SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Epstein–Barr virus (EBV), a ubiquitous human herpes virus, is associated with an increasing number of lymphoid and epithelial malignancies. The ability of the virus to establish life-long persistent infections and induce growth transformation is related to the function of a set of viral proteins that are variously expressed in both normal and malignant cells. Recent evidence indicates that these viral proteins are able to usurp cellular pathways that promote the cell growth and survival, while impairing anti-viral immune responses. Elucidation of the mechanisms by which EBV induces cell transformation and escapes host immune control provides the rational background for the design of new strategies of intervention for EBV-related malignancies. J. Cell. Physiol. 196: 207–218, 2003. © 2003 Wiley-Liss, Inc.

Epstein–Barr virus (EBV) is a ubiquitous human γ-herpesvirus that infects about 95% of the adult population world-wide. The majority of primary infections occurs in early childhood and are generally subclinical. However, when primary infection is delayed until adolescence or adulthood, as often occurs in countries with high socio-economic standards, it may cause infectious mononucleosis (IM), a self-limiting lymphoproliferative disorder characterized by increased numbers of EBV-infected B cells in peripheral blood and massive oligoclonal expansion of EBV-specific CD8+ T cells (reviewed in International Agency for Research on Cancer, 1997). EBV transmission is believed to occur mainly by contact with oropharyngeal secretions that usually contain infectious virus (Yao et al., 1985). These observations, together with the finding of intense EBV replication in the oral “hairy” leukoplakia lesions of HIV-infected patients (Greenspan et al., 1985), suggested that epithelial cells may be primary site of permissive infection by EBV. However, conclusive evidence demonstrates that B-lymphocytes and not epithelial cells are the site of EBV persistence. Thus, EBV can be eradicated in bone marrow transplant recipients by administration of high-dose chemotherapy which ablates the patient's lymphocytes without affecting the oropharyngeal epithelium (Gratama et al., 1988). Moreover, in tonsils from IM patients, EBV is detected in B-lymphocytes but not in epithelial cells (Niedobitek et al., 1997) and patients with X-linked agammaglobulinemia, a disorder characterized by a defect in B-cell maturation, are resistant to EBV infection (Faulkner et al., 1999).

TARGETS OF EBV INFECTION

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

The presence of EBV DNA and the constant expression of EBV-encoded gene products strongly indicate that the virus has a likely role in the pathogenesis of an increasing number of human malignancies. These include lymphomas of B, T, and NK origin such as endemic Burkitt's lymphoma (BL), immunoblastic lymphomas of immunocompromised patients, Hodgkin's lymphoma, a proportion of peripheral T-cell lymphomas, and nasal T/NK lymphomas (reviewed in Rickinson and Kieff, 1996) but also non-lymphoid malignancies, such as undifferentiated nasopharyngeal carcinomas (NPC) (reviewed in Dolcetti and Menezés, 2003), a fraction of gastric cancers (Iezzoni et al., 1995; Osato and Imai, 1996), and smooth muscle tumors in AIDS or transplant patients (Jenson et al., 1997) (Table 1). This spectrum of EBV-associated tumors reflects the predominant but not exclusive tropism of EBV for two distinct cell types: B-lymphocytes and epithelial cells. EBV infection of these and other cellular targets occurs through different and still partly obscure mechanisms and is followed by the expression of distinct sets of viral proteins. Elucidation of the modalities of infection and definition of the latency programs activated in these cellular backgrounds are crucial for understanding the contribution of EBV to the development of a broad variety of malignancies.

Table 1. EBV-associated benign and malignant disorders and the corresponding forms of viral latency
Disease% of EBV-related casesViral proteins expressed (Latency type)
Infectious mononucleosis (IM)> 99EBNA-1, -2, -3, -4, -5, -6, LMP-1, -2A, -2B
Immunoblastic B-cell lymphoma, AIDS-related≈ 95EBNA-1, -2, -3, -4, -5, -6, LMP-1, -2A, -2B
Post transplant lymphoproliferative disorder (PTLD)≈ 95EBNA-1, -2, -3, -4, -5, -6, LMP-1, -2A, -2B
Burkitt's lymphoma (BL), African> 95EBNA-1
BL, North American and AIDS-related≈ 20–30EBNA-1
Peripheral T-cell lymphoma≈ 40EBNA-1, LMP-1, -2A, -2B
Nasal T/NK cell lymphoma> 95EBNA-1, LMP-1, -2A, -2B
Hodgkin's disease (HD), general population≈ 40EBNA-1, LMP-1, -2A, -2B
HD, AIDS-related> 95EBNA-1, LMP-1, -2A, -2B
Nasopharyngeal carcinoma (NPC), Asian> 95EBNA-1, LMP-1, -2A, -2B
NPC, North American≈ 70EBNA-1, LMP-1, -2A, -2B
Lymphoepitheliomas (stomach, thymus)≈ 80EBNA-1, LMP-1, -2A, -2B
Gastric adenocarcinoma≈ 10EBNA-1, LMP-2A

B-LYMPHOCYTES

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

EBV has a pronounced tropism for human B-lymphocytes, which are readily infected and immortalized in vitro (reviewed in Kieff, 1996). Virus entry in these cells is mainly mediated by binding of the major viral envelope glycoprotein, gp350/220 (gp350) to CD21, the C3d complement receptor, that is expressed on the surface of all B-lymphocytes (Fingeroth et al., 1984). Unique for B-lymphocytes is the ability to support productive or latent EBV infections depending on their state of activation/differentiation. Long-lived memory B cells are the main site of latency where the virus hides from the immune system while terminally differentiated plasma cells, that are committed to die, support productive infection, favoring the spreading of the virus both within and outside the host (Fig. 1) (reviewed in Thorley-Lawson, 2001). Healthy EBV-seropositive adults carry between 1 and 50 EBV-infected B-lymphocytes per million cells in the peripheral blood (Babcock et al., 1998). Long-lived EBV-carrying memory B-lymphocytes may express a putative Latency 0, characterized by complete silencing of the viral genome, or a Latency I, in which LMP-2A alone or together with EBNA-1 may be expressed (Chen et al., 1995; Miyashita et al., 1997). As discussed below, the expression of these viral proteins is crucial for establishing and maintaining EBV persistence in B-lymphocytes. EBNA-1 has a pivotal role for the replication and partitioning of EBV episomes in proliferating virus cells (Yates et al., 1984), whereas LMP-2 promotes the survival of EBV-infected B-lymphocytes by blocking virus reactivation (Longnecker, 2000).

thumbnail image

Figure 1. Similarities between EBV infection and antigen-driven B-cell response. Depending on the location and differentiation status of EBV-infected B-lymphocytes, four different programs of viral gene expression can be observed in vivo. The “growth program” is activated following EBV infection of B cells and is responsible for the generation of proliferating B immunoblasts, similarly to what occurs to a resting naive B cell after interaction with cognate antigen. Within the germinal center, survival of B immunoblasts is strictly dependent on stimuli from the microenvironment (mainly CD40 triggering) provided by follicular dendritic cells and antigen-specific T-helper lymphocytes. These signals are surrogated by a “rescue program” of viral gene expression in EBV-infected cells. The evolution to a terminally differentiated plasma cell may lead to activation of the “lytic program” allowing spreading of the virus. Alternatively, the cells leave the follicle as resting memory B cells, which are almost invisible to the immune system due to the limited, or totally absent, viral gene expression of the “hiding program” (adapted from Masucci and Ernberg, 1994).

Download figure to PowerPoint

In the absence of effective immune surveillance, as observed in vitro or in vivo in immunosuppressed patients, EBV-infected B cells show a different latency program, called Latency III, characterized by the expression of six EBV nuclear antigens (EBNA-1-6) and three latent membrane proteins (LMP-1, -2A, and -2B) (reviewed in Rickinson and Kieff, 1996). This pattern has been called the “growth program” due to its association with autonomous B-cell proliferation, as exemplified by EBV carrying lymphoblastoid cell lines (LCLs). These cells express high levels of B-cell activation markers and adhesion molecules and resemble normal immunoblasts activated by interaction with the cognate antigen (Fig. 1). Activation of the growth program probably contributes to the expansion of the initial pool of EBV-infected B cells, increasing the likelihood of access to the memory B-cell compartment.

An intermediate form of latency has been identified in B-lymphocytes that home to the germinal centers of lymphoid follicles (Babcock et al., 2000; Babcock and Thorley-Lawson, 2000). In these cells, the expression of EBV proteins is restricted to EBNA-1 and the three LMPs (Latency II), a “rescue” program that provides signals allowing infected lymphoblasts to survive and differentiate into memory B cells (Fig. 1) (Thorley-Lawson, 2001). A similar pattern of viral gene expression is also found in EBV-associated Hodgkin's Disease (HD), which was recently identified as a B-cell malignancy (reviewed in Young et al., 2000). Elegant studies have demonstrated that Hodgkin and Reed–Sternberg cells of HD carry rearranged immunoglobulin genes with high loads of somatic mutations, suggesting that these cells represent clonal populations of transformed germinal center-related B-lymphocytes (Staudt, 2000).

