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.

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 Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

2001 ◽  
Vol 75 (1) ◽  
pp. 90-99 ◽  
Author(s):  
Rozenn Dalbiès-Tran ◽  
Evelyn Stigger-Rosser ◽  
Travis Dotson ◽  
Clare E. Sample
Keyword(s):  
Amino Acids ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Biological Significance ◽  
Evolutionary Conservation ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Amino Terminal ◽  
Gene Assay ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes.

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 High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

1989 ◽  
Vol 9 (6) ◽  
pp. 2738-2742 ◽  
Author(s):  
A S Kimball ◽  
G Milman ◽  
T D Tullius
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Binding Domain ◽  
Nuclear Antigen ◽  
Dna Binding Domain ◽  
Major Groove ◽  
Barr Virus ◽  
Dna Binding Site ◽  
Epstein Barr ◽  
Hydroxyl Radical Footprinting

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.

High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1

1989 ◽  
Vol 9 (6) ◽  
pp. 2738-2742
Author(s):  
A S Kimball ◽  
G Milman ◽  
T D Tullius
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Binding Domain ◽  
Nuclear Antigen ◽  
Dna Binding Domain ◽  
Major Groove ◽  
Barr Virus ◽  
Dna Binding Site ◽  
Epstein Barr ◽  
Hydroxyl Radical Footprinting

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.

scholarly journals Epstein-Barr Virus Nuclear Antigen 3C Facilitates Cell Proliferation by Regulating Cyclin D2

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Yonggang Pei ◽  
Rajnish Kumar Singh ◽  
Sanket Kumar Shukla ◽  
Fengchao Lang ◽  
Shengwei Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Epstein Barr Virus ◽  
Cell Cycle Progression ◽  
Nuclear Antigen ◽  
Cyclin D2 ◽  
Cycle Progression ◽  
Barr Virus ◽  
Epstein Barr ◽  
Related Proteins

ABSTRACTCell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. Epstein-Barr virus (EBV)-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process, thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2 and they colocalize together in nuclear compartments. We show that EBNA3C regulates the promoter of cyclin D2 through cooperation with master transcription factor Bcl6 and enhances its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of cyclin D2, suggesting that cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors, which drives the oncogenic process.IMPORTANCEEpstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they colocalize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell proliferation in the presence of cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.

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.

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