Cellular and Epstein-Barr virus specific DNA polymerases in virus-producing Burkitt's lymphoma cell lines.

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.     

The effects of mitogens on the expression of Epstein-Barr virus antigens in human lymphoid cell lines.

Treatment of human lymphoblastoid cells with either phytohemagglutinin (PHA), concanavalin A, Staphylococcus protein A, or polyinosinic acid-polycytidylic acid, in combination with 5-iodo-2' deoxyuridine (IUdR) markedly increased the expression of Epstein-Barr virus (EBV) early antigen (EA) relative to IUdR alone. Such treatment did not, however, modify the production of virus capsid antigen in any of the lymphoid cell lines tested. The effect of PHA on EA induction in Raji cells was not accompanied by changes in the incorporation of labeled precursors into cellular DNA, or in the intracellular concentration of either adenosine 3'5' cyclic monophosphate or guanosine 3'5' cyclic monophosphate. However, those mitogens that stimulated EA expression in Raji cells also increased the fluorescence polarization of 1,6 diphenyl 1,3,5-hexatriene-labeled Raji cells. The possible role of cell surface changes in the mitogen activation of latent EBV in human lymphoblastoid cells is discussed.     

Epstein-Barr virus, the TCL-1 oncogene and Burkitt's lymphoma

Epstein-Barr virus (EBV) is strongly implicated in the pathogenesis of several human malignancies, but notably in all endemic and some sporadic cases of Burkitt's lymphoma (BL). Although the virus's contribution to the development of BL remains unclear, evidence now suggests that a common feature of all BL tumours is the expression of the TCL-1 oncogene. A recent report shows that TCL-1 expression in virus-positive BL tumour cells is dependent on the presence of EBV. This finding suggests that the ability of EBV to induce TCL-1 would circumvent the need for the additional genetic or epigenetic changes that lead to the constitutive expression of this oncogene in EBV-negative BL tumours.     

Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.     

Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.     

Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma

We investigated whether Epstein--Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)-alpha. EBV-negative subclones isolated from EBV-positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN-alpha treatment. On the other hand, EBV-positive counterparts exhibited striking resistance against IFN-alpha-induced apoptosis. Transfection of an individual EBV latent gene into EBV-negative BL cells revealed that EBV-encoded poly(A)(-) RNAs (EBERs) were responsible for IFN resistance. EBERs bound double-stranded (ds) RNA-activated protein kinase (PKR), a key mediator of the antiviral effect of IFN-alpha, and inhibited its phosphorylation. Transfection of dominant-negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV-negative BL cells resistant to IFN-alpha-induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA-binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN-alpha-induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN-induced apoptosis in virus-associated tumors.     

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt's lymphoma B cell line BL30/B95-8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt's lymphoma B cells

IL-4 and IL-13, cytokines with similar biological effects may influence growth and progression of B-cell tumors through regulation of key cell surface molecules important in intercellular communications. In this study, we demonstrate that IL-4 and IL-13 exhibited differential effects on CD23 and CD44 expression and binding to hyaluronan in BL30/B95-8, a Burkitt's lymphoma (BL), and MK3.31, an Epstein-Barr virus transformed normal human B cell line (B-LCL). Studies conducted to understand the molecular mechanisms underlying this differential effect show that IL-4 induced phosphorylation of JAK1, JAK3, and STAT6 in BL30/B95-8 cells and of JAK3 and STAT6 in MK 3.31 cells. In contrast, IL-13 failed to induce the phosphorylation of JAK kinases or STAT6 proteins in these cell lines. The inability of BL30/B95-8 cells to respond to IL-13 was attributed to the loss of expression of IL-13R subunits alpha1 and alpha2, a finding confirmed for a number of other BL cell lines examined.     

Replacement of the Epstein-Barr virus plasmid with the EBER plasmid in Burkitt's lymphoma cells

Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.     

Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt's lymphoma cell line Akata

Our previous reports indicated that Epstein-Barr virus (EBV) contributes to the malignant phenotype and resistance to apoptosis in Burkitt's lymphoma (BL) cell line Akata (N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069-6073, 1994; J. Komano, M. Sugiura, and K. Takada, J. Virol. 72:9150-9156, 1998). Here we report that the EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. This is the first report which provides evidence that virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.     

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.