An additional type of interaction of EBV with B-lymphocytes is found in EBV-associated BL. Only EBNA-1 is selectively expressed in the tumor cells, while expression of the remaining EBNAs and LMPs is silenced by methylation of the relevant promoters (Masucci et al., 1989; Jansson et al., 1992). On the basis of differences in geographic distribution, two major subgroups of BLs can be distinguished: an endemic form, which is restricted to some regions of equatorial Africa and New Guinea, and a sporadic form, which occurs worldwide (reviewed in Hecht and Aster, 2000). Recently, the incidence of sporadic BL has markedly increased, mainly as a consequence of human immunodeficiency virus-related immunosuppression (Knowles et al., 1988). Although both endemic and sporadic BLs are characterized by the presence of chromosomal translocations activating the c-myc gene, the two BL types differ with regard to EBV association (Hecht and Aster, 2000). While nearly all the endemic BLs carry the EBV genome, the virus is found only in a proportion of sporadic BLs, with prevalence ranging from 10% to 85% (Hecht and Aster, 2000). BL cells display extensive phenotypic and functional similarities with germinal center centroblasts but these features are unstable in vitro, being detectable only in early passage BL lines (Group I BLs). During prolonged culture in vitro, some BL cell lines drift towards an LCL-like pattern of viral and cellular gene expression (Group III BLs). Group II cell lines exhibit an intermediate phenotype.

T CELLS AND NK CELLS

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Convincing evidence demonstrates that EBV may infect immature cortical thymocytes and mature T lymphocytes as well as natural killer (NK) cells (Watry et al., 1991; Paterson et al., 1995; Mitarnun et al., 2002). Although a variable proportion of T lymphocytes may express CD21 (Tsoukas and Lambris, 1993), the mode of virus entry in T and NK cells remains unclear. EBV-infected circulating T cells and the corresponding transformed T-cell lines invariably express a Latency II (EBNA-1+, LMP-1+, LMP-2+) (Imai et al., 1996; Groux et al., 1997). Recent advances in the molecular diagnosis of EBV-related diseases have led to the identification of an unexpectedly broad spectrum of EBV-associated lymphoproliferative disorders of T and NK cells. This includes fulminant EBV+ T-cell lymphoproliferative disorders following acute and chronic EBV infection (Imai et al., 1996; Quintanilla-Martinez et al., 2000), a subset of peripheral T-cell lymphomas (Mitarnun et al., 2002), extranodal NK/T cell lymphoma, nasal type (Tao et al., 1995a; Chan et al., 1999), enteropathy-type intestinal T-cell lymphoma (de Bruin et al., 1995), aggressive NK lymphoma (Abe et al., 2000), hepatosplenic and non hepatosplenic γδ T-cell lymphomas (Arnulf et al., 1998; Ohshima et al., 2000). The viral genome carried by the lymphoma cells is usually monoclonal, suggesting that infection precedes malignant transformation (Groux et al., 1997). These findings, together with the demonstration that these EBV-infected T cells are tumorigenic in immunodeficient mice (Groux et al., 1997) are consistent with a role of the virus in the pathogenesis of these disorders. Nevertheless, several perplexities still remain, concerning, for example, the consequences of LMP-1 expression in T cells. In contrast to LMP-1-expressing B cells, NF-kB or STAT are not activated in EBV-carrying T-cell lines (Knecht et al., 2001). Whether this is related to the prevalent nuclear localization of LMP-1 in transfected T cells (Xu et al., 2002) remains to be elucidated.

EPITHELIAL CELLS

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Besides lymphoid malignancies, EBV is also linked to several epithelial cell cancers including undifferentiated NPC (reviewed in Dolcetti and Menezés, 2003), lymphoepithelial carcinomas of the stomach, salivary glands, lungs, and thymus (reviewed in Iezzoni et al., 1995), and a proportion of conventional gastric adenocarcinomas (Imai et al., 1994). NPC is the tumor showing the strongest association with the EBV. The neoplastic cells of all NPC cases examined to date were found to be positive for EBV, regardless of geographical and/or ethnical origin of the patients (International Agency for Research on Cancer, 1997; Dolcetti and Menezés, 2003). EBV was also detected in high-grade precancerous lesions but not in the low-grade lesions and normal epithelia of the nasopharynx (Pathmanathan et al., 1995; Cheung et al., 1998; Chan et al., 2000). Moreover, both in situ and invasive NPC samples carry clonal EBV (Raab-Traub and Flynn, 1986; Pathmanathan et al., 1995), indicating that a monoclonal expansion of EBV-carrying cells is present in these lesions and supporting the assumption that EBV infection is an early event in the development of NPC. NPC cells were found to express EBNA-1, LMP-1, and LMP-2, but not EBNA-2 (reviewed in International Agency for Research on Cancer, 1997), a pattern of viral latency also observed in EBV positive HD and T-cell lymphomas. Similarly to NPC, most of lymphoepithelial carcinomas of the stomach arising worldwide are also EBV-associated (International Agency for Research on Cancer, 1997) (Table 1). In contrast, lymphoepithelial carcinomas of the salivary glands, lungs, and thymus are frequently EBV-related in areas where NPC is endemic, whereas similar tumors arising in Caucasians are usually EBV negative (International Agency for Research on Cancer, 1997). EBV was also found in 2–16% of conventional gastric adenocarcinomas, both from low- and high-incidence areas (Osato and Imai, 1996) (Table 1). The relatively high prevalence of this tumor makes EBV-associated gastric carcinomas a significant health problem in terms of absolute case number. Gastric carcinomas were found to express EBNA-1 and LMP-2A in the absence of EBNA-2 and LMP-1 (Osato and Imai, 1996). Therefore, the association with EBV and the pattern of virus latency expressed is heterogeneous among epithelial cell malignancies, suggesting that EBV probably contributes differently to the pathogenesis of these tumors.

Unlike B-lymphocytes, human epithelial cells cannot be easily infected or transformed by EBV and uncertainty still persists on the mechanism by which the virus enters these cells. Most of the available data suggest that CD21 is not expressed in human epithelial cells in vivo (International Agency for Research on Cancer, 1997; Burgos and Vera-Sempere, 2000; Speck et al., 2000). The observation that several epithelial cell lines were efficiently infected by co-cultivation with EBV-producing B-lymphocytes suggested that direct cell-to-cell contact might allow entry in CD21-negative cells (Imai et al., 1998). This may indeed occur in vivo, as suggested by the presence of B-lymphocytes undergoing lytic EBV infection in the nasopharyngeal mucosa (Tao et al., 1995b). Another possibility is that EBV may be transferred through uptake of apoptotic bodies, although available evidence is limited to EBV DNA integrated into chromosomes (Holmgren et al., 1999). Finally, in vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, by a mechanism involving the endocytosis of polymeric IgA–EBV complexes bound to the secretory component (SC) (Sixbey and Yao, 1992). Intriguingly, the SC protein is expressed on the basolateral membranes of epithelial cells localized in the fossa of Rosenmuller, where NPC usually develops (Nomori et al., 1985). The demonstration that the SC protein is expressed in NPC cells further supports the possibility that this mode of EBV infection may operate in vivo (Lin et al., 1997). More recently, evidence has been provided indicating that EBV virions produced in B cells efficiently infect epithelial cells whereas epithelial cell-derived virus preferentially infects B-lymphocytes (Borza and Hutt-Fletcher, 2002). The mechanism by which this switching occurs involves the viral glycoproteins gp350 and the gp42/gH/gL complex that bind to cell-surface receptors, CD21 and the HLA class II co-receptor, respectively. The EBV produced in B-lymphocytes lacks gp42 in its gH/gL complex due to intracellular entrapment of gp42 by HLA class II molecules (Borza and Hutt-Fletcher, 2002). B-cell-derived virus might then efficiently infect epithelial cells where a further cycle of EBV replication may occur. Since these cells do not express HLA class II they allow the production of gp42 containing virions that continue the cycle back into B-lymphocytes.

EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

The EBNA-2 protein is localized in the nucleus and is one of the first viral proteins expressed in EBV infected B-lymphocytes (Zimber-Strobl and Strobl, 2001). In co-operation with EBNA-5, EBNA-2 induces the transition of resting B cells from G0 to G1 (Zimber-Strobl and Strobl, 2001). EBNA-2 is a key regulator of viral gene expression, being able to stimulate transcription from the major latency BamHI-C promoter, which directs expression of all the EBNA genes, and the promoters of LMP-1 and LMP-2 (Zimber-Strobl and Strobl, 2001). In addition, EBNA-2 modulates the transcriptional activity of several cellular genes. C-fgr, c-myc, CD21, CD23, and EBI1/BLR2 are up-regulated whereas the immunoglobulin heavy chain genes are repressed in lymphocytes (Zimber-Strobl and Strobl, 2001). EBNA-2 does not bind to DNA directly and its transcriptional activity is mainly mediated by its interaction with the DNA-binding cellular protein RBP-J (also called RBP-Jκ, CBF1, KBF2, or CSL) (Grossman et al., 1994; Henkel et al., 1994; Waltzer et al., 1994; Zimber-Strobl et al., 1996). EBNA-2 is essential for EBV-induced immortalization of B-lymphocytes and complex formation with RBP-J is crucial for such activity (Yalamanchili et al., 1994). RBP-J is expressed ubiquitously and is an important component of the Notch signaling pathway, which regulates lymphoid development (Osborne and Miele, 1999). Notch proteins are a family of transmembrane receptors that, upon ligand binding, undergo proteolytic cleavage of their intracellular domain (Notch1 IC) (Fig. 2) (Osborne and Miele, 1999). The released Notch1 IC fragment is transported to the nucleus where it interacts with RBP-J and modulates the activity of target promoters (Fig. 2) (Osborne and Miele, 1999). Although Notch1 IC and ENBA-2 share the ability to transactivate genes by interacting with RBP-J, the set of promoters regulated by Notch1 IC and EBNA-2 is overlapping but not identical (Zimber-Strobl and Strobl, 2001). On these grounds, EBNA-2 may be regarded as a functional homologue of a constitutively activated Notch receptor. However, several lines of evidence indicate that EBV modifies RBP-J-dependent signaling in a highly regulated manner. Other EBV-encoded proteins (EBNA-3 and -6) also bind to RBP-J, but this interaction results in inhibition of Notch signaling (Robertson et al., 1995; Johannsen et al., 1996; Waltzer et al., 1996; Zhao et al., 1996).

thumbnail image

Figure 2. EBV proteins interfere with cellular signaling and manipulate the ubiquitin/proteasome system. Schematic representation of the signaling cascades usurped by LMP-1, LMP-2, and EBNA-2. EBNA2 mimics the function of activated Notch while LMP-1 acts as a constitutively activated CD40/TNF receptor. LMP-2 acts as a decoy protein which sequesters the Lyn and Syk kinases and target them for proteasomal degradation. The GAr of EBNA-1 inhibits processing by the ubiquitin/proteasome system. (U, ubiquitin; E3, ubiquitin ligase).

