Activated Notch1 Can Transiently Substitute for EBNA2 in the Maintenance of Proliferation of LMP1-Expressing Immortalized B Cells

Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-J/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G 2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells. Epstein-Barr virus (EBV), a lymphotropic human gammaherpesvirus, is associated with several human malignancies. It infects primary resting B cells and induces unlimited proliferation of virus-infected cells in vitro (so called EBV-immortalized lymphoblastoid cell lines). In these cell lines, only a subset of viral genes that code for six EBV nuclear proteins (EBNA1, -2, -3A, -3B, -3C, and -LP) and three latent membrane protein antigens (LMP1, -2A, and -2B) are expressed. Five of these, EBNA1, -2, -3A, and -3C and LMP1, appear to be absolutely required for B-cell immortalization (33).EBNA2 and LMP1 most likely contribute to B-cell immortalization by activation of cellular pathways physiologically dependent on ligand-receptor interactions. LMP1 mimics a constitutively activated receptor of the tumor necrosis factor receptor family (11,14,47) and shares functional characteristics with CD40 (35,64,70). Both molecules induce NF-B (5, 16, 37), stress-activated protein kinases (4,11,34), and the JAK/STAT pathway (13,18,30). Through activation of these pathways, LMP1 induces B-cell activation markers and cell adhesion molecules (33) and enhances the viability of nonproliferating B cells (70).EBNA2 is essential for initiation and maintenance of B-cell immortalization (7,17,31). Acting as a transcriptional activator, it modulates the transcription of different cellular genes, including the B-cell activation markers CD21 and CD23 and the proto-oncogenes c-fgr (33) and c-myc (28). In addition, it activates transcription of the viral RNAs coding for the various EBV nuclear and membrane proteins (33). EBNA2 does not bind to DNA directly (41, 68), but is recruited to EBNA2-responsive elements by interacting with the transcription factors RBP-J (also called ...

EBNA2 and Activated Notch Induce Expression of BATF

“…The ability of cellular Notch proteins to activate gene transcription is also facilitated by its interaction with CBF1 (72; reviewed in references 2 and 51), implying that there may be overlap between the genes induced by Notch signaling and those up-regulated during EBV latency (26). In support of this, constitutively active Notch can substitute for EBNA2 in B-cell immortalization (16,22). Interestingly, of the identified cellular genetic targets of EBNA2 in B cells (8,30,32,38,69), only CD21 is a common target for up-regulation by EBNA2 and activated Notch (71).…”

“…The Notch family of proteins is comprised of transmembrane receptor proteins that are processed into nucleartargeted transcriptional regulators following activation (reviewed in references 2 and 51). In recent years, a number of studies have shown that activated Notch functions in a way similar to that of EBNA2 to positively impact cellular gene expression via an interaction with CBF1 (26,45,70,72) and can function as a substitute for EBNA2 in B-cell immortalization (16,22). To examine if Notch activates BATF gene expression, a constitutively activated derivative of Notch1 (mNotch IC) was expressed in BJAB cells.…”

Maintenance of Long Term γ-Herpesvirus B Cell Latency Is Dependent on CD40-Mediated Development of Memory B Cells

“…LMP2a is a B cell receptor surrogate that is expressed in latently infected memory cells and is thought to mimic the normal B cell receptor-mediated survival signals (43). EBNA2 blocks differentiation and takes over cellular control of cell cycling (44,45). MHV-68 does not encode structural homologs of these EBV genes (28) and appears to be dependent on activation of naive B cells via normal Ag and T celldependent signaling mechanisms.…”

γ-Secretase inhibitors suppress the growth of leukemia and lymphoma cells

“…Three Burkitt lymphoma cell lines, Ramos, Daudi and Raji, were supplied by the Japanese Cancer Research Resources Bank. Raji is positive for EBNA2 protein, which is reported to activate the Notch signaling pathway bypassing Notch protein (10,11). A diffuse large B-cell lymphoma cell line, MD901, was donated by Dr T. Miki.…”