scholarly journals An Epstein-Barr Virus Isolated from a Lymphoblastoid Cell Line Has a 16-Kilobase-Pair Deletion Which Includes gp350 and the Epstein-Barr Virus Nuclear Antigen 3A

2001 ◽  
Vol 75 (18) ◽  
pp. 8556-8568 ◽  
Author(s):  
Wonkeun Lee ◽  
Yoon-Ha Hwang ◽  
Suk-Kyeong Lee ◽  
Chitra Subramanian ◽  
Erle S. Robertson
Keyword(s):  
Cell Line ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Lymphoblastoid Cell Line ◽  
Phorbol Esters ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.

scholarly journals The ATM/ATR Signaling Effector Chk2 Is Targeted by Epstein-Barr Virus Nuclear Antigen 3C To Release the G2/M Cell Cycle Block

2007 ◽  
Vol 81 (12) ◽  
pp. 6718-6730 ◽  
Author(s):  
Tathagata Choudhuri ◽  
Subhash C. Verma ◽  
Ke Lan ◽  
Masanao Murakami ◽  
Erle S. Robertson
Keyword(s):  
Cell Cycle ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Cell Cycle Checkpoint ◽  
Nuclear Antigen ◽  
M Cell ◽  
Barr Virus ◽  
Cycle Checkpoint ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects most of the human population and persists in B lymphocytes for the lifetime of the host. The establishment of latent infection by EBV requires the expression of a unique repertoire of genes. The product of one of these viral genes, the EBV nuclear antigen 3C (EBNA3C), is essential for the growth transformation of primary B lymphocytes in vitro and can regulate the transcription of a number of viral and cellular genes important for the immortalization process. This study demonstrates an associated function of EBNA3C which involves the disruption of the G2/M cell cycle checkpoint. We show that EBNA3C-expressing lymphoblastoid cell lines treated with the drug nocodazole, which is known to block cells at the G2/M transition, did not show a G2/M-specific checkpoint arrest. Analyses of the cell cycles of cells expressing EBNA3C demonstrated that the expression of this essential EBV nuclear antigen is capable of releasing the G2/M checkpoint arrest induced by nocodazole. This G2/M arrest in response to nocodazole was also abolished by caffeine, suggesting an involvement of the ATM/ATR signaling pathway in the regulation of this cell cycle checkpoint. Importantly, we show that the direct interaction of EBNA3C with Chk2, the ATM/ATR signaling effector, is responsible for the release of this nocodazole-induced G2/M arrest and that this interaction leads to the serine 216 phosphorylation of Cdc25c, which is sequestered in the cytoplasm by 14-3-3. Overall, our data suggest that EBNA3C can directly regulate the G2/M component of the host cell cycle machinery, allowing for the release of the checkpoint block.

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 Epstein-Barr Virus Nuclear Antigen 3C and Prothymosin Alpha Interact with the p300 Transcriptional Coactivator at the CH1 and CH3/HAT Domains and Cooperate in Regulation of Transcription and Histone Acetylation

2002 ◽  
Vol 76 (10) ◽  
pp. 4699-4708 ◽  
Author(s):  
Chitra Subramanian ◽  
Sameez Hasan ◽  
Martin Rowe ◽  
Michael Hottiger ◽  
Rama Orre ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Regulation Of Transcription ◽  
Transcriptional Coactivator ◽  
Prothymosin Alpha ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr ◽  
Coactivator P300

ABSTRACT The Epstein-Barr virus nuclear antigen 3C (EBNA3C), encoded by Epstein-Barr virus (EBV), is essential for mediating transformation of human B lymphocytes. Previous studies demonstrated that EBNA3C interacts with a small, nonhistone, highly acidic, high-mobility group-like nuclear protein prothymosin alpha (ProTα) and the transcriptional coactivator p300 in complexes from EBV-infected cells. These complexes were shown to be associated with histone acetyltransferase (HAT) activity in that they were able to acetylate crude histones in vitro. In this report we show that ProTα interacts with p300 similarly to p53 and other known oncoproteins at the CH1 amino-terminal domain as well as at a second domain downstream of the bromodomain which includes the CH3 region and HAT domain. Similarly, EBNA3C also interacts with p300 at regions which include the CH1 and CH3/HAT domains, suggesting that ProTα and EBNAC3C may interact in a complex with p300. We also show that ProTα activates transcription when targeted to promoters by fusion to the GAL4 DNA binding domain and that this activation is enhanced by the addition of an exogenous source of p300 under the control of a heterologous promoter. This overall activity is down-modulated in the presence of EBNA3C. These results further establish the interaction of cellular coactivator p300 with ProTα and demonstrate that the associated activities resulting from this interaction, which plays a role in acetylation of histones and coactivation, can be regulated by EBNA3C. Furthermore, this study establishes for the first time a transcriptional role for ProTα in recruitment or stabilization of coactivator p300, as well as other basal transcription factors, at the nucleosomes for regulation of transcription.

scholarly journals Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

2015 ◽  
Vol 90 (6) ◽  
pp. 2906-2919 ◽  
Author(s):  
Anqi Wang ◽  
Rene Welch ◽  
Bo Zhao ◽  
Tram Ta ◽  
Sündüz Keleş ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Binding Specificity ◽  
Nuclear Antigen ◽  
Lymphoblastoid Cell Lines ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTLatent infection of B lymphocytes by Epstein-Barr virus (EBV)in vitroresults in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors.IMPORTANCEEpstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell linesin vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity.

scholarly journals Epstein-Barr virus susceptibility of normal human B lymphocyte populations.

1984 ◽  
Vol 159 (1) ◽  
pp. 208-220 ◽  
Author(s):  
P Aman ◽  
B Ehlin-Henriksson ◽  
G Klein
Keyword(s):  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
B Lymphocyte ◽  
High Density ◽  
Nuclear Antigen ◽  
Virus Binding ◽  
Density Fractions ◽  
Barr Virus ◽  
Epstein Barr

Human blood and tonsil B lymphocytes were fractionated on density gradients and tested for virus binding and penetration into the cells. Epstein-Barr Virus (EBV) transformation was detected by immunofluorescence staining for EBV-determined nuclear antigen (EBNA). EBV bound to and penetrated all B cell populations, but only the high density populations were transformed. Activated B lymphocytes were found in the low density fractions and these cells were resistant to EBV infection. Infected and noninfected B lymphocytes were density-analyzed during in vitro culture. A spontaneous, not virus-induced, density decrease was found to precede the production of EBNA. Cells remaining at high density never expressed EBNA. The results suggest that EBV can transform only small resting B lymphocytes and that a virus-independent activation of the infected cells induces the EBNA production and transformation.

scholarly journals The Proto-Oncogene c-myc Is a Direct Target Gene of Epstein-Barr Virus Nuclear Antigen 2

1999 ◽  
Vol 73 (5) ◽  
pp. 4481-4484 ◽  
Author(s):  
Carmen Kaiser ◽  
Gerhard Laux ◽  
Dirk Eick ◽  
Nicola Jochner ◽  
Georg W. Bornkamm ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
De Novo ◽  
Nuclear Antigen ◽  
Resting Cells ◽  
Cell Cycle Regulators ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT 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.

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