Persistence of the Epstein–Barr Virus and the Origins of Associated Lymphomas
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《新英格兰医药杂志》
Epstein–Barr virus (EBV) is perhaps best known for its ability to immortalize human B lymphocytes in culture.1 This property makes it a candidate for causing human disease, particularly cancer and autoimmune disease.2,3 Recent work, however, has shown that EBV has evolved strategies that reduce its potential to become pathogenic.4,5,6 These new findings have encouraged a reassessment of how and when EBV may cause human disease. In this article, we review current knowledge of the ways in which EBV establishes and maintains a persistent infection at the same time that it minimizes its pathogenicity; we also discuss how these characteristics influence the understanding of the role of EBV in lymphomagenesis.
EBV Infection
In Vitro and in Vivo Infection
In vitro, EBV promiscuously infects resting B cells and almost always transforms them into proliferating blasts. The result is unregulated polyclonal expansion of latently infected lymphoblasts.7,8 The mechanism of this remarkable effect depends on the expression of nine viral latent proteins that are under the control of a master transcription factor, EBV nuclear antigen 2 (EBNA-2).1 The pattern of viral gene expression that drives this process is called the growth program5 (Table 1). In vitro events are very different from what occurs in the blood of healthy carriers of the virus.3,5 In healthy carriers, the B cells are also latently infected with EBV, but because these cells are all memory cells9 that are in a resting state,10 they express no viral proteins.11 Cells that express the growth program are found only in the lymph nodes.12,13 This restriction of the virus in the blood to resting memory B cells is maintained even in immunosuppressed patients, in whom the number of virus-infected cells, on average, is 50 times as high as in immunocompetent patients.14
Table 1. Five Transcription Programs Used by EBV to Establish and Maintain Infection.
Primary EBV infection in vivo generally occurs at an early age15 and is usually asymptomatic.16 However, if the infection is acquired during adolescence or later, it can result in infectious mononucleosis.17 During the early stages of infectious mononucleosis, extremely large numbers of EBV-infected B cells circulate in the blood, but they are all resting memory cells (up to 50 percent of such cells may be infected).18 They are not proliferating blasts and do not enter the growth program.11
Therefore, even under the extreme conditions of infectious mononucleosis or suppression of the immune response, the proliferating-lymphoblastoid stage of viral infection does not occur in the blood, and the infected cells remain in a nonpathogenic resting state. Why, then, has EBV developed the capacity to transform B cells by means of the growth program when such cells pose a potential risk to the host? What is the relation of these transformed B cells to the latently infected, resting memory cells in the peripheral blood? Central to the discussion in this article is the idea that EBV uses a strategy of transforming latently infected B cells into proliferating blasts because only in this way can the virus convert these cells into long-lived memory cells and thereby make the cells nonpathogenic.
The Persistence of EBV
The current model of persistent EBV infection holds that the growth program of the virus activates B cells to become proliferating blasts so that they can then differentiate into resting memory B cells through the process of the germinal-center reaction (Figure 1).20,21 It is in the germinal center that an activated naive B-cell blast that is responding to a foreign antigen during an immune response undergoes the transition into a long-lived memory B cell.20,22 The model stipulates that the difference between the immune-activated B-cell blast and the virus-infected blast that expresses the growth program is that viral genes, not antigens, provide all or some of the signals required to effect the transition to a memory B cell.
Figure 1. How EBV May Establish and Maintain Persistent Infection in Memory B Cells.
EBV infects naive B cells that are in the resting state in the lymphoid tissue of Waldeyer's ring and uses the growth program to activate these cells to become proliferating blasts. This process parallels the activation of a naive B cell on exposure to an antigen. The antigen-activated B-cell blast is rescued through entry into the pool of memory B cells when it receives signals from antigen and antigen-specific helper T cells. The virus switches from the growth program to the default program in order to deliver these rescue signals to the latently infected blast. Then the memory cells exit the cell cycle and enter the peripheral circulation. For infected cells, entry into the peripheral circulation results in the shutdown of all protein-encoding genes — known as the latency program. Memory B cells occasionally divide, as part of the homeostatic mechanism for maintaining stable numbers of cells. When a cell that is carrying the virus undergoes division, it expresses EBV nuclear antigen 1 (EBNA-1) alone to allow the viral genome to divide along with the cell. In response to unknown signals (perhaps bystander T-cell help19), memory cells may differentiate into plasma cells and secrete antibody. This differentiation may be related to the mechanisms that sustain lifetime production of antibody. If such a cell contains the virus, it will reactivate viral replication and infectious virus will be produced. GC denotes germinal center.
Consistent with this model are the findings that lymphoblasts produced by the growth program resemble antigen-activated B cells both in their cell-surface phenotype23,24 and in their morphologic features25 and that the only type of EBV-infected B cell that expresses the growth program in the Waldeyer's ring in healthy carriers is the naive B cell.12,13 The virus is thought to push infected naive B cells into the memory state by switching the cell from the growth program to another pattern of transcription called the default program (Table 1).5,26 The default program involves the expression of only three latent proteins, two of which, latent membrane protein 1 (LMP-1) and LMP-2, are able to produce the signals of the germinal center27,28 that cause latently infected B-cell blasts to form germinal centers29 and make the transition into the memory compartment (Figure 1).
The latently infected memory cells that have been produced shut down the expression of viral proteins, enter the latency program11 (Table 1), and circulate primarily between the peripheral blood and Waldeyer's ring.30 An exception occurs when the latently infected memory cells divide, in which case they express the EBNA-1 protein (Table 1),11 thereby allowing viral DNA to replicate.31 In this case, cell division is not driven by the virus, because none of the growth-promoting latent proteins are present, but is instead regulated by the cell as part of the normal mechanism of memory B-cell homeostasis.
