Epstein-Barr Virus-Encoded Bcl-2 Homologue Functions as a Survival Factor in Wp-Restricted Burkitt Lymphoma Cell Line P3HR-1

Burkitt lymphoma (BL) is etiologically associated with Epstein-Barr virus (EBV). EBV-positive BL tumors display two latent forms of infection. One is referred to as latency I infection, in which EBV expresses the virus genome maintenance protein EBNA1 as the only viral protein. The other is referred to as Wp-restricted latency and was recently identified in a subset of BL tumors. In these tumors, EBV expresses EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the viral Bcl-2 homologue BHRF1, all of which are driven by the BamHI W promoter (Wp). To investigate the role of EBV in Wp-restricted BL, we conditionally expressed a dominant-negative EBNA1 (dnEBNA1) mutant which interrupts the virus genome maintenance function of EBNA1 in the P3HR-1 BL cell line. Induction of dnEBNA1 expression caused loss of the EBV genome and resulted in apoptosis of P3HR-1 cells in the absence of exogenous apoptosis inducers, indicating that P3HR-1 cells cannot survive without EBV. Stable transfection of the BHRF1 gene into P3HR-1 cells rescued the cells from the apoptosis induced by dnEBNA1 expression, whereas stable transfection of truncated EBNA-LP, EBNA3A, or EBNA3C did not. Moreover, knockdown of BHRF1 expression in P3HR-1 cells resulted in increased cell death. These results indicate that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor. Our finding implies a critical contribution of BHRF1 to the pathogenesis of Wp-restricted BLs.Epstein-Barr virus (EBV), a gammaherpesvirus with B-cell growth-transforming ability, is causally implicated in Burkitt lymphoma (BL) (see reference 40 for a review). In vitro, EBV infection efficiently transforms human B cells into lymphoblastoid cell lines (LCLs), which display a transcription program called latency III, during which the virus expresses 6 nuclear proteins (EBNA1, -2, -3A, -3B, -3C, and -LP), 3 integral membrane proteins (LMP1, -2A, and -2B), 2 small nonpolyadenylated EBV-encoded RNAs (EBERs) (EBER1 and EBER2), BamHI A rightward transcripts (BARTs), and microRNAs (see reference 24 for a review). However, EBV-positive BL tumors express a limited set of viral genes and do not express EBNA2 or LMP1, which play essential roles in B-cell growth transformation. Two latency forms of infection are known in EBV-positive BLs. One is referred to as latency I infection, in which EBV expresses only the virus genome maintenance protein EBNA1 (whose expression is driven by the Qp promoter), EBERs, and BARTs. In addition to the classical latency I BLs, another subset of BLs that express EBNA1, EBNA3A, EBNA3B, EBNA3C, truncated EBNA-LP, EBERs, and BARTs was recently identified, and this form of infection is referred to as Wp-restricted latency because the expression of the EBNAs is driven by the Wp promoter (19). These Wp-restricted BLs are characterized by infection with an EBV that carries a deletion of the EBNA2 gene and its surrounding sequences (19). A more recent report has shown that Wp-restricted BLs express the EBV-encoded Bcl-2 homologue BHRF1 as a latent gene (21). BHRF1 was primarily identified as a member of the early antigen complex (38). However, it was also reported that BHRF1 could be expressed as a latent gene (3).The hallmark of BL tumors is reciprocal translocation between the c-myc gene and one of the immunoglobulin (Ig) loci, which leads to constitutive and deregulated expression of c-myc (26). The c-myc/Ig translocation appears to play a critical role in the pathogenesis of BL (30, 49). Activation of c-myc not only stimulates cell proliferation, but also increases susceptibility to apoptosis (6). In BLs, EBV is postulated to counteract the proapoptotic effects that are probably induced by c-myc activation, because several lines of evidence have demonstrated that EBV provides a survival advantage to BL cells. It has been shown that EBV-positive BL lines are more resistant to apoptotic triggers than EBV-negative BL lines (22, 28, 31, 36, 41). EBERs confer resistance to apoptosis induced by various stimuli in BL lines (27, 36). EBNA1 is also reported to have an antiapoptotic function. Inhibition of EBNA1 by a retrovirus vector expressing a dominant-negative derivative of EBNA1 (dnEBNA1) decreased survival and cloning efficiency in BL lines (23). Enforced expression of EBNA1 in EBV-negative cells inhibits apoptosis induced by p53 (23, 42). Furthermore, Wp-restricted BL lines are more resistant to apoptosis than latency I BL lines (20, 22). Some viral proteins that are expressed in Wp-restricted but not in latency I BLs are reported to have antiapoptotic functions. For example, the truncated form of EBNA-LP that is specifically expressed in Wp-restricted BLs confers resistance to apoptosis induced by verotoxin 1 or staurosporine (10). EBNA3A and EBNA3C cooperate to repress expression of the proapoptotic tumor suppressor Bim and contribute to resistance to apoptosis induced by cytotoxic agents, such as nocadazole and cisplatin (1). Enforced BHRF1 expression protects latency I BL cells from ionomycin-induced apoptosis (21). These previous reports demonstrate that various EBV gene products have the ability to confer resistance to apoptosis induced by exogenous apoptotic triggers. However, it is still unclear whether EBV is essential for the survival of EBV-positive BL cells in the absence of exogenous apoptotic stimuli and, if so, which viral gene plays a central role in the survival.The experiments reported here focus on the role of EBV in the growth and survival of Wp-restricted BL cells. We established a Wp-restricted P3HR-1 BL cell line in which dnEBNA1 was conditionally expressed in a tetracycline-regulated manner. The EBV genome disappeared from the cells after induction of dnEBNA1 expression because dnEBNA1 interrupted the virus genome maintenance function of EBNA1 (23, 25, 32, 37). The growth and survival of P3HR-1 cells in the absence and presence of dnEBNA1 expression were compared. Our data presented here clearly show that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor.