Epstein‐Barr virus nuclear antigen 2 (EBNA‐2) plays a crucial role in B cell immortalization by Epstein‐Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes. Recently, we showed that EBNA‐2 interacts with the TP1 promoter of EBV through a cellular protein. In this report we provide evidence that this protein is recombination signal binding protein (RBP)‐J kappa, highly conserved in evolution, and originally isolated by its ability to bind to the J kappa‐type V(D)J recombination signal sequence. To identify the cellular protein interacting with the TP1 promoter, we performed electrophoretic mobility shift assays using binding sequences of known transcription factors, that carry partial homology to the crucial sequences of the EBNA‐2 responsive element (EBNA‐2RE), as competitor. Competition assays revealed the RBP‐J kappa recognition site as a very efficient competitor of cellular TP1 promoter binding protein. In parallel, we purified the protein to homogeneity from Raji cells by two ion‐exchange columns and affinity purification using the EBNA‐2RE coupled to magnetic beads. Affinity purified fractions separated on SDS‐PAGE revealed a single predominant band after silver staining which was recognized by anti‐RBP‐J kappa monoclonal antibody. These purified fractions exhibited binding specificity for EBNA‐2RE and EBNA‐2. In vitro‐translated murine RBP‐2 cDNA reacted with EBNA‐2RE and EBNA‐2 in the same fashion as the affinity purified protein. The interaction between RBP‐J kappa and EBNA‐2 is a prerequisite for EBNA‐2‐mediated transactivation of the TP1 promoter.

RBP-J is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-J-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-J. For simplicity, we propose to call RBP-J RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.RBP-J is a 60-kDa DNA binding protein recognizing a consensus sequence (C/T)GTGGGAA although it has no typical DNA binding motif (19,23,38,52). The structure of the RBP-J protein is strongly conserved during evolution among nematode, fruit fly, mouse, and human (1, 5, 13, 38).We and others have shown that the Drosophila RBP-J gene is identical to Suppressor of Hairless [Su(H)] (14, 46), a member of the neurogenic gene family including Notch, Delta, Enhancer of split [E(spl)], and Hairless. Genetic analyses have shown that the neurogenic genes, including Su(H), participate in lateral inhibition to single out a sensory mother cell from its precursor cells during peripheral nervous system development (2, 40). RBP-J/Su(H) binding sites were identified in the 5Ј-flanking regions of the E(spl) complex [E(spl)-C] neurogenic genes of Drosophila melanogaster, and the transactivation of the E(spl) m8 enhancer promoter by Su(H) was demonstrated in the Schneider cell line (16). Transactivation of the E(spl)-C promoters by Su(H) was also demonstrated in vivo by using transgenic flies carrying E(spl)-C promoter-␤-galactosidase gene constructs (3,15,16,33).A breakthrough in the elucidation of the RBP-J function in vertebrates came from studies on transcriptional regulation of human DNA tumor viruses. The adenovirus capsid protein polypeptide IX (pIX) promoter contains the RBP-J targetlike TGGGAAAGAA sequence between the SP1 binding site and the TATA box. Repression of the pIX promoter by RBP-J was shown by in vitro as well as in vivo experiments (9). Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is essential for transformation of primary human B lymphocytes (7, 21, 58) and acts as a transcriptional activator of latent viral as well as cellular genes by interacting with 22,54,61). Thus, RBP-J is essential to B-lymphocyte transformation by EBV.RBP-J knockout mice die before 10.5 days of gestation and show severe developmental defects in somites and neural tube...

SUMMARYAll Epstein-Barr virus (EBV) isolates can be classified as type A or type B depending upon the identity of their EBV nuclear antigen (EBNA) 2 protein. The great majority of isolates examined to date encode an EBNA 2A protein like that of the reference type A strain B95-8. Type B virus strains, encoding an antigenically distinct EBNA 2B protein, have as yet only been rescued from rare Burkitt's lymphoma (BL) cell lines of African origin (Jijoye, AG876). Our recent finding that type B isolates are less efficient than type A in in vitro transformation assays prompted us to determine (i) the relative contribution the two types of virus make to the incidence of BL in endemic areas of Africa (Kenya) and New Guinea and (ii) the relative incidence of infection with these two types in the normal population in these same areas. On the first point, EBNA 2 gene typing using specific DNA probes showed that four of ten recently established Kenyan BL cell lines and two of four BL cell lines from New Guinea carried type B virus isolates. To address the second point, spontaneous lymphoblastoid cell lines were established from the blood of normal virus carriers and typed for EBNA 2 at the protein level; a significant proportion (> 20 ~) of the normal population in both the above BLendemic areas were infected with type B isolates. This is the first indication of the widespread nature of type B virus infection in any community and the first isolation of such viruses from a non-BL source. The reproducible size of the EBNA 2B protein encoded by all type B isolates irrespective of their geographical origin, and of the EBNA 1 protein encoded by all type B isolates from one area, contrasted markedly with the extreme variability in the size both of EBNA 2A and of EBNA 1 seen generally among type A isolates. This suggests that the number of type B virus strains in existence worldwide could be quite limited. Most importantly, the data suggest that type B viruses, despite their relatively poor performance in in vitro transformation assays, can contribute at least as efficiently as can type A viruses to the pathogenesis of BL.

Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein.Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2-and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.Epstein-Barr virus (EBV) infection is associated with several human malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and lymphomas in the immunosuppressed host (53). EBV latent infection of human B lymphocytes in vitro induces expression of B-cell activation markers, proliferation, and eventual outgrowth of continuously growing lymphoblastoid cell lines (LCLs) (36). LCLs phenotypically resemble physiologically activated primary B cells (36). The ability of EBV to ...

Cotton-top tamarins were inoculated with sufficient Epstein-Barr virus to induce multiple tumors in each animal within 14 to 21 days. The tumors consisted of large-cell lymphomas that contained multiple copies of the Epstein-Barr virus genome and generated Epstein-Barr virus-carrying cell lines showing no detectable consistent chromosomal abnormality. Hybridization of tumor DNA with immunoglobulin gene probes revealed that each lymphoma was oligo- or monoclonal in origin and that individual tumors from the same animal arose from different B-cell clones. Thus the virus induced multiple transformation events in tamarins in vivo to cause malignant tumors resembling the Epstein-Barr virus-associated lymphomas of patients with organ transplants.