Ultimately, the latent virus in memory B cells is reactivated and replicates at a site that allows it to spread to new hosts. Recent studies have shown that infectious virus is produced when memory cells in Waldeyer's ring differentiate into plasma cells (Figure 1 and unpublished data). This event closes the cycle of viral infection and persistence (Figure 2) and underscores how extensively EBV makes use of the biology of normal B cells.
Figure 2. The Cycle of Persistent EBV Infection.
Every element of the cycle of infection is susceptible to attack by the host's immune system, with the exception of resting memory B cells, in which the virus is quiescent. Cytotoxic T cells recognize all other types of infected cells, and antibody neutralizes the virus. Purple lines with bars denote blocking.
In summary, EBV uses its growth program to activate newly infected B cells so that they can differentiate into resting memory B cells. In memory cells the virus finds a perfect niche. It can persist in them for long periods, because memory B cells rarely die, and it is safe in these cells because they express no viral proteins that can be detected by the immune system. Moreover, in cells that are in the resting state, the virus poses no threat to its host, because the growth-promoting genes are no longer expressed.
Resolution of the Infection
Infection by EBV is controlled by both cellular and humoral immune mechanisms (Figure 2). Antibody limits the spread of infectious virus,32 and cytotoxic T cells destroy infected cells that express viral proteins.33 The cellular response can be extremely vigorous; in infectious mononucleosis, up to 50 percent of all CD8-positive cells in the blood are cytotoxic T cells that are directed against cells in which EBV is replicating.34 The cytotoxic T cells are the main component of the classic lymphocytosis (atypical lymphocytes) in infectious mononucleosis.35 It is likely that the major targets for control of EBV by the immune system are memory cells that have initiated viral replication. By killing these cells and preventing the spread of infectious virus by antibody, the immune response reduces the level of infection (Figure 2). However, the immune system is unable to eliminate the virus completely, and as a consequence, viral shedding and virus-infected cells persist at a low level, approximately 1 in 10,000 to 100,000 memory B cells.30
Minimizing the Pathogenic Effect of EBV
A central tenet of the ideas discussed above is that EBV uses the strategy of activating latently infected B cells to become proliferating blasts because this is the only way the virus can convert these cells into long-lived memory cells. This strategy has important implications for the pathogenesis of EBV-associated diseases. On the one hand, activation of the newly infected cell is dangerous to both the host and the virus, because it risks the development of a potentially fatal neoplastic disease that could limit the period of time in which the virus can spread to new hosts. On the other hand, the virus has the means to ensure that the proliferating lymphoblasts are short-lived. In the case of newly infected naive B cells in Waldeyer's ring, EBV rapidly pushes the cells out of the cell cycle and into a resting memory state.
A risk to the host arises if EBV infects a B cell under conditions in which the infected cell cannot differentiate out of the cell cycle or if memory cells are accidentally triggered into expressing the growth program (as is the case with bystander B cells, Figure 3). Both cases could lead to uncontrolled growth and the development of a tumor. Ordinarily, these possibilities are prevented, because EBV has the apparently paradoxical property of conserving the viral targets that the cytotoxic T cells recognize on infected lymphoblasts.6 The conservation of these antigens by EBV is very different from the continuous mutations that allow influenza virus and the human immunodeficiency virus to avoid recognition by the immune system.36,37 Consequently, EBV in a proliferating lymphoblast is a sure target for the immune response, and conservation of the targets guarantees that lymphoblastoid cells that express the growth program but have not differentiated or cannot differentiate out of the cell cycle will be destroyed.
Figure 3. Putative Checkpoints in the EBV Life Cycle That Might Give Rise to Lymphoma.
EBV normally infects naive B cells in Waldeyer's ring, which differentiate into memory B cells, exit the cell cycle (thick red arrows), and are therefore not pathogenic. Hodgkin's disease arises from a virus-infected cell that is blocked at the germinal-center stage, which results in constitutive expression of the default program. Burkitt's lymphoma arises from a germinal-center cell that is entering the memory compartment but is stuck at the point of proliferation owing to the activated c-myc oncogene. Consequently, the cell expresses EBNA-1 only. In both Hodgkin's disease and Burkitt's lymphoma, the critical event may be a cellular mutation during the immunologic disturbance associated with acute EBV infection. Because the number of infected cells is so high at this point, there is a reasonable possibility that the cell undergoing mutation will have the virus in it by chance. Any cell other than the naive B cell in Waldeyer's ring that becomes infected (thin red arrows) and expresses the growth program will continue to proliferate, because it cannot differentiate out of the cell cycle (thin dashed purple arrows). The rarity of such an event highlights the extent to which EBV infection is carefully controlled. Normally, bystander B-cell blasts would be destroyed by cytotoxic T cells (CTL, blue arrow), but if the CTL response is suppressed, the blasts can lead to post-transplantation lymphoproliferative disease (PTLD).
Other Interpretations
The ideas discussed here are based on a large body of evidence that, in vivo, EBV uses different transcription programs in different types of B cells (the growth program in lymphoblasts, the default program in germinal-center cells, and the latency program in memory B cells). The relation between such cells has been inferred from parallels with the biology of normal B cells but has yet to be demonstrated experimentally. Furthermore, the model may be oversimplified in several respects. For example, it implies that viral genes completely replace antigen signaling during the germinal-center reaction. However, the relative contributions of antigen and viral genes have not been established. Similarly, cells with a germinal-center phenotype that express the default program have been described, but it is not known where they reside and whether they expand to form a true germinal center.