Download figure to PowerPoint

Constitutive activation of Notch receptors can lead to the development of different types of cancer (Zimber-strobl and Strobl, 2001). However, due to the relatively strong immunogenicity of EBNA-2 (reviewed in Rickinson et al., 1996), this mechanism of transformation may operate only in the context of profound immune suppression. In fact, EBNA-2 protein expression is detected only in tumors with the broader form of EBV latency, such as post-transplant lymphoproliferative disorders and AIDS-related non-Hodgkin's lymphomas (International Agency for Research on Cancer, 1997).

LMP-1: A PLEIOTROPIC VIRAL ONCOGENE

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Early studies have identified LMP-1 as a viral oncoprotein on the basis of its ability to transform rodent cells. Fibroblasts constitutively expressing LMP-1 demonstrate reduced serum requirements, increased growth in soft agar, loss of contact inhibition, and tumorigenic potential in nude mice (Wang et al., 1985; Dawson et al., 1990; Fahraeus et al., 1990). Moreover, studies with EBV mutants have demonstrated that LMP-1 is critical for the immortalization of primary B-lymphocytes (Kaye et al., 1993). LMP-1 alone can induce many of the phenotypic and functional changes associated with EBV infection including increased homotypic adhesion and up-regulation of adhesion molecules (LFA-1, ICAM-1, LFA-3; Moorthy and Thorley-Lawson, 1993; Zimber-Strobl et al., 1996), B-cell activation markers (CD23, CD30, CD40, CD71; Calender et al., 1987), and anti-apoptotic genes (Bcl-2, BclxL, Mcl1, A20; Henderson et al., 1991; Laherty et al., 1992; Wang et al., 1996). Moreover, expression of LMP-1 as a transgene in mice under the control of the immunoglobulin promoter/enhancer results in increased frequency of B-cell lymphomas, indicating that this viral protein has oncogenic properties in vivo (Kulwichit et al., 1998).

LMP-1 has pleiotropic effects also in epithelial cells. In particular, LMP-1 transforms immortalized epithelial cells to tumorigenic lines (Dawson et al., 1990) and induces epidermal hyperplasia when expressed in the skin of transgenic mice (Wilson et al., 1990). In addition, LMP-1 was shown to alter morphology and cytokeratin expression in immortalized human keratinocyte cell lines (Dawson et al., 1990; Fahraeus et al., 1990) whereas conflicting results have been reported on the effect of LMP-1 on epithelial cell differentiation (Dawson et al., 1990; Fahraeus et al., 1992; Niedobitek et al., 1992; Nicholson et al., 1997). Furthermore, LMP-1 induces in vitro the expression of molecules involved in cell-to-cell adhesion (ICAM-1), mediating growth and/or survival signals (CD40, CD70, EGF-receptor, A20; Dawson et al., 1990; Miller et al., 1995; Fries et al., 1996) as well as cytokines such as IL-6 (Eliopoulos et al., 1997) and IL-8 (Yoshizaki et al., 2001).

Recent studies have shed some light into the molecular mechanisms underlying the function of LMP-1. This is a phosphoprotein of 386 amino acids characterized by a short amino-terminal sequence, six membrane-spanning domains, and a long carboxy-terminal cytoplasmic tail (reviewed in Kieff, 1996). The N-terminus and transmembrane domains form aggregates in the cytoplasmic membrane, allowing LMP-1 to act like a constitutively activated receptor (reviewed in Kieff, 1996; Eliopoulos and Rickinson, 1998; Young et al., 2000). Molecular and biochemical approaches have demonstrated that LMP-1 recruits several tumor necrosis factor (TNF)-receptor associated factors (TRAFs) in two distinct C-terminus activating domains, interfering thus with TNF-receptor-mediated cellular signaling pathways (Mosialos et al., 1995) (Fig. 2). Binding of TRAFs to LMP-1 results in the induction of the NF-κB and AP-1 transcription factors (Eliopoulos and Rickinson, 1998; Young et al., 2000), confirming that LMP-1 shares functional properties with members of the TNF-receptor superfamily, particularly CD40 (Fig. 2) (Eliopoulos et al., 1996; Eliopoulos and Rickinson, 1998; Busch and Bishop, 1999; Young et al., 2000). Consistently, it has been shown that LMP-1 can partly restore the wild-type phenotype of mice deficient in CD40 (Uchida et al., 1999). However, unlike the TNF-receptor, LMP-1 engages at least part of the CD40 pathway in a ligand-independent manner. The constitutive activity of LMP-1 correlates with altered turnover of TRAFs. Recruitment of TRAF-2 and -3 to CD40 activates the signaling molecules and their subsequent ubiquitin/proteasome-dependent degradation, a mechanism that accounts for the transient nature of CD40 triggering (Brown et al., 2001). In contrast, binding of TRAF-2 and -3 to LMP-1 leads to their activation but fails to target them for proteasomal degradation (Brown et al., 2001). A further C-terminus domain of LMP-1 triggers a STAT-dependent signaling cascade by binding to JAK3, which is known to be involved in the control of cellular proliferation (Fig. 2) (Gires et al., 1999). The hijack of these cellular signaling pathways by LMP-1 is likely to contribute to the pathogenesis of most EBV-associated disorders through the simultaneous or sequential activation of signals involved in the promotion of cell activation, growth, and survival.

LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Unlike LMP-1, the LMP-2 protein is not essential for B-cell transformation in vitro (Longnecker et al., 1992). Nevertheless, the constant expression of this viral gene in EBV-carrying memory B cells from healthy individuals suggests that LMP-2 probably plays an important role in mediating virus persistence (Babcock et al., 1998). Expression of LMP-2A in transgenic mice was shown to provide survival signals that allow immature B cells to progress through developmental checkpoints that would normally result in cell death (Caldwell et al., 2000). This has been related to the ability of LMP-2A to activate the serine–threonine kinase Akt resulting in the constitutive delivery of an anti-apoptotic signal (Fig. 2) (Scholle et al., 2000; Swart et al., 2000). Akt is a multifunctional mediator of phosphatidylinositol 3-kinase (PI3-K) activity, a pathway that is also involved in the control of B-cell proliferation as demonstrated by the finding that chemical inhibition of PI3-K induce growth arrest of EBV-immortalized B cells (Brennan et al., 2002). It is presently unknow whether the constitutive activation of Akt induced by LMP-2A also promotes B-cell proliferation. LMP-2A was shown to inhibit the switch from latency to lytic EBV replication induced by BCR triggering (Longnecker, 2000). This effect has been related to the ability of LMP-2A to interfere with BCR signaling and may play a major role in mediating EBV persistence in the infected host.

LMP-2A contains multiple membrane spanning domains and cytoplasmic N- and C-terminal domains and forms aggregates in the plasma membrane of latently infected B cells (Longnecker, 2000). The amino-terminal domain contains motifs that bind the tyrosine kinases Lyn and Syk through their SH2-domains (Fig. 2) (Longnecker, 2000). These kinases are recruited to the BCR following antigen cross-linking and their subsequent activation stimulates downstream events resulting in B-cell differentiation and proliferation (DeFranco, 1997). LMP-2A works as a decoy protein sequestering Lyn and Syk and inhibiting thus BCR signaling (Fig. 2). Recent evidence indicates that the ubiquitin/proteasome pathway plays a crucial role in LMP-2A-mediated BCR silencing. The N-terminus of LMP-2A contains proline-rich motifs, which can bind to WW domain-containing ubiquitin-protein ligases of the Nedd-4 family (AIP4, WWP2/AIP2, and KIAA0439) (Ikeda et al., 2000; Winberg et al., 2000). Binding of these ubiquitin ligases to LMP-2A induces ubiquitination of LMP-2A, Lyn, and Syk. Thus, LMP-2A probably functions as a molecular scaffold for the recruitment of both BCR-associated tyrosine kinases and E3 protein-ubiquitin ligases (Fig. 2) behaving similarly to the E6 protein of the oncogenic HPV-16 and -18. HPV-E6 mediates the interaction between the tumor suppressor p53 and the ubiquitin ligase E6-AP, which results in ubiquitination and enhanced proteasomal degradation of p53 (Scheffner et al., 1993). These findings suggest that the expression of scaffold proteins for ubiquitin-dependent proteolysis is probably a common strategy accomplished by unrelated viruses to inactivate critical cellular signaling.

