scholarly journals Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein–Barr virus nuclear antigen 3C

Virology ◽  
2006 ◽  
Vol 346 (2) ◽  
pp. 379-384
Author(s):  
Nikhil Sharma ◽  
Jason S. Knight ◽  
Erle S. Robertson
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Epstein Barr ◽  
Amino Terminal Domain ◽  
Regulatory Properties

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 Retinoids irreversibly inhibit in vitro growth of Epstein-Barr virus- immortalized B lymphocytes

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 3147-3159 ◽  
Author(s):  
F Pomponi ◽  
R Cariati ◽  
P Zancai ◽  
P De Paoli ◽  
S Rizzo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Antigen Expression ◽  
Inhibitory Effect ◽  
Lymphoproliferative Disorders ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Drug Concentrations ◽  
Epstein Barr

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study, we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs), since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity, but 9-cis-retinoic acid (RA), 13-cis-RA, and all-trans-RA (ATRA) were markedly more efficacious than Ro40–8757, Ro13–6298, and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs, thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1, a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems, the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact, retinoid-induced modifications of cell morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and increased expression of CD38 and c-Ig), and IgM production were late events, highly heterogeneous, and often slightly relevant, being therefore only partially indicative of a drug-related differentiative process. Moreover, EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids, indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients.

scholarly journals SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen

2005 ◽  
Vol 25 (5) ◽  
pp. 1749-1763 ◽  
Author(s):  
Jason S. Knight ◽  
Nikhil Sharma ◽  
Erle S. Robertson
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Kinase Inhibitor ◽  
Cyclin A ◽  
Cell Cycle Checkpoint ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Human Cancers ◽  
Cycle Checkpoint ◽  
Epstein Barr

ABSTRACT The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.

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 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 Nuclear Import of Epstein-Barr Virus Nuclear Antigen 1 Mediated by NPI-1 (Importin α5) Is Up- and Down-Regulated by Phosphorylation of the Nuclear Localization Signal for Which Lys379 and Arg380 Are Essential

2006 ◽  
Vol 80 (4) ◽  
pp. 1979-1991 ◽  
Author(s):  
Ryo Kitamura ◽  
Toshihiro Sekimoto ◽  
Sayuri Ito ◽  
Shizuko Harada ◽  
Hideo Yamagata ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
Nuclear Transport ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Amino Terminal ◽  
Localization Signal ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin α NPI-1 (importin α5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin α1) bound only weakly and Qip1 (importin α3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

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 1 interacts with regulator of chromosome condensation 1 dynamically throughout the cell cycle

2017 ◽  
Vol 98 (2) ◽  
pp. 251-265 ◽  
Author(s):  
Thibaut Deschamps ◽  
Quentin Bazot ◽  
Derek M Leske ◽  
Ruth MacLeod ◽  
Dimitri Mompelat ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Chromosome Condensation ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP

2003 ◽  
Vol 77 (22) ◽  
pp. 11992-12001 ◽  
Author(s):  
Zhong Deng ◽  
Constandache Atanasiu ◽  
John S. Burg ◽  
Dominique Broccoli ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Dominant Negative ◽  
Full Length ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr ◽  
Dyad Symmetry

ABSTRACT Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5- to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G1/S while TRF1 binding peaks in G2/M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.

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