An alternative explanation for the persistence of EBV infection has been suggested by Kurth et al.,38,39 who have proposed that EBV-infected cells do not participate in the germinal-center reaction but, rather, that EBV directly infects memory B cells. This idea is based on studies in which EBV-infected cells undergoing clonal expansion were identified in the germinal centers of tonsils from patients with infectious mononucleosis. The cells expressed EBNA-2, which is characteristic of the growth program. Although this idea is attractive because of its simplicity, it provides no mechanism to explain how the proliferating memory cells exit the cell cycle, what the role of the default program may be, and why latently infected germinal-center and memory cells from the tonsils of healthy carriers do not express EBNA-2.12 They always express the default program.
In fact, the observations of Kurth et al.38,39 are consistent with and predicted by the model shown in Figure 1 and Figure 2, which holds that latently infected naive B cells rapidly differentiate out of the cell cycle by means of the default program to become resting memory cells. Owing to the high level of viremia and the disruption of lymphoid tissue in infectious mononucleosis, germinal-center or memory B cells may incidentally become directly infected. These bystander infected cells (Figure 3) will express the growth program (that is, they will be EBNA-2–positive), not the default program.12 They will then expand rapidly because they cannot differentiate out of the cell cycle and will therefore be the dominant population of infected cells in the tonsils of patients with infectious mononucleosis. Expanding populations of such infected cells are exactly what Kurth et al. observed.38,39 Eventually, the cytotoxic T-cell response destroys these cells, leaving behind only the small number of infected germinal-center cells that express the default program — as is seen in healthy carriers of the virus.
It has also been suggested that EBV-driven differentiation occurs in the extrafollicular regions of the tonsil, rather than in the germinal centers. This idea is not inconsistent with the model and was based on studies of transgenic mice in which expression of LMP-1 from a constitutive promoter resulted in lymphoma and blocked germinal-center development.40 However, LMP-1 is not expressed from a constitutive promoter in the virus,41,42 it is not oncogenic in healthy carriers, and in germinal-center cells it is not expressed alone but, rather, expressed in the presence of LMP-2,12 which, from experiments in transgenic mice, is known to cause gut mucosal B cells to undergo germinal-center development.29 For these reasons, the phenotype of these mice is difficult to interpret, and it can be concluded only that constitutive expression of LMP-1 by itself blocks germinal-center development.
EBV and Epithelium
Although the focus of this review is on the infection of B lymphocytes, it is worth noting that there is increasing evidence to suggest that the epithelium of Waldeyer's ring also has a role in both primary infection and viral shedding.43,44 Although the participation of epithelium remains to be clearly established, it might have major implications for understanding the role of EBV in carcinoma of the mucosal epithelium (nasopharyngeal and gastric carcinoma2,3).
EBV and Disease
EBV has been associated with a number of diseases (Table 2), particularly autoimmune disease and cancer.2,3,45,46 Demonstration of a causative role of EBV in autoimmune disease has been difficult, however, because worldwide, more than 90 percent of people are infected with EBV by the time they are adults,15 most of them in early childhood, and EBV persists in them for the rest of their lives. In order to associate EBV with a particular disease, it must be explained why the virus causes disease in only a few persons, when almost everyone is infected with it. Furthermore, because the virus is carried in the blood by infected memory B cells,9,47 sensitive tests will detect it in any inflamed tissue, regardless of the role of the virus in causing the inflammation. The model of EBV infection discussed here adds a further complication in that EBV is exquisitely sensitive to changes in the immune system. Changes in the viral burden or atypical behavior of the virus may be an indirect effect of a compromised immune system that results from the autoimmune disease, rather than evidence that the virus has a role in the disease.
Table 2. Diseases in Which EBV May Have a Causative Role.
The evidence of an association between EBV and cancer is stronger than is the case for autoimmune disease, and the ability of the virus to drive cellular proliferation identifies it as a potential carcinogen.
Lymphoma in Immunosuppressed Patients
Immunosuppressed patients are at risk for B-cell lymphoproliferative diseases, such as post-transplantation lymphoproliferative disease. These diseases are a heterogeneous collection of disorders48 that usually carry the virus and express the growth program.49 The obvious explanation for post-transplantation lymphoproliferative disease is that an impaired cytotoxic T-cell response permits uninhibited growth of EBV-infected cells, but the situation is not so simple. Two events must occur for lymphoblastoid cells that express the growth program to survive and evolve into a lymphoma. First, the EBV-infected cell must be unable to exit the cell cycle and become a resting memory B cell. Second, the cytotoxic T-cell response must be impeded so that the lymphoblasts are not killed.
Post-transplantation lymphoproliferative disease may be initiated when a type of B cell other than a naive B cell in Waldeyer's ring becomes infected and expresses the growth program (as in the case of bystander cells, Figure 3). These cells cannot exit the growth program, and they continue to proliferate owing to the absence of effective T-cell immunity. This mechanism may explain the heterogeneity of the disease.50 Even in immunosuppressed patients, this event must be rare, because only one or two of the millions of EBV-infected cells in the human body develop into tumors. That every infected B cell does not simply proliferate out of control further attests to the tight regulation of EBV-driven proliferation in vivo, even in the presence of a crippled immune response.
