Mutational analysis of the J recombination signal sequence binding protein (RBP-J)/Epstein-Barr virus nuclear antigen 2 (EBNA2) and RBP-J/Notch interaction.

Epstein-Barr virus nuclear antigen 2 (EBNA2) and the Notch protein both function within the nucleus as transcriptional adaptor proteins. EBNA2 plays a key role during the immortalization of primary B-cells by Epstein-Barr virus (EBV). Notch proteins are involved in lymphomagenesis as well as in multiple cell fate decisions during tissue differentiation and development. Both, EBNA2 and Notch interact with the DNA binding protein RBP-J and thereby gain access to the promoter of their target genes. In order to identify regions within the J recombination signal sequence binding protein (RBP-J), that are relevant for either the Notch or the EBNA2 interaction, we have performed a mutational analysis of RBP-J. A library of RBP-J mutants was screened by a reverse two-hybrid system for alleles that fail to bind to either EBNA2 or Notch. The sequence analysis of these alleles reveals that a limited and particularly distinct number of amino-acid positions are relevant for either interaction only. Given the important role of RBP-J in B-cell immortalization, the EBNA2/RBP-J protein-protein interaction could be a candidate target for therapeutic intervention in EBV related diseases.

Notch1IC partially replaces EBNA2 function in B cells immortalized by Epstein-Barr virus.

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.

Direct and indirect regulation of cytokine and cell cycle proteins by EBNA-2 during Epstein-Barr virus infection.

We have studied the pathways of regulation of cytokine and cell cycle control proteins during infection of human B lymphocytes by Epstein-Barr virus (EBV). Among 30 cytokine RNAs analyzed by the RNase protection assay, tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor, lymphotoxin (LT), and LTbeta were found to be regulated within 20 h of EBV infection of primary B cells. Similar results were obtained using the estrogen-regulated EBNA-2 cell line EREB2.5, in which RNAs for LT and TNF-alpha were induced within 6 h of activation of EBNA-2. Expression of Notch also caused an induction of TNF-alpha RNA. The induction of TNF-alpha RNA by EBNA-2 was indirect, and constitutive expression of either LMP-1 or c-myc proteins did not substitute for EBNA-2 in induction of TNF-alpha RNA. Cyclin D2 is also an indirect target of EBNA-2-mediated transactivation. EBNA-2 was found to activate the cyclin D2 promoter in a transient-transfection assay. A mutant of EBNA-2 that does not bind RBP-Jkappa retained some activity in this assay, and activation did not depend on the presence of B-cell-specific factors. Deletion analysis of the cyclin D2 promoter revealed that removal of sequences containing E-box c-myc consensus DNA binding sequences did not reduce EBNA-2-mediated activation of the cyclin D2 promoter in the transient-transfection assay. The results indicate that cytokines are an early target of EBNA-2 and that EBNA-2 can regulate cyclin D2 transcription in EBV-infected cells by mechanisms additional to the c-myc pathway.

Cell cycle activation by c-myc in a burkitt lymphoma model cell line.

The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.

Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses.

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Neoplastic transformation by Notch is independent of transcriptional activation by RBP-J signalling.

Signalling through the transmembrane receptor Notch is triggered by ligand binding, which induces the proteolytic cleavage of the Notch protein. This cleavage generates an intracellular fragment of the Notch protein (Notch-IC), which translocates into the nucleus and modifies transcription of target genes through its association with the RBP-J protein. Thus, the isolated Notch-IC protein represents the constitutively activated receptor. We have performed a deletion analysis of Notch IC in order to identify the transferable transactivation domain of Notch-IC and the minimal domain of Notch-IC required for RBP-J dependent transactivational activation. Functionally, Notch-IC has been linked to cell fate decision in development and oncogenesis in vivo. In vitro, Notch-IC can cooperate in neoplastic transformation of baby rat kidney cells with the adenoviral E1A protein. We have defined the minimal domain of Notch-IC required for E1A cotransformation. This domain, consisting of the ankyrin repeats of Notch-IC only, can neither activate RBP-J dependent transcription nor does it carry a transactivation domain. Therefore, the ankyrin repeat domain of Notch-IC might trigger novel pathways relevant for transformation but unrelated to RBP-J signalling.