Although the biological effects of LMP-2A in epithelial cells are still poorly defined, the available data indicate a role distinct from that in B-lymphocytes. In particular, it has been demonstrated that the interaction of epithelial cells with extracellular matrix proteins triggers LMP-2A phosphorylation, suggesting that this EBV protein is involved in signaling pathways activated by cell adhesion (Scholle et al., 1999). More recently, ectopic expression of LMP-2A in a human keratinocyte cell line resulted in enhanced proliferation, clonogenicity in soft agar, and inhibition of differentiation. Moreover, LMP-2A-expressing keratinocytes were highly tumorigenic in nude mice, inducing aggressive tumors that frequently developed distant metastases (Scholle et al., 2000). These properties were related to the ability of LMP-2A to activate the PI3-K/Akt pathway (Scholle et al., 2000). In a different epithelial cell system, LMP-2A was shown to promote the mobility and invasion through activation of MAPK kinases (Chen et al., 2002), further supporting the possibility that LMP-2A might contribute to the metastatic spreading of carcinomas.

IMMUNOLOGICAL CONTROL OF TRANSFORMATION

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Asymptomatic EBV persistence in the infected host is normally secured by high numbers of memory EBV-specific CD8+ T cells that continuously patrol the whole body. Several lines of evidence indicate that the involvement of EBV in malignancies requires not only the coordinate activity of latent viral proteins but also to the ability of the virus to inhibit host immune responses directed towards EBV-carrying cells.

DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

The down-regulation of viral proteins that provide target epitopes for cytotoxic T-cell (CTL)-mediate rejection is a key feature of life-long persistence in immunocompetent carriers. The virus detected in circulating memory cells is either transcriptionally silent or only LMP-2A, and sometimes EBNA-1 mRNAs are detected (Chen et al., 1995; Miyashita et al., 1997; Babcock et al., 1998, 1999). It remains to be determined whether these latently infected cells express immunologically significant amounts of these proteins. Furthermore, these cells express relatively low levels of MHC class I and co-stimulatory molecules (Miyashita et al., 1997; Babcock et al., 1998), and are characterized by an antigen processing machinery that produces antigenic peptides with limited efficiency (Frisan et al., 2000). As discussed above, in healthy individuals EBV may express a broader spectrum of viral proteins: B immunoblasts express the EBNA-2-driven growth program while memory and germinal center B-lymphocytes are EBNA-2-negative and express EBNA-1, LMP-1, and LMP-2 only (Fig. 1). These less restricted latency programs are likely expressed only transiently in the immunocompetent host, due to the ability of EBNAs and LMPs to elicit protective CTL responses (Rickinson et al., 1996). These same forms of latency, however, are “crystallized” in some EBV-related malignancies, particularly in those occurring in patients with severely impaired immune responses when the full set of EBV latency proteins may be expressed and therefore directly contribute to B-cell transformation. With regard to EBV-associated tumors with the Latency II phenotype, it has been suggested that stable expression of EBNA-1, LMP-1, and LMP-2 may occur in B cells that are unable to differentiate as a consequence of underlying genetic alterations. This is the case of Reed–Sternberg cells of HD, which are unable to exit from the germinal center stage due to mutations leading to constitutive activation of the NF-κB signaling pathway (Krappmann et al., 1999; Staudt, 2000). Alternatively, a relatively stable Latency II may be established after inappropriate infection of proliferating epithelial cells, as in the case of NPC. Since these cells are unable to undergo terminal differentiation, LMP-1 expression can not be switched off, contributing thus to the transformed phenotype.

EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

With the possible exception of circulating EBV-infected memory B cells, the EBNA-1 protein is expressed in all EBV latency states and all EBV-associated malignancies, clearly indicating that the biologic properties of this protein are critical for both virus persistence and EBV-mediated transformation (Leight and Sugden, 2000). Evaluation of the putative role of EBNA-1 in oncogenesis is however difficult, because this protein is essential for efficient maintenance and expression of EBV episomes in dividing cells (Leight and Sugden, 2000). This function is accomplished through the binding of EBNA-1 dimers with the Dyad Symmetry and Family of Repeat sequences that are located within the origin of plasmid replication (oriP) region of the EBV genome (Fig. 2) (Leight and Sugden, 2000). EBNA-1 is also a transcriptional regulator that modulates the activity of the two EBNA promoters: Wp and Cp and its own latent promoter Qp (Leight and Sugden, 2000). The structure of the EBNA-1 protein is peculiar because most of its amino terminal part is composed of glycine and alanine repeats (GAr) that vary in length among different EBV isolates (Falk et al., 1995). Apart from the demonstration that this long repetitive sequence is the major target of EBNA specific antibody responses (Dillner et al., 1984), the function of this domain remained unresolved until it was shown that GAr inhibits the presentation of antigenic epitopes from EBNA-1 (Fig. 2). Expression of an EBNA-1 chimera containing a characterized epitope from an immunodominant EBV protein successfully elicited CTL responses only after GAr removal (Levitskaya et al., 1995). This effect was related to the ability of GAr to hamper the proteasome-mediated processing of EBNA-1, thus preventing the efficient generation of peptides that can bind to HLA class I molecules (Levitskaya et al., 1997). Notably, the GAr was shown to act as a transferable element that confers resistance to proteasomal degradation when expressed in the context of both viral and cellular proteins. These findings were confirmed also in a mouse model where CTLs generated by immunization with a GAr deletion mutant were unable to kill cells expressing full-length EBNA-1 (Mukherjee et al., 1998). Interestingly, when EBNA-1 is supplied as an exogenous antigen, the protein is efficiently presented in the context of HLA class I molecules by an alternative pathway that is independent of the TAP transporter (Blake et al., 1997, 2000). These cells sensitized by exogenous EBNA-1 can be lysed by CD8+ CTLs specific for HLA class I-restricted EBNA-1 epitopes that can be isolated from the memory pool of healthy EBV-seropositive individuals (Blake et al., 1997). These EBNA-1-specific CD8+ CTLs probably originate in vivo by cross-priming mechanisms involving phagocytosis of EBNA-1-containing cell debris by professional antigen presenting cells. The contribution of these cells to the control of EBV infection remains unclear, as they do not target EBV-carrying cells. A closer look at the mechanisms underlying GAr function showed that IκB-α-GAr chimeras failed to form stable complexes with the proteasome, whereas their ubiquitination was similar to that of wild-type proteins (Sharipo et al., 1998). Moreover, the use of more sensitive biochemical approaches allowed the demonstration that, although polyubiquitinated p53-GAr chimeras can interact with the polyubiquitin-binding subunit of the proteasome, the outcome of this interaction may be affected by the GAr in a way that may lead to the rapid release of functionally intact proteins (Heessen et al., 2002). Nevertheless, the molecular mechanisms underlying this effect remain still unclear.

Selective blockage of EBNA-1 degradation by the proteasome and failure to present endogenous EBNA-1 derived epitopes allow latently infected cells to successfully escape cell-mediated immune responses. This immune evasion strategy is particularly effective, considering that EBNA-1 expression is strictly required for the maintenance of the viral episome in proliferating EBV-infected cells. Furthermore, the protective effects of the GAr makes EBNA-1 an extremely stable protein, allowing the persistence of the viral episome also under low metabolic conditions. The peculiar biologic properties conferred by the GAr to EBNA-1, therefore, may at least partly explain how this protein contributes to virus persistence and EBV-mediated cell transformation. Available evidence however indicates that EBNA-1 probably also exerts other non-immunologic functions that may be relevant to the transformed phenotype. In fact, mice transgenic for EBNA-1 within the B cell compartment showed a high incidence of B-cell lymphomas (Wilson et al., 1996). Moreover, expression of EBNA-1 in an EBV-negative NPC cell line resulted in increased tumorigenic and metastatic capability, suggesting that EBNA-1 may enhance malignant progression of EBV-associated epithelial tumors (Sheu et al., 1996).

LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Besides exerting oncogenic properties by interfering with the TNF-receptor signaling, LMP-1 also has additional functions that may affect host immune responses directed towards EBV-carrying cells. In particular, LMP-1 has been shown to up-regulate HLA class I molecules, as well as components of the antigen presentation machinery, such as peptide transporters and some interferon-γ-inducible subunits of the immunoproteasome (Frisan et al., 1998). Considering that these LMP-1-mediated effects could result in an increased sensitivity of EBV-carrying cells to immune surveillance, it is not clear what advantage the virus has in up-regulating the expression of antigen processing components. One possible explanation is that allowing an uncontrolled expansion of EBV-carrying immunoblasts with a Latency III program may put the human host at risk, being ultimately detrimental also for the virus. In this respect, the effects of LMP-1 on antigen presentation may favor an efficient recognition and elimination of EBV+ immunoblasts by CTLs. It is noteworthy that LMP-1 is not expressed in the pool of circulating memory B cells and therefore recognition of LMP-1 would not affect this virus reservoir.