Hodgkin's Disease
The first recognition of an association between EBV and Hodgkin's disease came from the observation that infectious mononucleosis is a risk factor for this form of lymphoma.15 Subsequently, Reed–Sternberg cells were found in some cases of infectious mononucleosis,51 and approximately 40 percent of Hodgkin's tumors were shown to contain clonal EBV (up to 80 percent in developing countries).52,53 Although there are no obvious differences to indicate that EBV-positive and EBV-negative Hodgkin's disease are distinct entities, there is evidence that infectious mononucleosis is a risk factor only for EBV-positive Hodgkin's disease.54
EBV-positive tumors express the viral genes EBNA-1, LMP-1, and LMP-255,56,57,58 of the default transcription program, which is the program that is used by latently infected germinal-center B cells. The immunoglobulin genes of Reed–Sternberg cells are hypermutated to the same extent as germinal-center B cells.59 Thus, the immunoglobulin mutations and the data on viral gene expression independently support the idea that Hodgkin's disease arises from an EBV-infected germinal-center B cell.
The presence of EBV in approximately 40 percent of tumors would seem to rule out a chance association of the virus with Hodgkin's disease, but this view does not take into account the fact that the number of EBV-infected cells is extremely high in infectious mononucleosis (up to 50 percent of all memory B cells are infected18). Therefore, if the immunologic disruption of infectious mononucleosis, rather than EBV itself, is the risk factor for Hodgkin's disease, there is a high probability that the premalignant germinal-center cell will have EBV in it by chance.
A reasonable scenario for the association of EBV with Hodgkin's disease is that a germinal-center B cell acquires a mutation during infectious mononucleosis that blocks its differentiation.60 If that cell happens also to contain EBV, it will become a germinal-center cell that constitutively expresses LMP-1 and LMP-2 (Figure 3 and Table 3). The virus may then be carried as a chance passenger, or, more likely, the constitutive expression of LMP-1 and LMP-2 will provide growth and survival signals that enhance tumor growth.
Table 3. Putative Infected Cell of Origin and Viral Role for the Three EBV-Associated Lymphomas.
Burkitt's Lymphoma
EBV was discovered 40 years ago in tumor cells from patients who had Burkitt's lymphoma,61 yet its contribution to the development of this tumor remains enigmatic. The consistent genetic lesion in Burkitt's lymphoma is deregulated activation of the c-myc oncogene owing to reciprocal translocation with an immunoglobulin gene.62,63,64 Burkitt's lymphoma has the same pattern of immunoglobulin gene hypermutation as germinal-center and memory B cells,65 but it has the cellular phenotype of a germinal-center cell.66
The most compelling evidence of the involvement of EBV in Burkitt's lymphoma is the high frequency of tumors that carry the virus67 in endemic areas (98 percent) and the presence of clonal EBV in all the tumor cells.68 There is no satisfactory explanation of how EBV participates in the pathogenesis of Burkitt's lymphoma.69,70,71 None of the growth-promoting latent genes are expressed, and the only latent protein of the virus present is EBNA-1.72 According to current knowledge, an EBNA-1–only phenotype is present in nontumor cells only when a latently infected memory cell that expresses the latency program divides.11 This mechanism raises the possibility that Burkitt's lymphoma arises if a translocation in the c-myc gene occurs in an EBV-infected germinal-center cell that is on its way to becoming a memory cell. This cell would normally express the latency program, but owing to the activated c-myc, it is instead stuck in the proliferating mode and therefore constitutively expresses only EBNA-1 (Figure 3 and Table 3). The maintenance of the germinal-center phenotype in this presumptive memory-cell tumor can be explained by the observation that an activated c-myc gene will push an EBV-infected cell toward the germinal-center phenotype if the genes that promote viral growth are not expressed.73 This explanation implies that the virus is present in the tumor cells solely by chance, as a passenger. It is difficult, however, to decipher the origin of tumors on the basis of the phenotype of the end-stage tumor. Given that tumorigenesis is a multistep process that occurs over long periods of time, it is virtually impossible to know how directly the final cellular or viral phenotype of Burkitt's lymphoma relates to the original infected precursor.
Conclusions
We are beginning to develop a comprehensive understanding of how EBV persists in vivo, and this knowledge may provide insights into the origin of EBV-associated diseases. However, the virus has evolved strategies to minimize or eliminate its pathogenic potential, in the interest of maintaining infection and the survival of the host in which it persists. Therefore, causal relationships between the virus and disease should be interpreted with care.
Source Information
From the Department of Pathology, Tufts University School of Medicine (D.A.T.-L.), and the Department of Rheumatology, Tufts–New England Medical Center (A.G.) — both in Boston.
Address reprint requests to Dr. Thorley-Lawson at the Department of Pathology, Jaharis Bldg., Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111, or at david.thorley-lawson@tufts.edu.
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Deacon EM, Pallesen G, Niedobitek G, et al. Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells. J Exp Med 1993;177:339-349.
Herbst H, Dallenbach F, Hummel M, et al. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc Natl Acad Sci U S A 1991;88:4766-4770.
Niedobitek G, Kremmer E, Herbst H, et al. Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis. Blood 1997;90:1664-1672.
Kuppers R, Rajewsky K. The origin of Hodgkin and Reed/Sternberg cells in Hodgkin's disease. Annu Rev Immunol 1998;16:471-493.
Staudt LM. The molecular and cellular origins of Hodgkin's disease. J Exp Med 2000;191:207-212.
Epstein MA, Achong BG, Barr YM. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 1964;1:702-703.
Klein G. Specific chromosomal translocations and the genesis of B-cell-derived tumors in mice and men. Cell 1983;32:311-315.
Leder P. Translocations among antibody genes in human cancer. In: Lenoir GM, O'Conor GT, Olweny CLM, eds. Burkitt's lymphoma: a human cancer model. Lyons, France: International Agency for Research on Cancer, 1985:341-71. (IARC publications no. 60.)
Manolov G, Manolova Y. Marker band in one chromosome 14 from Burkitt lymphomas. Nature 1972;237:33-34.