Induction of autophagy and inhibition of tumorigenesis by beclin 1.

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2.

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.

The Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein required for B lymphocyte immortalization, induces the synthesis of type I interferon in Burkitt's lymphoma cell lines.

Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein involved in cell transformation, interferes with the cellular response to type I interferons (IFN-alpha/beta). We investigated the function of conditionally expressed EBNA2 in the context of the IFN response in Burkitt's lymphoma cell lines. Expression of EBNA2 led to the transcriptional activation of both endogenous or transfected IFN-stimulated genes (ISGs), genes which contain within their promoters either the interferon-stimulated response element (ISRE) or the gamma interferon activation site (GAS). In search of a molecular mechanism for the transcriptional induction of ISGs, we observed an EBNA2-dependent synthesis of IFN-beta mRNA at low levels and the secretion of low amounts of IFN. A transfected IFN-beta promoter responded to EBNA2 activation, and a sequence closely resembling a RBP-Jkappa binding site was pinpointed as a potential target of EBNA2 activity. EBNA2-dependent transcriptional induction of the IFN-beta promoter occurred in EBV-negative Burkitt's lymphoma cells, indicating that other EBV genes were not required for the induction of IFN-beta synthesis.

Epstein-Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival.

Epstein-Barr virus (EBV) infects human primary B lymphocytes and induces and maintains proliferation of these cells efficiently in vitro. Mutants of Epstein-Barr virus which express EBV nuclear antigen 2 (EBNA2) in a conditional fashion allow dissection of individual contributions of viral genes to B cell immortalization. EBNA2 is a transcriptional activator of cellular and viral genes, including the viral latent membrane protein 1 (LMP1), which is essential for B cell immortalization and has oncogenic effects in non-lymphoid cells. To analyze the role of this gene in B cell immortalization, LMP1 was constitutively expressed in B cells infected with EBV carrying a conditional EBNA2 allele. In the absence of functional EBNA2, LMP1 was incapable of sustaining B cell proliferation in two independent assays but induced a phenotype consistent with prolonged cell viability. Activation of CD40 displayed a comparable phenotype. These data indicate that both CD40 activation and LMP1 expression may use a common pathway for B cell activation. Proliferation of human B cells, however, requires one or more additional signals triggered by EBNA2.

Mosaicicm in bcr-abl protein expression in B cells in chronic myelogenous leukemia.

Chronic myelogenous leukemia is a disease of the pluripotent stem cell that involves the myeloid and, to a varying degree, the lymphoid compartment. We studied the involvement of B cells in chronic myelogenous leukemia at diagnosis and during treatment. B lymphocytes were immortalized by infection with Epstein-Barr virus. B-lymphoid cell lines could be established from 25 patients suffering from Philadelphia-chromosome (Ph1)-positive chronic myelogenous leukemia. The cell lines were tested for expression of the typical 210-kDa fusion protein, p210, using Western-blot analysis, and/or for mRNA expression of bcr-abl fusion genes, using reverse transcriptase polymerase chain reaction analysis. At diagnosis, mosaicism of B cells was demonstrated in every patient. During treatment with interferon alpha, p210-expressing B-lymphoid cell lines could not be established from 8 of 8 patients. Following discontinuation of IFN-alpha therapy, p210-positive cell lines were found early, even before cytogenetic recurrence. Resistance to IFN-alpha therapy and progression of the disease were both associated with the appearance of p210-positive cell lines. Cell lines established from 3 healthy individuals and from patients suffering from Ph1-negative diseases did not show p210 expression in Western blots. Our data suggest that B lymphocytes are involved early in the disease, and that B-cell mosaicism may be a sensitive marker for resistance to IFN-alpha therapy and disease progression.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

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 kappa locus composed 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 of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Epstein-Barr virus nuclear antigen 2 (EBNA2)-oestrogen receptor fusion proteins complement the EBNA2-deficient Epstein-Barr virus strain P3HR1 in transformation of primary B cells but suppress growth of human B cell lymphoma lines.

To develop a transformation system with a conditional Epstein-Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines.

Epstein-Barr virus nuclear antigen 2 is a transcriptional suppressor of the immunoglobulin mu gene: implications for the expression of the translocated c-myc gene in Burkitt's lymphoma cells.