Another puzzling question is why LMP-1 expression by neoplastic cells is tolerated in patients with EBV-associated malignancies with the Latency II program. This mainly occurs in NPC and HD patients, despite the fact that LMP-1 carries CTL target epitopes restricted through common HLA alleles (Rickinson et al., 1996). Analysis of virus-specific CTL responses at the tumor site of EBV-positive HD patients showed that infiltrating CTLs are functionally impaired and unable to eliminate neoplastic cells (Frisan et al., 1995). Available evidence indicates that this is not due to defects in antigen processing or presentation since NPC cells and Reed–Sternberg cells of HD usually express high levels of HLA class I molecules and are functionally competent in endogenously processing and presenting EBV epitopes for class I-restricted CTL killing (Oudejans et al., 1996; Khanna et al., 1998; Lee et al., 1998, 2000; Murray et al., 1998). One possible explanation comes from the recent identification of two short sequences in the first transmembrane domain of LMP-1 that display a high degree of homology to an immunosuppressive domain in a retroviral transmembrane protein (Dukers et al., 2000). Administration of recombinant peptides corresponding to these sequences resulted in a strong inhibition of both CTL and NK cell responses in vitro (Dukers et al., 2000). To exert immunosuppressive effects in vivo, however, LMP-1 has to be excreted from the cells. Indeed, LMP-1 was detected in exosomes purified from the supernatant of EBV-positive B-cell lines, suggesting a possible mechanism by which LMP-1 could affect local immune responses to EBV-infected cells (Dukers et al., 2000). LMP-1 is a short-lived protein (Martin and Sugden, 1991) and is efficiently degraded by the ubiquitin/proteasome machinery (Aviel et al., 2000). This may favor a sustained production of these immunosuppressive peptides, the size of which is within the range of the fragments usually generated by the proteasome (Kisselev et al., 1999). Due to their hydrophobicity, these peptides could traverse cell membranes and impair immune responses locally.

CTL ESCAPE MUTANTS

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

Mutations preventing binding of viral epitopes to HLA molecules is a common mechanism of immunoevasion for genetically unstable viruses. Although EBV is a genetically stable virus providing highly conserved immunodominant epitopes, rare instances of CTL escape mutants have been described. Immunodominant EBNA-4 epitopes restricted by HLA A11 were frequently mutated in isolates from South East Asia where HLA A11 is expressed in more than 50% of the population (de Campos-Lima et al., 1993). All mutations identified so far targeted anchor residues that are important for binding to HLA A11 (de Campos-Lima et al., 1993, 1994; Levitsky et al., 1997). By contrast, mutations within the same EBNA-4 epitopes were found at a markedly lower prevalence in isolates from other regions where HLA A11 was less represented (de Campos-Lima et al., 1993, 1994). These findings suggested that CTLs could exert an “immune” pressure on the EBV genome and select for mutants that are no longer recognized by HLA class I-restricted CTLs. Subsequent studies carried out on isolates from different geographic areas questioned the correlation between mutations disrupting immunodominant EBV epitopes and the prevalence of defined HLA alleles in a given population (Burrows et al., 1996; Khanna et al., 1997), suggesting that sequence variation within CTL epitopes is probably due to genetic drift or fortuitous events. The issue is far from being resolved due to our poor understanding of the population dynamics of EBV strains and possible founder effects related to human migration.

Recent evidence supports the view that immune pressure may select CTL escape mutants that have growth advantages in vivo, even in the setting of immune suppression. Treatment of an immunocompromised patient with adoptive transfer of EBV-specific CTLs primarily directed to EBNA-4 epitopes induced the selection of a CTL escape mutant lacking these immunodominant epitopes (Gottschalk et al., 2001). The fatal outcome of this therapeutic escape indicates that selection of EBV mutants may constitute a serious clinical problem in the setting of the EBV-associated disorders of immunosuppressed patients, particularly when CTLs targeting a limited number of EBV epitopes is used.