Klein U, Klein G, Ehlin-Henriksson B, Rajewsky K, Kuppers R. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes. Mol Med 1995;1:495-505.
Gregory CD, Tursz T, Edwards CF, et al. Identification of a subset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype. J Immunol 1987;139:313-318.
de-Thé G. Epstein-Barr virus and Burkitt's lymphoma worldwide: the causal relationship revisited. In: Lenoir GM, O'Conor GT, Olweny CLM, eds. Burkitt's lymphoma: a human cancer model. Lyons, France: International Agency for Research on Cancer, 1985:165-76. (IARC publications no. 60.)
Gulley ML, Raphael M, Lutz CT, Ross DW, Raab-Traub N. Epstein-Barr virus integration in human lymphomas and lymphoid cell lines. Cancer 1992;70:185-191.
Lenoir GM, Bornkamm GW. Burkitt's lymphoma, a human cancer model for the study of the multistep development of cancer: proposal for a new scenario. In: Klein G, ed. Advances in viral oncology. Vol. 7. New York: Raven Press, 1987:173-206.
Klein G. In defense of the "old" Burkitt lymphoma scenario. In: Klein G, ed. Advances in viral oncology. Vol. 7. New York: Raven Press, 1987:207-11.
Kelly G, Bell A, Rickinson A. Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2. Nat Med 2002;8:1098-1104.
Gregory CD, Rowe M, Rickinson AB. Different Epstein-Barr virus-B cell interactions in phenotypically distinct clones of a Burkitt's lymphoma cell line. J Gen Virol 1990;71:1481-1495.
Polack A, Hortnagel K, Pajic A, et al. c-myc Activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1. Proc Natl Acad Sci U S A 1996;93:10411-10416.
Related Letters:
EBV and Burkitt's Lymphoma
Rossi G., Bonetti F., Thorley-Lawson D. A.(David A. Thorley-Lawson, )
EBV Infection
In Vitro and in Vivo Infection
In vitro, EBV promiscuously infects resting B cells and almost always transforms them into proliferating blasts. The result is unregulated polyclonal expansion of latently infected lymphoblasts.7,8 The mechanism of this remarkable effect depends on the expression of nine viral latent proteins that are under the control of a master transcription factor, EBV nuclear antigen 2 (EBNA-2).1 The pattern of viral gene expression that drives this process is called the growth program5 (Table 1). In vitro events are very different from what occurs in the blood of healthy carriers of the virus.3,5 In healthy carriers, the B cells are also latently infected with EBV, but because these cells are all memory cells9 that are in a resting state,10 they express no viral proteins.11 Cells that express the growth program are found only in the lymph nodes.12,13 This restriction of the virus in the blood to resting memory B cells is maintained even in immunosuppressed patients, in whom the number of virus-infected cells, on average, is 50 times as high as in immunocompetent patients.14
Table 1. Five Transcription Programs Used by EBV to Establish and Maintain Infection.
Primary EBV infection in vivo generally occurs at an early age15 and is usually asymptomatic.16 However, if the infection is acquired during adolescence or later, it can result in infectious mononucleosis.17 During the early stages of infectious mononucleosis, extremely large numbers of EBV-infected B cells circulate in the blood, but they are all resting memory cells (up to 50 percent of such cells may be infected).18 They are not proliferating blasts and do not enter the growth program.11
Therefore, even under the extreme conditions of infectious mononucleosis or suppression of the immune response, the proliferating-lymphoblastoid stage of viral infection does not occur in the blood, and the infected cells remain in a nonpathogenic resting state. Why, then, has EBV developed the capacity to transform B cells by means of the growth program when such cells pose a potential risk to the host? What is the relation of these transformed B cells to the latently infected, resting memory cells in the peripheral blood? Central to the discussion in this article is the idea that EBV uses a strategy of transforming latently infected B cells into proliferating blasts because only in this way can the virus convert these cells into long-lived memory cells and thereby make the cells nonpathogenic.
The Persistence of EBV
The current model of persistent EBV infection holds that the growth program of the virus activates B cells to become proliferating blasts so that they can then differentiate into resting memory B cells through the process of the germinal-center reaction (Figure 1).20,21 It is in the germinal center that an activated naive B-cell blast that is responding to a foreign antigen during an immune response undergoes the transition into a long-lived memory B cell.20,22 The model stipulates that the difference between the immune-activated B-cell blast and the virus-infected blast that expresses the growth program is that viral genes, not antigens, provide all or some of the signals required to effect the transition to a memory B cell.
Figure 1. How EBV May Establish and Maintain Persistent Infection in Memory B Cells.
EBV infects naive B cells that are in the resting state in the lymphoid tissue of Waldeyer's ring and uses the growth program to activate these cells to become proliferating blasts. This process parallels the activation of a naive B cell on exposure to an antigen. The antigen-activated B-cell blast is rescued through entry into the pool of memory B cells when it receives signals from antigen and antigen-specific helper T cells. The virus switches from the growth program to the default program in order to deliver these rescue signals to the latently infected blast. Then the memory cells exit the cell cycle and enter the peripheral circulation. For infected cells, entry into the peripheral circulation results in the shutdown of all protein-encoding genes — known as the latency program. Memory B cells occasionally divide, as part of the homeostatic mechanism for maintaining stable numbers of cells. When a cell that is carrying the virus undergoes division, it expresses EBV nuclear antigen 1 (EBNA-1) alone to allow the viral genome to divide along with the cell. In response to unknown signals (perhaps bystander T-cell help19), memory cells may differentiate into plasma cells and secrete antibody. This differentiation may be related to the mechanisms that sustain lifetime production of antibody. If such a cell contains the virus, it will reactivate viral replication and infectious virus will be produced. GC denotes germinal center.
