Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key gene expressed in EBV type III latent infection that can transactivate numerous promoters, including those for all the other type III viral latency genes as well as cellular genes responsible for cell proliferation. EBNA-2 is essential for EBV-mediated immortalization of primary B lymphocytes. We now report that EBNA-2, a phosphoprotein, is hyperphosphorylated specifically in mitosis. Evidence that the cyclin-dependent kinase p34cdc2 may be involved in this hyperphosphorylation includes (i) coimmunoprecipitation of EBNA-2 and p34cdc2, suggesting physical association; (ii) temporal correlation between hyperphosphorylation of EBNA-2 and an increase in p34cdc2 kinase activity; and (iii) ability of purified p34cdc2/cyclin B1 kinase to phosphorylate EBNA-2 in vitro. Hyperphosphorylation of EBNA-2 appears to suppress its ability to transactivate the latent membrane protein 1 (LMP-1) promoter by about 50%. The association between EBNA-2 and PU.1 is also decreased by about 50% in M-phase-arrested cells, as shown by coimmunoprecipitation from cell lysates, suggesting that hyperphosphorylation of EBNA-2 impairs its affinity for PU.1. Finally, endogenous LMP-1 mRNA levels in M phase are around 55% of those in asynchronously growing cells. These results suggest that regulation of gene expression during type III latency may be regulated in a cell-cycle-related manner.

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A possible function of Epstein-Barr virus-determined nuclear antigen (EBNA): Stimulation of chromatin template activity in vitro

Virology ◽

10.1016/0042-6822(79)90419-7 ◽

1979 ◽

Vol 95 (1) ◽

pp. 222-226 ◽

Cited By ~ 5

Author(s):

Tohru Kamata ◽

Shigeaki Tanaka ◽

Shogo Aikawa ◽

Yorio Hinuma ◽

Yasushi Watanabe

Keyword(s):

Epstein Barr Virus ◽

Nuclear Antigen ◽

Template Activity ◽

Barr Virus ◽

Chromatin Template ◽

Epstein Barr ◽

Stimulation Of

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Curcumin Inhibits Proliferation of Epstein–Barr Virus-Associated Human Nasopharyngeal Carcinoma Cells by Inhibiting EBV Nuclear Antigen 1 Expression

BioMed Research International ◽

10.1155/2019/8592921 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10

Author(s):

Limei Liu ◽

Jiaomin Yang ◽

Wuguang Ji ◽

Chao Wang

Keyword(s):

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Nuclear Antigen ◽

Mrna Levels ◽

Curcumin Treatment ◽

Antitumor Effects ◽

Barr Virus ◽

Cycle Arrest ◽

Epstein Barr

This investigation aims to study the effect of curcumin on the proliferation, cycle arrest, and apoptosis of Epstein–Barr virus- (EBV-) positive nasopharyngeal carcinoma (NPC) cells. EBV+ NPC cells were subjected to curcumin treatment. The cell viability was evaluated with the CCK-8. Cell cycle and apoptosis were analyzed by flow cytometry analysis. Expression (protein and mRNA) levels were detected with western blotting and quantitative real-time PCR, respectively. Curcumin efficiently reduced the viability of EBV+ NPC cells. Curcumin induced the cycle arrest of the HONE1 and HK1-EBV cells positive for EBV. Moreover, curcumin treatment promoted the NPC cell apoptosis, via the mitochondria- and death receptor-mediated pathways. Furthermore, curcumin decreased the expression of EBNA1 in the HONE1 and HK1-EBV cells and inhibited the transcriptional level of EBNA1 in the HeLa cells. Curcumin induced EBNA1 degradation via the proteasome-ubiquitin pathway. In addition, curcumin inhibited the proliferation of HONE1 and HK1-EBV cells positive for EBV, probably by decreasing the expression level of EBNA1. In both the HONE1 and HK1-EBV cells, curcumin inhibited the EBV latent and lytic replication. Curcumin could reduce the EBNA1 expression and exert antitumor effects against NPC in vitro.

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

Journal of Virology ◽

10.1128/jvi.00053-07 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6718-6730 ◽

Cited By ~ 56

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.

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

Blood ◽

10.1182/blood.v88.8.3147.bloodjournal8883147 ◽

1996 ◽

Vol 88 (8) ◽

pp. 3147-3159 ◽

Cited By ~ 24

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.

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Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals

Journal of General Virology ◽

10.1099/vir.0.81927-0 ◽

2006 ◽

Vol 87 (10) ◽

pp. 2879-2884 ◽

Cited By ~ 7

Author(s):

Marion Buck ◽

Anita Burgess ◽

Roslynn Stirzaker ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Viral Proteins ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Epstein Barr

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.

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Epstein-Barr Virus Nuclear Antigen 3C Activates the Latent Membrane Protein 1 Promoter in the Presence of Epstein-Barr Virus Nuclear Antigen 2 through Sequences Encompassing an Spi-1/Spi-B Binding Site

Journal of Virology ◽

10.1128/jvi.74.11.5151-5160.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5151-5160 ◽

Cited By ~ 81

Author(s):

Bo Zhao ◽

Clare E. Sample

Keyword(s):

Dna Binding ◽

Binding Site ◽

Binding Sites ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Nuclear Antigen ◽

Latent Membrane Protein 1 ◽

Barr Virus ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.

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IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5748 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5748-5757 ◽

Cited By ~ 190

Author(s):

L Zhang ◽

J S Pagano

Keyword(s):

Transcriptional Activation ◽

Epstein Barr Virus ◽

Nuclear Antigen ◽

Type I ◽

Blood Leukocytes ◽

Type Iii ◽

Barr Virus ◽

Virus Latency ◽

Restricted Type ◽

Epstein Barr

The Epstein-Barr virus (EBV) BamHI Q promoter (Qp) is the only promoter used for the transcription of Epstein-Barr virus nuclear antigen 1 (EBNA-1) mRNA in cells in the most restricted (type I) latent infection state. However, Qp is inactive in type III latency. With the use of the yeast one-hybrid system, a new cellular gene has been identified that encodes proteins which bind to sequence in Qp. The deduced amino acid sequence of the gene has significant homology to the interferon regulatory factors (IRFs). This new gene and products including two splicing variants are designated IRF-7A, IRF-7B, and IRF-7C. The expression of IRF-7 is predominantly in spleen, thymus, and peripheral blood leukocytes (PBL). IRF-7 proteins were identified in primary PBL with specific antiserum against IRF-7B protein. IRF-7s can bind to interferon-stimulated response element (ISRE) sequence and repress transcriptional activation by both interferon and IRF-1. Additionally, a functional viral ISRE sequence, 5'-GCGAAAACGAAAGT-3', has been identified in Qp. Finally, the expression of IRF-7 is consistently high in type III latency cells and almost undetectable in type I latency, corresponding to the activity of endogenous Qp in these latency states and the ability of the IRF-7 proteins to repress Qp-reporter constructs. The identification of a functional viral ISRE and association of IRF-7 with type III latency may be relevant to the mechanism of regulation of Qp.

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Epstein–Barr virus nuclear antigen (EBNA) 3A induces the expression of and interacts with a subset of chaperones and co-chaperones

Journal of General Virology ◽

10.1099/vir.0.83414-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 866-877 ◽

Cited By ~ 38

Author(s):

Paul Young ◽

Emma Anderton ◽

Kostas Paschos ◽

Rob White ◽

Martin J. Allday

Keyword(s):

Epstein Barr Virus ◽

Expression System ◽

Nuclear Antigen ◽

Human Diploid Fibroblasts ◽

Barr Virus ◽

Cellular Genes ◽

Infected Cells ◽

Epstein Barr ◽

Chaperone Complex

Viral nuclear oncoproteins EBNA3A and EBNA3C are essential for the efficient immortalization of B cells by Epstein–Barr virus (EBV) in vitro and it is assumed that they play an essential role in viral persistence in the human host. In order to identify cellular genes regulated by EBNA3A expression, cDNA encoding EBNA3A was incorporated into a recombinant adenoviral vector. Microarray analysis of human diploid fibroblasts infected with either adenovirus EBNA3A or an empty control adenovirus consistently showed an EBNA3A-specific induction of mRNA corresponding to the chaperones Hsp70 and Hsp70B/B′ and co-chaperones Bag3 and DNAJA1/Hsp40. Analysis of infected fibroblasts by real-time quantitative RT-PCR and Western blotting confirmed that EBNA3A, but not EBNA3C, induced expression of Hsp70, Hsp70B/B′, Bag3 and DNAJA1/Hsp40. This was also confirmed in a stable, inducible expression system. EBNA3A activated transcription from the Hsp70B promoter, but not multimerized heat-shock elements in transient transfection assays, consistent with specific chaperone and co-chaperone upregulation. Co-immunoprecipitation experiments suggest that EBNA3A can form a complex with the chaperone/co-chaperone proteins in both adenovirus-infected cells and EBV-immortalized lymphoblastoid cell lines. Consistent with this, induction of EBNA3A resulted in redistribution of Hsp70 from the cytoplasm to the nucleus. EBNA3A therefore specifically induces (and then interacts with) all of the factors necessary for an active Hsp70 chaperone complex.

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