scholarly journals Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Gene Deletion Is Consistently Linked with EBNA3A, -3B, and -3C Expression in Burkitt's Lymphoma Cells and with Increased Resistance to Apoptosis

2005 ◽  
Vol 79 (16) ◽  
pp. 10709-10717 ◽  
Author(s):  
Gemma L. Kelly ◽  
Anne E. Milner ◽  
Rosemary J. Tierney ◽  
Debbie S. G. Croom-Carter ◽  
Markus Altmann ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Cell Phenotype ◽  
Parent Cell ◽  
Wild Type ◽  
Apoptosis Resistance ◽  
Viral Genomes ◽  
Barr Virus ◽  
Restricted Form ◽  
Epstein Barr

ABSTRACT Most Epstein-Barr virus (EBV)-positive Burkitt's lymphomas (BLs) carry a wild-type EBV genome and express EBV nuclear antigen 1 (EBNA1) selectively from the BamHI Q promoter (latency I). Recently we identified a distinct subset of BLs carrying both wild-type and EBNA2 gene-deleted (transformation-defective) viral genomes. The cells displayed an atypical “BamHI W promoter (Wp)-restricted” form of latency where Wp (rather than Qp) was active and EBNA1, -3A, -3B, -3C, and -LP were expressed in the absence of EBNA2 or latent membrane proteins 1 and 2. Here we present data strongly supporting the view that the EBNA2-deleted genome is transcriptionally active in these cells and the wild-type genome is silent. Single-cell cloning of three parental Wp-restricted BL lines generated clones carrying either both viral genomes or the EBNA2-deleted genome only, never clones with the wild-type genome only. All rescued clones displayed the Wp-restricted form of latency characteristic of the parent line and retained the original parent cell phenotype. Interestingly, Wp-restricted parent lines and derived clones were markedly more resistant to inducers of apoptosis than standard latency I BL lines. Furthermore, in vitro infection of EBV-negative BL lines with an EBNA2 gene-deleted virus generated EBV-positive converts with Wp-restricted latency and a similarly marked apoptosis resistance. We postulate that, in the subset of BLs displaying Wp-restricted latency, infection of a tumor progenitor cell with an EBNA2 gene-deleted virus has provided that cell with a survival advantage through broadening antigen expression to include the EBNA3 proteins.

scholarly journals The EBNA2 Polyproline Region Is Dispensable for Epstein-Barr Virus-Mediated Immortalization Maintenance

2002 ◽  
Vol 76 (14) ◽  
pp. 7349-7355 ◽  
Author(s):  
Alexey V. Gordadze ◽  
David Poston ◽  
Paul D. Ling
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Gene Dosage ◽  
Nuclear Antigen ◽  
Striking Difference ◽  
Loss Of Function ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that ΔPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing ΔPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in ΔPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant ΔPPR-EBNA2 EBVs to immortalize B cells.

scholarly journals Epstein-Barr Virus Contributes to the Malignant Phenotype and to Apoptosis Resistance in Burkitt’s Lymphoma Cell Line Akata

1998 ◽  
Vol 72 (11) ◽  
pp. 9150-9156 ◽  
Author(s):  
Jun Komano ◽  
Makoto Sugiura ◽  
Kenzo Takada
Keyword(s):  
Epstein Barr Virus ◽  
Burkitt’S Lymphoma ◽  
Soft Agar ◽  
Burkitt's Lymphoma ◽  
Nuclear Antigen ◽  
Apoptosis Resistance ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

ABSTRACT In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.

scholarly journals Epstein-Barr Virus Nuclear Protein EBNA3A Is Critical for Maintaining Lymphoblastoid Cell Line Growth

2003 ◽  
Vol 77 (19) ◽  
pp. 10437-10447 ◽  
Author(s):  
Seiji Maruo ◽  
Eric Johannsen ◽  
Diego Illanes ◽  
Andrew Cooper ◽  
Elliott Kieff
Keyword(s):  
Epstein Barr Virus ◽  
Critical Role ◽  
Growth Arrest ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Barr Virus ◽  
Fusion Protein Expression ◽  
Epstein Barr ◽  
Biochemical Analyses

ABSTRACT To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

scholarly journals EBNA3A Association with RBP-Jκ Down-Regulates c-myc and Epstein-Barr Virus-Transformed Lymphoblast Growth

2003 ◽  
Vol 77 (2) ◽  
pp. 999-1010 ◽  
Author(s):  
Andrew Cooper ◽  
Eric Johannsen ◽  
Seiji Maruo ◽  
Ellen Cahir-McFarland ◽  
Diego Illanes ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epstein Barr Virus ◽  
Cell Cycle Progression ◽  
Binding Domain ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Interaction Domain ◽  
Barr Virus ◽  
The Core ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jκ/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jκ, did not change EBNA3C association with RBP-Jκ or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jκ, decreased c-myc expression, and caused G0/G1 growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jκ-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jκ binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jκ binding domain. The dependence of EBNA3A effects on the core RBP-Jκ interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jκ association strongly implicates RBP-Jκ in c-myc promoter regulation.

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