Consistent with this model are the findings that lymphoblasts produced by the growth program resemble antigen-activated B cells both in their cell-surface phenotype23,24 and in their morphologic features25 and that the only type of EBV-infected B cell that expresses the growth program in the Waldeyer's ring in healthy carriers is the naive B cell.12,13 The virus is thought to push infected naive B cells into the memory state by switching the cell from the growth program to another pattern of transcription called the default program (Table 1).5,26 The default program involves the expression of only three latent proteins, two of which, latent membrane protein 1 (LMP-1) and LMP-2, are able to produce the signals of the germinal center27,28 that cause latently infected B-cell blasts to form germinal centers29 and make the transition into the memory compartment (Figure 1).
The latently infected memory cells that have been produced shut down the expression of viral proteins, enter the latency program11 (Table 1), and circulate primarily between the peripheral blood and Waldeyer's ring.30 An exception occurs when the latently infected memory cells divide, in which case they express the EBNA-1 protein (Table 1),11 thereby allowing viral DNA to replicate.31 In this case, cell division is not driven by the virus, because none of the growth-promoting latent proteins are present, but is instead regulated by the cell as part of the normal mechanism of memory B-cell homeostasis.
Ultimately, the latent virus in memory B cells is reactivated and replicates at a site that allows it to spread to new hosts. Recent studies have shown that infectious virus is produced when memory cells in Waldeyer's ring differentiate into plasma cells (Figure 1 and unpublished data). This event closes the cycle of viral infection and persistence (Figure 2) and underscores how extensively EBV makes use of the biology of normal B cells.
Figure 2. The Cycle of Persistent EBV Infection.
Every element of the cycle of infection is susceptible to attack by the host's immune system, with the exception of resting memory B cells, in which the virus is quiescent. Cytotoxic T cells recognize all other types of infected cells, and antibody neutralizes the virus. Purple lines with bars denote blocking.
In summary, EBV uses its growth program to activate newly infected B cells so that they can differentiate into resting memory B cells. In memory cells the virus finds a perfect niche. It can persist in them for long periods, because memory B cells rarely die, and it is safe in these cells because they express no viral proteins that can be detected by the immune system. Moreover, in cells that are in the resting state, the virus poses no threat to its host, because the growth-promoting genes are no longer expressed.
Resolution of the Infection
Infection by EBV is controlled by both cellular and humoral immune mechanisms (Figure 2). Antibody limits the spread of infectious virus,32 and cytotoxic T cells destroy infected cells that express viral proteins.33 The cellular response can be extremely vigorous; in infectious mononucleosis, up to 50 percent of all CD8-positive cells in the blood are cytotoxic T cells that are directed against cells in which EBV is replicating.34 The cytotoxic T cells are the main component of the classic lymphocytosis (atypical lymphocytes) in infectious mononucleosis.35 It is likely that the major targets for control of EBV by the immune system are memory cells that have initiated viral replication. By killing these cells and preventing the spread of infectious virus by antibody, the immune response reduces the level of infection (Figure 2). However, the immune system is unable to eliminate the virus completely, and as a consequence, viral shedding and virus-infected cells persist at a low level, approximately 1 in 10,000 to 100,000 memory B cells.30
Minimizing the Pathogenic Effect of EBV
A central tenet of the ideas discussed above is that EBV uses the strategy of activating latently infected B cells to become proliferating blasts because this is the only way the virus can convert these cells into long-lived memory cells. This strategy has important implications for the pathogenesis of EBV-associated diseases. On the one hand, activation of the newly infected cell is dangerous to both the host and the virus, because it risks the development of a potentially fatal neoplastic disease that could limit the period of time in which the virus can spread to new hosts. On the other hand, the virus has the means to ensure that the proliferating lymphoblasts are short-lived. In the case of newly infected naive B cells in Waldeyer's ring, EBV rapidly pushes the cells out of the cell cycle and into a resting memory state.
A risk to the host arises if EBV infects a B cell under conditions in which the infected cell cannot differentiate out of the cell cycle or if memory cells are accidentally triggered into expressing the growth program (as is the case with bystander B cells, Figure 3). Both cases could lead to uncontrolled growth and the development of a tumor. Ordinarily, these possibilities are prevented, because EBV has the apparently paradoxical property of conserving the viral targets that the cytotoxic T cells recognize on infected lymphoblasts.6 The conservation of these antigens by EBV is very different from the continuous mutations that allow influenza virus and the human immunodeficiency virus to avoid recognition by the immune system.36,37 Consequently, EBV in a proliferating lymphoblast is a sure target for the immune response, and conservation of the targets guarantees that lymphoblastoid cells that express the growth program but have not differentiated or cannot differentiate out of the cell cycle will be destroyed.
Figure 3. Putative Checkpoints in the EBV Life Cycle That Might Give Rise to Lymphoma.
EBV normally infects naive B cells in Waldeyer's ring, which differentiate into memory B cells, exit the cell cycle (thick red arrows), and are therefore not pathogenic. Hodgkin's disease arises from a virus-infected cell that is blocked at the germinal-center stage, which results in constitutive expression of the default program. Burkitt's lymphoma arises from a germinal-center cell that is entering the memory compartment but is stuck at the point of proliferation owing to the activated c-myc oncogene. Consequently, the cell expresses EBNA-1 only. In both Hodgkin's disease and Burkitt's lymphoma, the critical event may be a cellular mutation during the immunologic disturbance associated with acute EBV infection. Because the number of infected cells is so high at this point, there is a reasonable possibility that the cell undergoing mutation will have the virus in it by chance. Any cell other than the naive B cell in Waldeyer's ring that becomes infected (thin red arrows) and expresses the growth program will continue to proliferate, because it cannot differentiate out of the cell cycle (thin dashed purple arrows). The rarity of such an event highlights the extent to which EBV infection is carefully controlled. Normally, bystander B-cell blasts would be destroyed by cytotoxic T cells (CTL, blue arrow), but if the CTL response is suppressed, the blasts can lead to post-transplantation lymphoproliferative disease (PTLD).