NEW THERAPEUTIC STRATEGIES

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED

The recent advances in our understanding of the mechanisms by which EBV induces cell transformation and escapes the host immune control provide the rationale for new strategies of intervention for EBV-related malignancies. The success of the adoptive transfer of polyclonal EBV-specific CTLs for the prophylaxis and treatment of PTLDs (reviewed in Rooney et al., 2001) warrants testing the efficacy of CTL-based therapies also in other EBV-related diseases such as HD and NPC. This is going to be a challenging task due to the low immunogenicity of LMP-1 and LMP-2 compared to the EBNAs expressed in PTLDs. The demonstration that LMP-1- and -2-specific CTL precursors can be detected in HD and NPC patients (Frisan et al., 1995; Lee et al., 2000; Chapman et al., 2001) suggests that boosting these responses could be of therapeutic benefit in this setting. Among the new immunotherapeutic approaches are the attempts to increase the immunogenicity of LMP-1 or LMP-2 by using autologous dendritic cells pulsed with these antigens; the latter approach is currently under investigation in humans. Considering the crucial role of EBNA-1 in the pathogenesis of all EBV-associated disorders, strategies able to induce EBNA-1 processing and degradation in vivo would be highly effective since they would trigger CTL responses to all EBV-carrying cells. The identification of EBNA-1-specific CTLs and the demonstration that covalent linking of ubiquitin to EBNA-1 restores degradation suggest that this approach may be feasible. Agents able to selectively block the interaction between LMP-2A and the Lyn/Syk kinases could release the LMP-2A-mediated inhibition of BCR signaling, allowing the initiation of virus replication upon physiological and/or pharmacological stimulation. Moreover, the ability of LMP-2 to activate the PI3-K/Akt pathway in both lymphoid and epithelial cells suggests that this signaling cascade may constitute a potentially relevant target for innovative therapies (Vivanco and Sawyers, 2002). However, the available PI3-K/AKT inhibitors (wortmannin and LY294002) act also on related kinases and show unfavorable pharmacokinetic properties in vivo, indicating to the need to develop more selective drugs. If the generation of LMP-1-derived immunosuppressive peptides requires efficient proteasomal degradation, pharmacologic inhibition of this machinery may contribute to restore intratumoral LMP-1-specific CTL responses in LMP-1-positive malignancies. Finally, the demonstration that EBNA-2 contributes to EBV-induced transformation by mimicking intracellular Notch/RBP-J activity suggests that drugs able to prevent the binding of EBNA-2 to RBP-J or specifically inhibit Notch signaling may be of therapeutic relevance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. TARGETS OF EBV INFECTION
  4. B-LYMPHOCYTES
  5. T CELLS AND NK CELLS
  6. EPITHELIAL CELLS
  7. MECHANISMS OF TRANSFORMATION
  8. EBNA-2: A KEY REGULATOR OF VIRAL AND CELLULAR GENE EXPRESSION
  9. LMP-1: A PLEIOTROPIC VIRAL ONCOGENE
  10. LMP-2: A SURVIVAL SIGNAL THAT PREVENTS EBV REACTIVATION
  11. IMMUNOLOGICAL CONTROL OF TRANSFORMATION
  12. DOWN-REGULATION OF IMMUNOGENIC VIRAL PROTEINS
  13. EBNA-1: IMMUNE ESCAPE THROUGH BLOCKAGE OF PROTEASOMAL DEGRADATION
  14. LMP-1: ENHANCED PRODUCTION OF IMMUNOSUPPRESSIVE PEPTIDES?
  15. CTL ESCAPE MUTANTS
  16. NEW THERAPEUTIC STRATEGIES
  17. Acknowledgements
  18. LITERATURE CITED
  • Abe Y, Muta K, Ohshima K, Yasumoto S, Shiratsuchi M, Saito R, Tsujita J, Shibata T, Furue M, Kikuchi M, Nishimura J, Nawata H. 2000. Subcutaneous panniculitis by Epstein–Barr virus-infected natural killer (NK) cell proliferation terminating in aggressive subcutaneous NK cell lymphoma. Am J Hematol 64: 221225.
  • Arnulf B, Copie-Bergman C, Delfau-Larue MH, Lavergne-Slove A, Bosq J, Wechsler J, Wassef M, Matuchansky C, Epardeau B, Stern M, Bagot M, Reyes F, Gaulard P. 1998. Nonhepatosplenic gammadelta T-cell lymphoma: A subset of cytotoxic lymphomas with mucosal or skin localization. Blood 91: 17231731.
  • Aviel S, Winberg G, Massucci M, Ciechanover A. 2000. Degradation of the Epstein–Barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue. J Biol Chem 275: 2349123499.
  • Babcock GJ, Thorley-Lawson DA. 2000. Tonsillar memory B cells, latently infected with Epstein–Barr virus, express the restricted pattern of latent genes previously found only in Epstein–Barr virus-associated tumors. Proc Natl Acad Sci USA 97: 1225012255.
  • Babcock GJ, Decker LL, Volk M, Thorley-Lawson DA. 1998. EBV persistence in memory B cells in vivo. Immunity 9: 395404.
  • Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. 1999. Epstein–Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 190: 567576.
  • Babcock GJ, Hochberg D, Thorley-Lawson AD. 2000. The expression pattern of Epstein–Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell. Immunity 13: 497506.
  • Blake N, Lee S, Redchenko I, Thomas W, Steven N, Leese A, Steigerwald-Mullen P, Kurilla MG, Frappier L, Rickinson A. 1997. Human CD8 + T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 6: 791802.
  • Blake N, Haigh T, Shaka'a G, Croom-Carter D, Rickinson A. 2000. The importance of exogenous antigen in priming the human CD8 + T cell response: Lessons from the EBV nuclear antigen EBNA1. J Immunol 165: 70787087.
  • Borza CM, Hutt-Fletcher LM. 2002. Alternate replication in B cells and epithelial cells switches tropism of Epstein–Barr virus. Nat Med 8: 594599.
  • Brennan P, Mehl AM, Jones M, Rowe M. 2002. Phosphatidylinositol 3-kinase is essential for the proliferation of lymphoblastoid cells. Oncogene 21: 12631271.
  • Brown KD, Hostager BS, Bishop GA. 2001. Differential signaling and tumor necrosis factor receptor-associated factor (TRAF) degradation mediated by CD40 and the Epstein–Barr virus oncoprotein latent membrane protein 1 (LMP1). J Exp Med 193: 943954.
  • Burgos JS, Vera-Sempere FJ. 2000. Immunohistochemical absence of CD21 membrane receptor in nasopharyngeal carcinoma cells infected by Epstein–Barr virus in Spanish patients. Laryngoscope 110: 20812084.
  • Burrows JM, Burrows SR, Poulsen LM, Sculley TB, Moss DJ, Khanna R. 1996. Unusually high frequency of Epstein–Barr virus genetic variants in Papua New Guinea that can escape cytotoxic T-cell recognition: Implications for virus evolution. J Virol 70: 24902496.
  • Busch LK, Bishop GA. 1999. The EBV transforming protein, latent membrane protein 1, mimics and cooperates with CD40 signaling in B lymphocytes. J Immunol 162: 25552561.
  • Caldwell RG, Brown RC, Longnecker R. 2000. Epstein–Barr virus LMP2A-induced B-cell survival in two unique classes of EmuLMP2A transgenic mice. J Virol 74: 11011113.
  • Calender A, Billaud M, Aubry JP, Banchereau J, Vuillaume M, Lenoir GM. 1987. Epstein–Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells. Proc Natl Acad Sci USA 84: 80608064.
  • Chan AC, Ho JW, Chiang AK, Srivastava G. 1999. Phenotypic and cytotoxic characteristics of peripheral T-cell and NK-cell lymphomas in relation to Epstein–Barr virus association. Histopathology 34: 1624.
  • Chan AS, To KF, Lo KW, Mak KF, Pak W, Chiu B, Tse GM, Ding M, Li X, Lee JC, Huang DP. 2000. High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. Cancer Res 60: 53655370.
  • Chapman AL, Rickinson AB, Thomas WA, Jarrett RF, Crocker J, Lee SP. 2001. Epstein–Barr virus-specific cytotoxic T lymphocyte responses in the blood and tumor site of Hodgkin's disease patients: Implications for a T-cell-based therapy. Cancer Res 61: 62196226.
  • Chen F, Zou JZ, di Renzo L, Winberg G, Hu LF, Klein E, Klein G, Ernberg I. 1995. A subpopulation of normal B cells latently infected with Epstein–Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1. J Virol 69: 37523758.
  • Chen SY, Lu J, Shih YC, Tsai CH. 2002. Epstein–Barr virus latent membrane protein 2A regulates c-Jun protein through extracellular signal-regulated kinase. J Virol 76: 95569561.
  • Cheung F, Pang SW, Hioe F, Cheung KN, Lee A, Yau TK. 1998. Nasopharyngeal carcinoma in situ, two cases of an emerging diagnostic entity. Cancer 83: 10691073.
  • Dawson CW, Rickinson AB, Young LS. 1990. Epstein–Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature 344: 777780.
  • de Bruin PC, Jiwa NM, Oudejans JJ, Radaszkiewicz T, Meijer CJ. 1995. Epstein–Barr virus in primary gastrointestinal T cell lymphomas. Association with gluten-sensitive enteropathy, pathological features, and immunophenotype. Am J Pathol 146: 861867.
  • de Campos-Lima PO, Gavioli R, Zhang QJ, Wallace LE, Dolcetti R, Rowe M, Rickinson AB, Masucci MG. 1993. HLA-A11 epitope loss isolates of Epstein–Barr virus from a highly A11 + population. Science 260: 98100.
  • de Campos-Lima PO, Levitsky V, Brooks J, Lee SP, Hu LF, Rickinson AB, Masucci MG. 1994. T cell responses and virus evolution: Loss of HLA A11-restricted CTL epitopes in Epstein–Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues. J Exp Med 179: 12971305.
  • DeFranco AL. 1997. The complexity of signaling pathways activated by the BCR. Curr Opin Immunol 9: 296308.
  • Dillner J, Sternas L, Kallin B, Alexander H, Ehlin-Henriksson B, Jornvall H, Klein G, Lerner R. 1984. Antibodies against a synthetic peptide identify the Epstein–Barr virus-determined nuclear antigen. Proc Natl Acad Sci USA 81: 46524656.
  • Dolcetti R, Menezés J. 2003. Epstein–Barr virus and undifferentiated nasopharyngeal carcinoma: New immunobiological and molecular insights on a long-standing etiopathogenic association. Adv Cancer Res (in press).
  • Dukers DF, Meij P, Vervoort MB, Vos W, Scheper RJ, Meijer CJ, Bloemena E, Middeldorp JM. 2000. Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol 165: 663670.
  • Eliopoulos AG, Rickinson AB. 1998. Epstein–Barr virus: LMP1 masquerades as an active receptor. Curr Biol 8: R196R198.
  • Eliopoulos AG, Dawson CW, Mosialos G, Floettmann JE, Rowe M, Armitage RJ, Dawson J, Zapata JM, Kerr DJ, Wakelam MJ, Reed JC, Kieff E, Young LS. 1996. CD40-induced growth inhibition in epithelial cells is mimicked by Epstein–Barr virus-encoded LMP1: Involvement of TRAF3 as a common mediator. Oncogene 13: 22432254.
