Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin K locus composed of a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype ofEBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern ofviral and cellular genes rather than its germinal center origin.

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Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) shares protein motifs with the B-cell receptor that play a role in B-cell receptor signalling and has been shown to mimic an activated B-cell receptor by providing a survival signal for mature B cells in transgenic mice. Conversely, LMP2A has been reported not to support but to inhibit B-cell receptor signalling with respect to virus reactivation and to block lytic virus induction after anti-Ig treatment of EBV-infected B cells. To solve this apparent paradox, the role of LMP2A in lytic-cycle induction was re-examined in B cells conditionally immortalized by EBV. It was shown that, in the absence of other stimuli, LMP2A expression alone could lead to induction of the virus lytic cycle. Similarly to B-cell receptor stimulation by anti-Ig treatment, this LMP2A-mediated reactivation was dependent on the mitogen-activated protein kinase pathway and could be inhibited by the viral LMP1. Our data reinforce the notion that LMP2A is a functional homologue of the B-cell receptor, not only with respect to B-cell survival but also with respect to regulation of the lytic cycle. INTRODUCTIONEpstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus. In the vast majority of cases, viral infection is benign, yet EBV is associated with a number of human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, lymphoproliferative diseases in immunocompromised individuals and Hodgkin's disease (Rickinson & Kieff, 2001). After primary infection, EBV persists for the life of the host in the peripheral blood in resting memory B cells (Thorley-Lawson, 2001). Virus reactivation from these cells is strictly controlled and mainly observed in immunocompromised individuals (Lam et al., 1991;Decker et al., 1996; Babcock et al., 1999). Latent membrane protein 2A (LMP2A) transcripts have been detected in memory B cells, the site of EBV persistence in the periphery, by RT-PCR (Qu & Rowe, 1992;Tierney et al., 1994;Miyashita et al., 1997;Babcock et al., 1998 Babcock et al., , 1999), yet analysis of the EBV latent geneexpression pattern at the single-cell level has revealed that none of the viral latent genes is expressed in latently infected memory B cells (Hochberg et al., 2004a, b). In contrast to peripheral EBV-positive B cells, memory B cells in tonsils exhibit consistent expression of LMP2A together with the latent viral nuclear protein EBNA1 and the viral LMP1 . The expression of LMP2A and LMP1 in tonsillar memory B cells is believed to provide a survival signal to peripheral latently EBV-infected cells and to rescue the cells from apoptosis when they migrate through secondary lymphoid organs. LMP1 and LMP2A may thus contribute significantly to long-term survival of EBV-positive memory B cells in vivo.LMP2A is an integral membrane protein with 12 hydrophobic transmembrane domains and cytoplasmic N and C termini (Laux et al., 1988(Laux et al., , 1989Rowe et al., 1990). Similarly to the intracellular domain of the B-cell receptor, the N terminus of ...

Epstein-Barr virus (EBV) transforms primary B cellsBurkitt's lymphoma (BL) is a human B-cell neoplasia that occurs classically as a childhood malignancy with high incidence in areas with holoendemic malarial infection (Nesbit et al., 1999). BL cells are characterized by reciprocal chromosomal translocations involving the gene loci of the protooncogene c-myc (myc) and of immunoglobulin genes. The juxtaposition of immunoglobulin gene enhancers results in the oncogenic activation of myc (Henriksson & Lu$ scher, 1996).

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