Other Interpretations
The ideas discussed here are based on a large body of evidence that, in vivo, EBV uses different transcription programs in different types of B cells (the growth program in lymphoblasts, the default program in germinal-center cells, and the latency program in memory B cells). The relation between such cells has been inferred from parallels with the biology of normal B cells but has yet to be demonstrated experimentally. Furthermore, the model may be oversimplified in several respects. For example, it implies that viral genes completely replace antigen signaling during the germinal-center reaction. However, the relative contributions of antigen and viral genes have not been established. Similarly, cells with a germinal-center phenotype that express the default program have been described, but it is not known where they reside and whether they expand to form a true germinal center.
An alternative explanation for the persistence of EBV infection has been suggested by Kurth et al.,38,39 who have proposed that EBV-infected cells do not participate in the germinal-center reaction but, rather, that EBV directly infects memory B cells. This idea is based on studies in which EBV-infected cells undergoing clonal expansion were identified in the germinal centers of tonsils from patients with infectious mononucleosis. The cells expressed EBNA-2, which is characteristic of the growth program. Although this idea is attractive because of its simplicity, it provides no mechanism to explain how the proliferating memory cells exit the cell cycle, what the role of the default program may be, and why latently infected germinal-center and memory cells from the tonsils of healthy carriers do not express EBNA-2.12 They always express the default program.
In fact, the observations of Kurth et al.38,39 are consistent with and predicted by the model shown in Figure 1 and Figure 2, which holds that latently infected naive B cells rapidly differentiate out of the cell cycle by means of the default program to become resting memory cells. Owing to the high level of viremia and the disruption of lymphoid tissue in infectious mononucleosis, germinal-center or memory B cells may incidentally become directly infected. These bystander infected cells (Figure 3) will express the growth program (that is, they will be EBNA-2–positive), not the default program.12 They will then expand rapidly because they cannot differentiate out of the cell cycle and will therefore be the dominant population of infected cells in the tonsils of patients with infectious mononucleosis. Expanding populations of such infected cells are exactly what Kurth et al. observed.38,39 Eventually, the cytotoxic T-cell response destroys these cells, leaving behind only the small number of infected germinal-center cells that express the default program — as is seen in healthy carriers of the virus.
It has also been suggested that EBV-driven differentiation occurs in the extrafollicular regions of the tonsil, rather than in the germinal centers. This idea is not inconsistent with the model and was based on studies of transgenic mice in which expression of LMP-1 from a constitutive promoter resulted in lymphoma and blocked germinal-center development.40 However, LMP-1 is not expressed from a constitutive promoter in the virus,41,42 it is not oncogenic in healthy carriers, and in germinal-center cells it is not expressed alone but, rather, expressed in the presence of LMP-2,12 which, from experiments in transgenic mice, is known to cause gut mucosal B cells to undergo germinal-center development.29 For these reasons, the phenotype of these mice is difficult to interpret, and it can be concluded only that constitutive expression of LMP-1 by itself blocks germinal-center development.
EBV and Epithelium
Although the focus of this review is on the infection of B lymphocytes, it is worth noting that there is increasing evidence to suggest that the epithelium of Waldeyer's ring also has a role in both primary infection and viral shedding.43,44 Although the participation of epithelium remains to be clearly established, it might have major implications for understanding the role of EBV in carcinoma of the mucosal epithelium (nasopharyngeal and gastric carcinoma2,3).
EBV and Disease
EBV has been associated with a number of diseases (Table 2), particularly autoimmune disease and cancer.2,3,45,46 Demonstration of a causative role of EBV in autoimmune disease has been difficult, however, because worldwide, more than 90 percent of people are infected with EBV by the time they are adults,15 most of them in early childhood, and EBV persists in them for the rest of their lives. In order to associate EBV with a particular disease, it must be explained why the virus causes disease in only a few persons, when almost everyone is infected with it. Furthermore, because the virus is carried in the blood by infected memory B cells,9,47 sensitive tests will detect it in any inflamed tissue, regardless of the role of the virus in causing the inflammation. The model of EBV infection discussed here adds a further complication in that EBV is exquisitely sensitive to changes in the immune system. Changes in the viral burden or atypical behavior of the virus may be an indirect effect of a compromised immune system that results from the autoimmune disease, rather than evidence that the virus has a role in the disease.
Table 2. Diseases in Which EBV May Have a Causative Role.
The evidence of an association between EBV and cancer is stronger than is the case for autoimmune disease, and the ability of the virus to drive cellular proliferation identifies it as a potential carcinogen.
Lymphoma in Immunosuppressed Patients
Immunosuppressed patients are at risk for B-cell lymphoproliferative diseases, such as post-transplantation lymphoproliferative disease. These diseases are a heterogeneous collection of disorders48 that usually carry the virus and express the growth program.49 The obvious explanation for post-transplantation lymphoproliferative disease is that an impaired cytotoxic T-cell response permits uninhibited growth of EBV-infected cells, but the situation is not so simple. Two events must occur for lymphoblastoid cells that express the growth program to survive and evolve into a lymphoma. First, the EBV-infected cell must be unable to exit the cell cycle and become a resting memory B cell. Second, the cytotoxic T-cell response must be impeded so that the lymphoblasts are not killed.