  • Eliopoulos AG, Stack M, Dawson CW, Kaye KM, Hodgkin L, Sihota S, Rowe M, Young LS. 1997. Epstein–Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors. Oncogene 14: 28992916.
  • Fahraeus R, Rymo L, Rhim JS, Klein G. 1990. Morphological transformation of human keratinocytes expressing the LMP gene of Epstein–Barr virus. Nature 345: 447449.
  • Fahraeus R, Chen W, Trivedi P, Klein G, Obrink B. 1992. Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells. Int J Cancer 52: 834838.
  • Falk K, Gratama JW, Rowe M, Zou JZ, Khanim F, Young LS, Oosterveer MA, Ernberg I. 1995. The role of repetitive DNA sequences in the size variation of Epstein–Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis. J Gen Virol 76: 779790.
  • Faulkner GC, Burrows SR, Khanna R, Moss DJ, Bird AG, Crawford DH. 1999. X-Linked agammaglobulinemia patients are not infected with Epstein–Barr virus: Implications for the biology of the virus. J Virol 73: 15551564.
  • Fingeroth JD, Weis JJ, Tedder TF, Strominger JL, Biro PA, Fearon DT. 1984. Epstein–Barr virus receptor of human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci USA 81: 45104514.
  • Fries KL, Miller WE, Raab-Traub N. 1996. Epstein–Barr virus latent membrane protein 1 blocks p53-mediated apoptosis through the induction of the A20 gene. J Virol 70: 86538659.
  • Frisan T, Sjoberg J, Dolcetti R, Boiocchi M, DeRe V, Carbone A, Brautbar C, Battat S, Biberfeld P, Eckman M, Ost O, Christensson B, Sundstrom C, Bjorkholm M, Pisa P, Masucci MG. 1995. Local suppression of Epstein–Barr virus (EBV)-specific cytotoxicity in biopsies of EBV-positive Hodgkin's disease. Blood 86: 14931501.
  • Frisan T, Levitsky V, Polack A, Masucci MG. 1998. Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines. J Immunol 160: 32813289.
  • Frisan T, Levitsky V, Masucci MG. 2000. Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells. Int J Cancer 88: 881888.
  • Gires O, Kohlhuber F, Kilger E, Baumann M, Kieser A, Kaiser C, Zeidler R, Scheffer B, Ueffing M, Hammerschmidt W. 1999. Latent membrane protein 1 of Epstein–Barr virus interacts with JAK3 and activates STAT proteins. EMBO J 18: 30643073.
  • Gottschalk S, Ng CY, Perez M, Smith CA, Sample C, Brenner MK, Heslop HE, Rooney CM. 2001. An Epstein–Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs. Blood 97: 835843.
  • Gratama JW, Oosterveer MA, Zwaan FE, Lepoutre J, Klein G, Ernberg I. 1988. Eradication of Epstein–Barr virus by allogeneic bone marrow transplantation: Implications for sites of viral latency. Proc Natl Acad Sci USA 85: 86938696.
  • Greenspan JS, Greenspan D, Lennette ET, Abrams DI, Conant MA, Petersen V, Freese UK. 1985. Replication of Epstein–Barr virus within the epithelial cells of oral “hairy” leukoplakia, an AIDS-associated lesion. N Engl J Med 313: 15641571.
  • Grossman SR, Johannsen E, Tong X, Yalamanchili R, Kieff E. 1994. The Epstein–Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein. Proc Natl Acad Sci USA 91: 75687572.
  • Groux H, Cottrez F, Montpellier C, Quatannens B, Coll J, Stehelin D, Auriault C. 1997. Isolation and characterization of transformed human T-cell lines infected by Epstein–Barr virus. Blood 89: 45214530.
  • Hecht JL, Aster JC. 2000. Molecular biology of Burkitt's lymphoma. J Clin Oncol 18: 37073721.
  • Heessen S, Leonchiks A, Issaeva N, Sharipo A, Selivanova G, Masucci MG, Dantuma NP. 2002. Functional p53 chimeras containing the Epstein–Barr virus Gly-Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis. Proc Natl Acad Sci USA 99: 15321537.
  • Henderson S, Rowe M, Gregory C, Croom-Carter D, Wang F, Longnecker R, Kieff E, Rickinson A. 1991. Induction of bcl-2 expression by Epstein–Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65: 11071115.
  • Henkel T, Ling PD, Hayward SD, Peterson MG. 1994. Mediation of Epstein–Barr virus EBNA2 transactivation by recombination signal-binding protein J kappa. Science 265: 9295.
  • Holmgren L, Szeles A, Rajnavolgyi E, Folkman J, Klein G, Ernberg I, Falk KI. 1999. Horizontal transfer of DNA by the uptake of apoptotic bodies. Blood 93: 39563963.
  • Iezzoni JC, Gaffey MJ, Weiss LM. 1995. The role of Epstein–Barr virus in lymphoepithelioma-like carcinomas. Am J Clin Pathol 103: 308315.
  • Ikeda M, Ikeda A, Longan LC, Longnecker R. 2000. The Epstein–Barr virus latent membrane protein 2A PY motif recruits WW domain-containing ubiquitin-protein ligases. Virology 268: 178191.
  • Imai S, Koizumi S, Sugiura M, Tokunaga M, Uemura Y, Yamamoto N, Tanaka S, Sato E, Osato T. 1994. Gastric carcinoma: Monoclonal epithelial malignant cells expressing Epstein–Barr virus latent infection protein. Proc Natl Acad Sci USA 91: 91319135.
  • Imai S, Sugiura M, Oikawa O, Koizumi S, Hirao M, Kimura H, Hayashibara H, Terai N, Tsutsumi H, Oda T, Chiba S, Osato T. 1996. Epstein–Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection. Blood 87: 14461457.
  • Imai S, Nishikawa J, Takada K. 1998. Cell-to-cell contact as an efficient mode of Epstein–Barr virus infection of diverse human epithelial cells. J Virol 72: 43714378.
  • International Agency for Research on Cancer. 1997. Epstein–Barr virus and Kaposi's sarcoma herpesvirus/human herpesvirus 8. IARC monographs on the evaluation of carcinogenic risks to humans. Vol. 70. Lyon, France: WHO.
  • Jansson A, Masucci M, Rymo L. 1992. Methylation of discrete sites within the enhancer region regulates the activity of the Epstein–Barr virus BamHI W promoter in Burkitt lymphoma lines. J Virol 66: 6269.
  • Jenson HB, Leach CT, McClain KL, Joshi VV, Pollock BH, Parmley RT, Chadwick EG, Murphy SB. 1997. Benign and malignant smooth muscle tumors containing Epstein–Barr virus in children with AIDS. Leuk Lymphoma 27: 303314.
  • Johannsen E, Miller CL, Grossman SR, Kieff E. 1996. EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein–Barr virus-transformed B lymphocytes. J Virol 70: 41794183.
  • Kaye KM, Izumi KM, Kieff E. 1993. Epstein–Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc Natl Acad Sci USA 90: 91509154.
  • Khanna R, Slade RW, Poulsen L, Moss DJ, Burrows SR, Nicholls J, Burrows JM. 1997. Evolutionary dynamics of genetic variation in Epstein–Barr virus isolates of diverse geographical origins: Evidence for immune pressure-independent genetic drift. J Virol 71: 83408346.
  • Khanna R, Busson P, Burrows SR, Raffoux C, Moss DJ, Nicholls JM, Cooper L. 1998. Molecular characterization of antigen-processing function in nasopharyngeal carcinoma (NPC): Evidence for efficient presentation of Epstein–Barr virus cytotoxic T-cell epitopes by NPC cells. Cancer Res 58: 310314.
  • Kieff E. 1996. Epstein–Barr virus and its replication. In: FieldsBN, KnipeDM, HowleyPM, editors. Virology. Philadelphia: Lippincott, Raven Publishers, pp 23432396.
  • Kisselev AF, Akopian TN, Woo KM, Goldberg AL. 1999. The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274: 33633371.
  • Knecht H, Berger C, Rothenberger S, Odermatt BF, Brousset P. 2001. The role of Epstein–Barr virus in neoplastic transformation. Oncology 60: 289302.
  • Knowles DM, Chamulak GA, Subar M, Burke JS, Dugan M, Wernz J, Slywotzky C, Pelicci G, Dalla-Favera R, Raphael B. 1988. Lymphoid neoplasia associated with the acquired immunodeficiency syndrome (AIDS). The New York University Medical Center experience with 105 patients (1981–1986). Ann Intern Med 108: 744753.
  • Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Dorken B, Scheidereit C. 1999. Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed–Sternberg cells. Oncogene 18: 943953.
  • Kulwichit W, Edwards RH, Davenport EM, Baskar JF, Godfrey V, Raab-Traub N. 1998. Expression of the Epstein–Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice. Proc Natl Acad Sci USA 95: 1196311968.
  • Laherty CD, Hu HM, Opipari AW, Wang F, Dixit VM. 1992. The Epstein–Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. J Biol Chem 267: 2415724160.
  • Lee SP, Constandinou CM, Thomas WA, Croom-Carter D, Blake NW, Murray PG, Crocker J, Rickinson AB. 1998. Antigen presenting phenotype of Hodgkin Reed–Sternberg cells: Analysis of the HLA class I processing pathway and the effects of interleukin 10 on Epstein–Barr virus-specific cytotoxic T cell recognition. Blood 92: 10201030.
  • Lee SP, Chan AT, Cheung ST, Thomas WA, CroomCarter D, Dawson CW, Tsai CH, Leung SF, Johnson PJ, Huang DP. 2000. CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells. J Immunol 165: 573582.
  • Leight ER, Sugden B. 2000. EBNA-1: A protein pivotal to latent infection by Epstein–Barr virus. Rev Med Virol 10: 83100.
  • Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen PM, Klein G, Kurilla MG, Masucci MG. 1995. Inhibition of antigen processing by the internal repeat region of the Epstein–Barr virus nuclear antigen-1. Nature 375: 685688.
  • Levitskaya J, Sharipo A, Leonchiks A, Ciechanover A, Masucci MG. 1997. Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1. Proc Natl Acad Sci USA 94: 1261612621.
  • Levitsky V, Zhang QJ, Levitskaya J, Kurilla MG, Masucci MG. 1997. Natural variants of the immunodominant HLA A11-restricted CTL epitope of the EBV nuclear antigen-4 are nonimmunogenic due to intracellular dissociation from MHC class I: Peptide complexes. J Immunol 159: 53835390.
  • Lin CT, Lin CR, Tan GK, Chen W, Dee AN, Chan WY. 1997. The mechanism of Epstein–Barr virus infection in nasopharyngeal carcinoma cells. Am J Pathol 150: 17451756.
  • Longnecker R. 2000. Epstein–Barr virus latency: LMP2, a regulator or means for Epstein–Barr virus persistence? Adv Cancer Res 79: 175200.
  • Longnecker R, Miller CL, Miao XQ, Marchini A, Kieff E. 1992. The only domain which distinguishes Epstein–Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential. J Virol 66: 64616469.
  • Martin J, Sugden B. 1991. Transformation by the oncogenic latent membrane protein correlates with its rapid turnover, membrane localization, and cytoskeletal association. J Virol 65: 32463258.
  • Masucci MG, Ernberg I. 1994. Epstein–Barr virus: Adaptation to a life within the immune system. Trends Microbiol 2: 125130.
  • Masucci MG, Contreras-Salazar B, Ragnar E, Falk K, Minarovits J, Ernberg I, Klein G. 1989. 5-Azacytidine up regulates the expression of Epstein–Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael. J Virol 63: 31353141.