A conditional mutant of Epstein-Barr virus nuclear antigen 2 (EBNA2) regulated by estrogen was employed to study the effect of EBNA2 on the cellular phenotype. Activation of EBNA2 in lymphoblastoid cell lines (LCLs) and in B cell lymphoma lines resulted in down-regulation of cell surface IgM and Ig-mu steady-state RNA expression. In LCLs, activation of EBNA2 is required for maintaining proliferation, whereas in Burkitt's lymphoma (BL) cell lines with t(8;14) translocations, activation of EBNA2 induces growth arrest. In these cells, Northern and nuclear run-on analyses revealed rapid simultaneous repression of Ig-mu and c-myc transcription as early as 30 min after activation of EBNA2. Since c-myc expression is under the control of the Ig heavy chain locus in BL cell lines with a t(8;14) translocation, we propose that Ig-mu and c-myc are down-regulated by EBNA2 through a common mechanism.

Epstein-Barr virus nuclear antigen 2-estrogen receptor fusion proteins transactivate viral and cellular genes and interact with RBP-J kappa in a conditional fashion.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transcriptional activator of viral and cellular genes involved in B cell transformation by EBV and is targeted to EBV responsive promoters through interaction with cellular DNA binding proteins such as RBP-J kappa. To develop a conditional system in which the function of EBNA2 can be switched on and off, we have fused the hormone binding domain of the estrogen receptor to the N- or C-terminus of EBNA2. Here we show that after transient or stable transfer of these chimerical EBNA2 genes into human B cell lymphoma lines, transactivation of LMP1, TP1, and TP2 promoter constructs, expression of the cell surface markers CD21 and CD23, and binding of EBNA2 to its cellular partner RBP-J kappa are dependent on the presence of estrogen. The EBNA2 fusion proteins proved to be virtually inactive in the absence of hormone.

Expression of the chemokine receptor BLR2/EBI1 is specifically transactivated by Epstein-Barr virus nuclear antigen 2.

In our attempt to identify chemokine receptors that are related to Burkitt's lymphoma receptor 1 (BLR1) and are expressed in activated lymphocytes we used RT-PCR resulting in the isolation of a cDNA encoding a seven transmembrane receptor termed BLR2. The protein shows significant sequence similarities to the family of G-protein coupled chemokine receptors and turned out to be identical to the recently described receptor EBI1. Northern blot analysis revealed that BLR2 mRNA could be highly stimulated in mitogen- and anti-CD3-treated peripheral blood lymphocytes. BLR2-specific mRNA could be detected in all Epstein-Barr virus positive B cell lines. We show that transcription of the BLR2 gene could be specifically induced in Epstein-Barr virus negative BL 41 cells via estrogen-mediated activation of Epstein-Barr virus nuclear antigen 2, a key regulator of viral and cellular genes in immortalized B cells. Our data suggest an involvement of BLR2 in the regulation of migration in activated lymphocytes and in viral pathogenesis.

Immortalization of human primary B lymphocytes in vitro with DNA.

Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy.

Expression of Epstein-Barr virus nuclear antigen-2 (EBNA2) induces CD21/CR2 on B and T cell lines and shedding of soluble CD21.

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) expressed as a fusion protein with the hormone-binding domain of the estrogen receptor was used to study expression of CD21 and other surface markers in different cell lines. Special emphasis was placed on cell lines with a normally low expression of CD21, especially on T cell lines. After induction of EBNA2, a substantial increase in CD21 mRNA was observed, as well as increased production of membrane CD21. This was found not only in cell lines of B cell origin, but also in the T cell line Jurkat. The amount of CD21 was quantitated by means of a fluorescence immunoassay, and found to correlate with the presence of EBNA2 protein. A decrease in EBNA2 abundance was associated with complete loss of cell-associated CD21. As we could also detect large amounts of soluble CD21 (sCD21) in the supernatant of the transfected cell lines, which exceeded the total amount contained in the respective cell lysates, this indicates considerable shedding of the newly synthesized receptor molecules induced by EBNA2, comparable to the situation described for CD23. It further provides an explanation of the recent findings of increased sCD21 levels in sera of patients with EBV-associated disease, and suggests a possible additional function of EBNA2 in vivo.

B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2.

Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B-cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B-cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors.