Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cel line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cels. These initial observations are now extended through an expanded study of EBNA-and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative Blymphoma cel line BL41 and the EBV-positive Burkitt's lymphoma cel line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2-and LMPl-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21. Si analyses revealed that BJAB cells express low levels of FcERIIa CD23 mRNA, and FceRIIb CD23 mRNA was not detectable. LMIP1 preferentially increases FceRIIb CD23 mRNA. EBNA-2 expression alone in BJAB increases the constitutively expressed FceRlla CD23 mRNA. However, when coexpressed with LMP1, EBNA-2 increases total CD23 mRNA without altering the high relative abundance of FcpRIIb to FcrRHla CD23 mRNA induced by LMP1. Thus, LMP1 likely activates the FcrRHb CD23 promoter, while EBNA-2 more likely transactivates a regulatory element common to bot...

There is a growing interest in using antigen-specific T cells for the treatment of human malignancy. For example, adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been effective prophylaxis and treatment of EBV-associated lymphoproliferative disease in immunocompromised patients. For all immunotherapies, however, there has been a hypothetical concern that mutations in tumor-specific antigens may lead to tumor escape. We now demonstrate that such events may indeed occur, with lethal outcome. A patient who developed lymphoma after marrow transplantation received donor-derived, EBV-specific CTLs but died with progressive disease. The tumor cells proved substantially less sensitive to cytolysis than the EBV-transformed B-cell line used for CTL generation. The major cytolytic activity of the donor CTL was directed against 2 HLA-A11-restricted epitopes in the viral EBNA-3B antigen. Sequence analysis of this gene in the tumor virus revealed a 245-base pair deletion, which removed these 2 CTL epitopes. Hence, the viral antigen in the tumor had mutated in a way that allowed escape from CTLs. Analysis of EBV polymorphisms demonstrated that before CTL infusion, more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant strain in vivo. Such an occurrence, even when polyclonal CTL lines are used against genetically stable virus antigens, suggests that escape mutants may be a serious problem when CTL therapy is directed against more unstable tumor cell-derived targets.

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein J, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. J DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind J activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of J. The human herpesvirus Epstein-Barr virus (EBV) establishes a latent infection within B lymphocytes that is maintained for the lifetime of the host. Since most EBV-related diseases occur years to decades after primary infection, the establishment of a latent infection is an essential step in the development of EBV-associated malignancies. Following EBV infection in vitro, primary B lymphocytes are immortalized and able to proliferate indefinitely in culture. Of the 12 viral genes expressed during latency in these cells, 6 encode proteins considered essential for efficient EBV-mediated immortalization in vitro: EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3A, EBNA-3C, EBNA-LP, and latent membrane protein 1 (LMP-1) (10,18,23,28,29,50,52).The molecular basis for the role of the EBV oncoprotein LMP-1 in transformation is its ability to constitutively activate the tumor necrosis factor receptor signal transduction pathway (36). While LMP-1 is capable of transforming immortal rodent cell lines (11), overexpression of LMP-1 in B cells results in cytotoxicity or cytostasis (15,31). The expression of LMP-1 in EBV-transformed B lymphocytes is regulated by the concerted actions of viral and cellular proteins through promoter elements targeted by ubiquitous as well as B-cell-specific proteins. One key regulator of LMP-1 expression, EBNA-2, activates transcription through interactions with cellular proteins, including J (for which there are two binding sites in the LMP-1 promoter, located in the regions from bp Ϫ298 to Ϫ290 and from bp Ϫ223 to Ϫ213) and Spi-1/Spi-B, related proteins of the ets family of transcription factors that bind to a single site in the LMP-1 promoter (bp Ϫ169 to Ϫ158) (17,20,22,24,25,27,48). Not only is J the downstream signaling protein of the Notch pathway, but it directly interacts with the intracellular domain of the Notch protein. Following activation of Notch, the intracellular domain is released by proteolysis and migrates to the nucleus to bind to DNA through its interaction with J (16).

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Epstein-Barr virus (EBV) expresses six nudear antigens (EBNAs) and three integral latent membrane proteins (LMPs) in latently infected growth-transformed B lymphoblastoid cell lines (LCLs). In contrast, EBV protein expression in Burkitt lymphoma tissue or in newly established Burkitt lymphoma cell lines is frequently restricted to the EBV genome maintenance protein, EBNA-1. EBNA-1 expression in the absence of other EBNAs and LMP-1 has been an enigma since, in LCLs, all EBNA mRNAs are processed from a single transcript. We now show that the basis for restricted EBV expression in Burkitt lymphoma cells is selective EBNA-1 mRNA transcription from a hitherto unrecognized promoter that is 50 kb closer to the EBNA-l-encoding exon than previously described EBNA-1 promoters. Infected cells with EBNA-1-restricted expression could preferentially persist in vivo in the face of EBV-immune T-cell responses, which are frequently directed against other EBNAs and are also dependent on LMP-1 expression.

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.Epstein-Barr virus | epithelial | organotypic culture | productive replication A lthough the association between Epstein-Barr virus and epithelial malignancies has been known for more than three decades, the EBV life cycle within the epithelial milieu is still only poorly understood. In contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and by the ability of some EBV-positive B-cell tumors to give rise to cell lines that maintain restricted programs of latency gene expression similar to those seen in vivo. Although in primary EBV infection the entire complement of EBV latencyassociated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP) and membrane proteins (LMPs 1, 2A, and 2B) promote cellular proliferation and survival (Latency III), EBV gene expression must be progressively silenced (Latency II; EBNA1 and LMPs 1 and 2) so that the most restricted program, Latency 0 (in which EBV gene expression is believed to be completely silenced

Epstein-Barr virus (EBV) antigens in tumor tissue define associations of virus with human malignancies and provide clues as to mechanisms of viral oncogenesis. In Burkitt's lymphoma, EBV markers are absent from 85% of sporadic cases and 4% of endemic (African) cases, raising questions as to the exact role EBV in the disease. Standard screening criteria may be insufficient to determine the EBV status of all tumors. One of 9 tumors from American patients expressed EBV nuclear antigen 1 (EBNA1) and contained standard episomal EBV DNA, making this series consistent with the 15% EBV association traditionally ascribed to sporadic Burkitt's lymphoma. Surprisingly, 3 tumors without detectable EBNA1 contained partial EBV genomes. Identification of defective, integrated viral DNA in some tumors indicates greater involvement of virus in sporadic Burkitt's lymphoma than previously documented and suggests a process of viral DNA rearrangement and loss during malignant progression most consistent with an initiating role for EBV in tumorigenesis.

The only member of the Epstein-Barr virus family of nuclear proteins (EBNAs) expressed during type I and type II latent infections is EBNA-1. This is in contrast to type III latency, during which all six nuclear proteins are expressed from a common transcription unit. The exclusive expression of EBNA-1 during type I and II latency is mediated through a recently identified promoter, Fp. The objective of this study was to characterize Fp in the Burkitt lymphoma cell background, where it is known to be differentially utilized. Using a short-term transfection assay and reporter gene plasmids containing Fp linked to the human growth hormone, we examined Fp activity in type I and type III latently infected and virus-negative Burkitt lymphoma cells. The data suggested that Fp is predominantly regulated through two distinct elements located between +24 and +270 relative to the transcription start site. One element positively mediates Fp activity, probably at the level of transcription, and acts in a virus-independent manner. The second element contains the EBNA-1 DNA binding domain III and negatively regulates Fp-directed gene expression in trans with EBNA-1 in type III as well as type I latency. Thus, we have identified a third function of EBNA-1, i.e., that of a repressor of gene expression, in addition to its known role in viral DNA replication and its ability to trans-activate gene expression. The overall activity of Fp in type I latently infected Burkitt cells was approximately sixfold lower than in virus-negative Burkitt cells, in which there is no autoregulation, suggesting that there is a fine balance between these two opposing regulatory elements during type I latency.