  • Miller WE, Earp HS, Raab-Traub N. 1995. The Epstein–Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor. J Virol 69: 43904398.
  • Mitarnun W, Suwiwat S, Pradutkanchana J, Saechan V, Ishida T, Takao S, Mori A. 2002. Epstein–Barr virus-associated peripheral T-cell and NK-cell proliferative disease/lymphoma: Clinicopathologic, serologic, and molecular analysis. Am J Hematol 70: 3138.
  • Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson DA. 1997. Identification of the site of Epstein–Barr virus persistence in vivo as a resting B cell. J Virol 71: 48824891.
  • Moorthy RK, Thorley-Lawson DA. 1993. All three domains of the Epstein–Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts. J Virol 67: 16381646.
  • Mosialos G, Birkenbach M, Yalamanchili R, Van Arsdale T, Ware C, Kieff E. 1995. The Epstein–Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80: 389399.
  • Mukherjee S, Trivedi P, Dorfman DM, Klein G, Townsend A. 1998. Murine cytotoxic T lymphocytes recognize an epitope in an EBNA-1 fragment, but fail to lyse EBNA-1-expressing mouse cells. J Exp Med 187: 445450.
  • Murray PG, Constandinou CM, Crocker J, Young LS, Ambinder RF. 1998. Analysis of major histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein–Barr virus-positive Hodgkin's disease. Blood 92: 24772483.
  • Nicholson LJ, Hopwood P, Johannessen I, Salisbury JR, Codd J, Thorley-Lawson D, Crawford DH. 1997. Epstein–Barr virus latent membrane protein does not inhibit differentiation and induces tumorigenicity of human epithelial cells. Oncogene 15: 275283.
  • Niedobitek G, Fahraeus R, Herbst H, Latza U, Ferszt A, Klein G, Stein H. 1992. The Epstein–Barr virus encoded membrane protein (LMP) induces phenotypic changes in epithelial cells. Virchows Arch B Cell Pathol Incl Mol Pathol 62: 5559.
  • Niedobitek G, Agathanggelou A, Herbst H, Whitehead L, Wright DH, Young LS. 1997. Epstein–Barr virus (EBV) infection in infectious mononucleosis: Virus latency, replication, and phenotype of EBV-infected cells. J Pathol 182: 151159.
  • Nomori H, Kameya T, Shimosato Y, Saito H, Ebihara S, Ono I. 1985. Nasopharyngeal carcinoma: Immunohistochemical study for keratin, secretory component, and leukocyte common antigen. Jpn J Clin Oncol 15: 95105.
  • Ohshima K, Haraoka S, Harada N, Kamimura T, Suzumiya J, Kanda M, Kawasaki C, Sugihara M, Kikuchi M. 2000. Hepatosplenic gammadelta T-cell lymphoma: Relation to Epstein–Barr virus and activated cytotoxic molecules. Histopathology 36: 127135.
  • Osato T, Imai S. 1996. Epstein–Barr virus and gastric carcinoma. Semin Cancer Biol 7: 175182.
  • Osborne B, Miele L. 1999. Notch and the immune system. Immunity 11: 653663.
  • Oudejans JJ, Jiwa NM, Kummer JA, Horstman A, Vos W, Baak JA, Kluin PM, Vandervalk P, Walboomers JM, Meijer CM. 1996. Analysis of major histocompatibility complex class I expression on Reed–Sternberg cells in relation to the cytotoxic T cell response in Epstein–Barr virus-positive and Epstein–Barr virus-negative Hodgkin's disease. Blood 87: 38443851.
  • Paterson RL, Kelleher C, Amankonah TD, Streib JE, Xu JW, Jones JF, Gelfand EW. 1995. Model of Epstein–Barr virus infection of human thymocytes: Expression of viral genome and impact on cellular receptor expression in the T-lymphoblastic cell line, HPB-ALL. Blood 85: 456464.
  • Pathmanathan R, Prasad U, Sadler R, Flynn K, Raab-Traub N. 1995. Clonal proliferations of cells infected with Epstein–Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med 333: 693698.
  • Quintanilla-Martinez L, Kumar S, Fend F, Reyes E, Teruya-Feldstein J, Kingma DW, Sorbara L, Raffeld M, Straus SE, Jaffe ES. 2000. Fulminant EBV+ T-cell lymphoproliferative disorder following acute/chronic EBV infection: A distinct clinicopathologic syndrome. Blood 96: 443451.
  • Raab-Traub N, Flynn K. 1986. The structure of the termini of the Epstein–Barr virus as a marker of clonal cellular proliferation. Cell 47: 883889.
  • Rickinson AB, Kieff E. 1996. Epstein–Barr virus. In: FieldsBN, HowleyPM, KnipeDM, editors. Fields virology. Philadelphia, PA: Lippincott-Raven Publishers, pp 23972446.
  • Rickinson AB, Lee SP, Steven NM. 1996. Cytotoxic T lymphocyte responses to Epstein–Barr virus. Curr Opin Immunol 8: 492497.
  • Robertson ES, Grossman S, Johannsen E, Miller C, Lin J, Tomkinson B, Kieff E. 1995. Epstein–Barr virus nuclear protein 3C modulates transcription through interaction with the sequence-specific DNA-binding protein J kappa. J Virol 69: 31083116.
  • Rooney CM, Aguilar LK, Huls MH, Brenner MK, Heslop HE. 2001. Adoptive immunotherapy of EBV-associated malignancies with EBV-specific cytotoxic T-cell lines. Curr Top Microbiol Immunol 258: 221229.
  • Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. 1993. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75: 495505.
  • Scholle F, Longnecker R, Raab-Traub N. 1999. Epithelial cell adhesion to extracellular matrix proteins induces tyrosine phosphorylation of the Epstein–Barr virus latent membrane protein 2: A role for C-terminal Src kinase. J Virol 73: 47674775.
  • Scholle F, Bendt KM, Raab-Traub N. 2000. Epstein–Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt. J Virol 74: 1068110689.
  • Sharipo A, Imreh M, Leonchiks A, Imreh S, Masucci MG. 1998. A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: A new mechanism for selective inhibition of proteolysis. Nat Med 4: 939944.
  • Sheu LF, Chen A, Meng CL, Ho KC, Lee WH, Leu FJ, Chao CF. 1996. Enhanced malignant progression of nasopharyngeal carcinoma cells mediated by the expression of Epstein–Barr nuclear antigen 1 in vivo. J Pathol 180: 243248.
  • Sixbey JW, Yao QY. 1992. Immunoglobulin A-induced shift of Epstein–Barr virus tissue tropism. Science 255: 15781580.
  • Speck P, Haan KM, Longnecker R. 2000. Epstein–Barr virus entry into cells. Virology 277: 15.
  • Staudt LM. 2000. The molecular and cellular origins of Hodgkin's disease. J Exp Med 191: 207212.
  • Swart R, Ruf IK, Sample J, Longnecker R. 2000. Latent membrane protein 2A-mediated effects on the phosphatidylinositol 3-Kinase/Akt pathway. J Virol 74: 1083810845.
  • Tao Q, Ho FC, Loke SL, Srivastava G. 1995a. Epstein–Barr virus is localized in the tumour cells of nasal lymphomas of NK, T or B cell type. Int J Cancer 60: 315320.
  • Tao Q, Srivastava G, Chan AC, Chung LP, Loke SL, Ho FC. 1995b. Evidence for lytic infection by Epstein–Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals. J Med Virol 45: 7177.
  • Thorley-Lawson DA. 2001. Epstein–Barr virus: Exploiting the immune system. Nat Rev Immunol 1: 7582.
  • Tsoukas CD, Lambris JD. 1993. Expression of EBV/C3d receptors on T cells: Biological significance. Immunol Today 14: 5659.
  • Uchida J, Yasui T, Takaoka-Shichijo Y, Muraoka M, Kulwichit W, Raab-Traub N, Kikutani H. 1999. Mimicry of CD40 signals by Epstein–Barr virus LMP1 in B lymphocyte responses. Science 286: 300303.
  • Vivanco I, Sawyers CL. 2002. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2: 489501.
  • Waltzer L, Logeat F, Brou C, Israel A, Sergeant A, Manet E. 1994. The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein–Barr virus EBNA2 protein to its DNA responsive elements. EMBO J 13: 56335638.
  • Waltzer L, Perricaudet M, Sergeant A, Manet E. 1996. Epstein–Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA. J Virol 70: 59095915.
  • Wang D, Liebowitz D, Kieff E. 1985. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43: 831840.
  • Wang S, Rowe M, Lundgren E. 1996. Expression of the Epstein–Barr virus transforming protein LMP1 causes a rapid and transient stimulation of the Bcl-2 homologue Mcl-1 levels in B-cell lines. Cancer Res 56: 46104613.
  • Watry D, Hedrick JA, Siervo S, Rhodes G, Lamberti JJ, Lambris JD, Tsoukas CD. 1991. Infection of human thymocytes by Epstein–Barr virus. J Exp Med 173: 971980.
  • Wilson JB, Weinberg W, Johnson R, Yuspa S, Levine AJ. 1990. Expression of the BNLF-1 oncogene of Epstein–Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell 61: 13151327.
  • Wilson JB, Bell JL, Levine AJ. 1996. Expression of Epstein–Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice. EMBO J 15: 31173126.
  • Winberg G, Matskova L, Chen F, Plant P, Rotin D, Gish G, Ingham R, Ernberg I, Pawson T. 2000. Latent membrane protein 2A of Epstein–Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases. Mol Cell Biol 20: 85268535.
  • Xu J, Ahmad A, Menezes J. 2002. Preferential localization of the Epstein–Barr virus (EBV) oncoprotein LMP-1 to nuclei in human T cells: Implications for its role in the development of EBV genome-positive T-cell lymphomas. J Virol 76: 40804086.
  • Yalamanchili R, Tong X, Grossman S, Johannsen E, Mosialos G, Kieff E. 1994. Genetic and biochemical evidence that EBNA 2 interaction with a 63-kDa cellular GTG-binding protein is essential for B lymphocyte growth transformation by EBV. Virology 204: 634641.
  • Yao QY, Rickinson AB, Epstein MA. 1985. A re-examination of the Epstein–Barr virus carrier state in healthy seropositive individuals. Int J Cancer 35: 3542.
  • Yates J, Warren N, Reisman D, Sugden B. 1984. A cis-acting element from the Epstein–Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci USA 81: 38063810.
  • Yoshizaki T, Horikawa T, Qing-Chun R, Wakisaka N, Takeshita H, Sheen TS, Lee SY, Sato H, Furukawa M. 2001. Induction of interleukin-8 by Epstein–Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma. Clin Cancer Res 7: 19461951.
  • Young LS, Dawson CW, Eliopoulos AG. 2000. The expression and function of Epstein–Barr virus encoded latent genes. J Clin Pathol Mol Pathol 53: 238247.
  • Zhao B, Marshall DR, Sample CE. 1996. A conserved domain of the Epstein–Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa. J Virol 70: 42284236.
  • Zimber-Strobl U, Strobl LJ. 2001. EBNA2 and Notch signalling in Epstein–Barr virus mediated immortalization of B lymphocytes. Semin Cancer Biol 11: 423434.
  • Zimber-Strobl U, Kempkes B, Marschall G, Zeidler R, Van Kooten C, Banchereau J, Bornkamm GW, Hammerschmidt W. 1996. Epstein–Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival. EMBO J 15: 70707078.