Post-transplantation lymphoproliferative disease may be initiated when a type of B cell other than a naive B cell in Waldeyer's ring becomes infected and expresses the growth program (as in the case of bystander cells, Figure 3). These cells cannot exit the growth program, and they continue to proliferate owing to the absence of effective T-cell immunity. This mechanism may explain the heterogeneity of the disease.50 Even in immunosuppressed patients, this event must be rare, because only one or two of the millions of EBV-infected cells in the human body develop into tumors. That every infected B cell does not simply proliferate out of control further attests to the tight regulation of EBV-driven proliferation in vivo, even in the presence of a crippled immune response.
Hodgkin's Disease
The first recognition of an association between EBV and Hodgkin's disease came from the observation that infectious mononucleosis is a risk factor for this form of lymphoma.15 Subsequently, Reed–Sternberg cells were found in some cases of infectious mononucleosis,51 and approximately 40 percent of Hodgkin's tumors were shown to contain clonal EBV (up to 80 percent in developing countries).52,53 Although there are no obvious differences to indicate that EBV-positive and EBV-negative Hodgkin's disease are distinct entities, there is evidence that infectious mononucleosis is a risk factor only for EBV-positive Hodgkin's disease.54
EBV-positive tumors express the viral genes EBNA-1, LMP-1, and LMP-255,56,57,58 of the default transcription program, which is the program that is used by latently infected germinal-center B cells. The immunoglobulin genes of Reed–Sternberg cells are hypermutated to the same extent as germinal-center B cells.59 Thus, the immunoglobulin mutations and the data on viral gene expression independently support the idea that Hodgkin's disease arises from an EBV-infected germinal-center B cell.
The presence of EBV in approximately 40 percent of tumors would seem to rule out a chance association of the virus with Hodgkin's disease, but this view does not take into account the fact that the number of EBV-infected cells is extremely high in infectious mononucleosis (up to 50 percent of all memory B cells are infected18). Therefore, if the immunologic disruption of infectious mononucleosis, rather than EBV itself, is the risk factor for Hodgkin's disease, there is a high probability that the premalignant germinal-center cell will have EBV in it by chance.
A reasonable scenario for the association of EBV with Hodgkin's disease is that a germinal-center B cell acquires a mutation during infectious mononucleosis that blocks its differentiation.60 If that cell happens also to contain EBV, it will become a germinal-center cell that constitutively expresses LMP-1 and LMP-2 (Figure 3 and Table 3). The virus may then be carried as a chance passenger, or, more likely, the constitutive expression of LMP-1 and LMP-2 will provide growth and survival signals that enhance tumor growth.
Table 3. Putative Infected Cell of Origin and Viral Role for the Three EBV-Associated Lymphomas.
Burkitt's Lymphoma
EBV was discovered 40 years ago in tumor cells from patients who had Burkitt's lymphoma,61 yet its contribution to the development of this tumor remains enigmatic. The consistent genetic lesion in Burkitt's lymphoma is deregulated activation of the c-myc oncogene owing to reciprocal translocation with an immunoglobulin gene.62,63,64 Burkitt's lymphoma has the same pattern of immunoglobulin gene hypermutation as germinal-center and memory B cells,65 but it has the cellular phenotype of a germinal-center cell.66
The most compelling evidence of the involvement of EBV in Burkitt's lymphoma is the high frequency of tumors that carry the virus67 in endemic areas (98 percent) and the presence of clonal EBV in all the tumor cells.68 There is no satisfactory explanation of how EBV participates in the pathogenesis of Burkitt's lymphoma.69,70,71 None of the growth-promoting latent genes are expressed, and the only latent protein of the virus present is EBNA-1.72 According to current knowledge, an EBNA-1–only phenotype is present in nontumor cells only when a latently infected memory cell that expresses the latency program divides.11 This mechanism raises the possibility that Burkitt's lymphoma arises if a translocation in the c-myc gene occurs in an EBV-infected germinal-center cell that is on its way to becoming a memory cell. This cell would normally express the latency program, but owing to the activated c-myc, it is instead stuck in the proliferating mode and therefore constitutively expresses only EBNA-1 (Figure 3 and Table 3). The maintenance of the germinal-center phenotype in this presumptive memory-cell tumor can be explained by the observation that an activated c-myc gene will push an EBV-infected cell toward the germinal-center phenotype if the genes that promote viral growth are not expressed.73 This explanation implies that the virus is present in the tumor cells solely by chance, as a passenger. It is difficult, however, to decipher the origin of tumors on the basis of the phenotype of the end-stage tumor. Given that tumorigenesis is a multistep process that occurs over long periods of time, it is virtually impossible to know how directly the final cellular or viral phenotype of Burkitt's lymphoma relates to the original infected precursor.
Conclusions
We are beginning to develop a comprehensive understanding of how EBV persists in vivo, and this knowledge may provide insights into the origin of EBV-associated diseases. However, the virus has evolved strategies to minimize or eliminate its pathogenic potential, in the interest of maintaining infection and the survival of the host in which it persists. Therefore, causal relationships between the virus and disease should be interpreted with care.
Source Information
From the Department of Pathology, Tufts University School of Medicine (D.A.T.-L.), and the Department of Rheumatology, Tufts–New England Medical Center (A.G.) — both in Boston.
Address reprint requests to Dr. Thorley-Lawson at the Department of Pathology, Jaharis Bldg., Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111, or at david.thorley-lawson@tufts.edu.
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