.S. Farstad I.N. Jahnsen F.L. Johansen F.E. Nilsen E.M. et al.Regional specialization in the mucosal immune system: what happens in the microcompartments?.Immunol Today. 1999; 20: 141-151Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar such a cell will be perfectly placed to release virus onto the mucosal surface, which, in the case of the tonsils, is saliva. Thus infectious virus is spread through saliva contact.33Golden H.D. Chang R.S. Prescott W. Simpson E. Cooper T.Y. Leukocyte-transforming agent: prolonged excretion by patients with mononucleosis and excretion by normal individuals.J Infect Dis. 1973; 127: 471-473Crossref PubMed Scopus (54) Google Scholar Although EBV is considered to be a B-lymphotropic virus, it can also infect epithelial cells because it is found in several important diseases of epithelial cells, including nasopharyngeal34Andersson-Anvret M. Forsby N. Klein G. Henle W. Relationship between the Epstein-Barr virus and undifferentiated nasopharyngeal carcinoma: correlated nucleic acid hybridization and histopathological examination.Int J Cancer. 1977; 20: 486-494Crossref PubMed Scopus (141) Google Scholar and gastric35Shibata D. Weiss L.M. Epstein-Barr virus-associated gastric adenocarcinoma.Am J Pathol. 1992; 140: 769-774PubMed Google Scholar carcinomas and oral hairy leukoplakia.36Greenspan J.S. Greenspan D. Lennette E.T. Abrams D.I. Conant M.A. Petersen V. et al.Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion.N Engl J Med. 1985; 313: 1564-1571Crossref PubMed Scopus (641) Google Scholar What is less clear is whether epithelial cells play a role in the normal biology of EBV. Early reports that claimed to find EBV in healthy nasopharyngeal epithelium have been discredited37Karajannis M.A. Hummel M. Anagnostopoulos I. Stein H. Strict lymphotropism of Epstein-Barr virus during acute infectious mononucleosis in nonimmunocompromised individuals.Blood. 1997; 89: 2856-2862PubMed Google Scholar; however, recent work has revisited this possibility. There is now evidence that normal epithelial cells in the nasopharynx express a distinct EBV receptor,38Tugizov S.M. Berline J.W. Palefsky J.M. Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells.Nat Med. 2003; 9: 307-314Crossref PubMed Scopus (218) Google Scholar that they can be infected in vitro, and that they are infected in vivo.39Pegtel D.M. Middeldorp J. Thorley-Lawson D.A. Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers.J Virol. 2004; 78: 12613-12624Crossref PubMed Scopus (94) Google Scholar However, it remains undetermined whether this infection occurs fortuitously because this epithelium is an area in which EBV happens to replicate or because it is an important component of the viral biology. The most likely role for epithelial cells is as a site for replication and amplification of the virus rather than as a site of persistent latent infection.40Gratama J.W. Oosterveer M.A. Zwaan F.E. Lepoutre J. Klein G. Ernberg I. Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implications for sites of viral latency.Proc Natl Acad Sci U S A. 1988; 85: 8693-8696Crossref PubMed Scopus (201) Google Scholar, 41Faulkner G.C. Burrows S.R. Khanna R. Moss D.J. Bird A.G. Crawford D.H. X-Linked agammaglobulinemia patients are not infected with Epstein-Barr virus: implications for the biology of the virus.J Virol. 1999; 73: 1555-1564PubMed Google Scholar Because the receptor is only expressed on the basolateral surface of epithelial cells, the virus can only infect from the lymphoid tissue and not from saliva. Thus if epithelial cells play an amplification role, it is during viral shedding and not primary infection. Perhaps the most compelling indirect evidence for epithelial cell infection comes from simple numbers. Estimates of the number of lymphocytes replicating EBV in the tonsils42Laichalk L.L. Thorley-Lawson D.A. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo.J Virol. 2005; 79: 1296-1307Crossref PubMed Scopus (395) Google Scholar indicates that there are not nearly enough to account for the rates of viral shedding found in saliva (Hadinoto and Thorley-Lawson, unpublished data). This suggests that there must be a location-mechanism for amplifying the virus shed from plasma cells. The obvious candidates are epithelial cells because, from studies on oral leukoplakia, we know that epithelial cells replicate EBV to high copy numbers. There are important unresolved questions relating to EBV persistence in memory B cells. First, what is the relative contribution of reinfection versus homeostatic cell division to the maintenance of stable levels of latently infected cells? We know that the host mounts a massive cytotoxic T-cell response against cells replicating EBV and newly infected cells43Khanna R. Moss D.J. Burrows S.R. Vaccine strategies against Epstein-Barr virus-associated diseases: lessons from studies on cytotoxic T-cell-mediated immune regulation.Immunol Rev. 1999; 170: 49-64Crossref PubMed Scopus (92) Google Scholar and a neutralizing antibody response against the virus.4Henle W. Henle G. Seroepidemiology of the virus.in: Epstein M.A. Achong B.G. The Epstein-Barr virus. Springer-Verlag, Berlin1979: 61-78Crossref Google Scholar It is therefore unclear whether newly infected cells are produced rapidly enough and survive long enough to contribute to the pool of latently infected memory cells once the immune response has begun. It is conceivable that new infection is only critical in establishing the pool of latently infected memory cells before the onset of the immune response and thereafter plays no role. A clue that this might be true comes from the observation that the epitopes recognized by cytotoxic T cells on newly infected B cells are conserved.44Khanna R. Slade R.W
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