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

2008 ◽  
Vol 89 (4) ◽  
pp. 866-877 ◽  
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.

scholarly journals Epstein-Barr Virus Nuclear Antigen 1 Interacts with Nm23-H1 in Lymphoblastoid Cell Lines and Inhibits Its Ability To Suppress Cell Migration

2005 ◽  
Vol 79 (3) ◽  
pp. 1559-1568 ◽  
Author(s):  
Masanao Murakami ◽  
Ke Lan ◽  
Chitra Subramanian ◽  
Erle S. Robertson
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Critical Role ◽  
Nuclear Antigen ◽  
Lymphoblastoid Cell Lines ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in the majority of latency programs in EBV-infected cells and is critical for the maintenance of EBV episomes in the infected cells. EBNA1 is also known to be involved in transcriptional activation and regulates expression of the EBV latent genes, including the EBNAs and LMP1. Thus, EBNA1 is a multifunctional protein with critical functions required for the persistence of the viral genome over successive generations, producing new daughter cells from the infected cell. We identify EBNA1 here as an interacting EBNA with the known suppressor of metastasis and cell migration, Nm23-H1. Nm23-H1 inhibits cell migration when expressed in cancer cells. We show that EBNA1 associates with Nm23-H1 in EBV-infected cells in vitro, as well as in lymphoblastoid cell lines (LCLs). Nm23-H1 predominantly localizes to the cytoplasm in BJAB and 293T cells; however, upon expression of EBNA1, Nm23-H1 is translocated to the nucleus in similar compartments to EBNA1, suggesting a potential functional role that is linked to EBNA1. Convincingly, in EBV-transformed LCLs Nm23-H1 is localized predominantly to the nucleus and colocalizes to similar compartment as EBNA1. Further, we tested the effects of EBNA1 on Nm23-H1-mediated suppression of cell migration and showed that EBNA1 rescues the suppression of cell migration mediated by Nm23-H1. These in vitro studies suggest that EBNA1 plays a critical role in regulating the activities of Nm23-H1, including cell migration, through a mechanism which involves direct interaction of this major regulator in EBV-infected cells.

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

Virology ◽  
1979 ◽  
Vol 95 (1) ◽  
pp. 222-226 ◽  
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

scholarly journals Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Tiffany R. Frey ◽  
Jozan Brathwaite ◽  
Xiaofan Li ◽  
Sandeepta Burgula ◽  
Ibukun A. Akinyemi ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Viral Spread ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Host Shutoff ◽  
Epstein Barr

ABSTRACT Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

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

2006 ◽  
Vol 87 (10) ◽  
pp. 2879-2884 ◽  
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.

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

2004 ◽  
Vol 78 (7) ◽  
pp. 3542-3552 ◽  
Author(s):  
Wei Yue ◽  
Matthew G. Davenport ◽  
Julia Shackelford ◽  
Joseph S. Pagano
Keyword(s):  
Epstein Barr Virus ◽  
Regulation Of Gene Expression ◽  
Cyclin B1 ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Type Iii ◽  
Barr Virus ◽  
M Phase ◽  
Epstein Barr

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.

scholarly journals 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

2000 ◽  
Vol 74 (11) ◽  
pp. 5151-5160 ◽  
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.

scholarly journals A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (23) ◽  
pp. 12857-12867 ◽  
Author(s):  
Jason S. Knight ◽  
Nikhil Sharma ◽  
Danielle E. Kalman ◽  
Erle S. Robertson
Keyword(s):  
Amino Acids ◽  
Epstein Barr Virus ◽  
Kinase Activity ◽  
Cyclin A ◽  
Carboxy Terminus ◽  
Barr Virus ◽  
Amino Terminal ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the α1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

scholarly journals Plasmacytic differentiation of circulating Epstein-Barr virus-infected B lymphocytes during acute infectious mononucleosis.

1981 ◽  
Vol 153 (2) ◽  
pp. 235-244 ◽  
Author(s):  
J E Robinson ◽  
D Smith ◽  
J Niederman
Keyword(s):  
Infectious Mononucleosis ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Phase 1 ◽  
Acute Disease ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Acute Infectious Mononucleosis

During the acute phase (1 wk of symptoms or less) of infectious mononucleosis (IM), 70--80% of circulating Epstein-Barr virus nuclear antigen (EBNA)-positive cells have differentiated toward plasma cells. Thus the characteristics of the infected cells in the majority of IM patients during early disease are indistinguishable from EBNA-positive tumor cells of a previously reported child who developed lymphoma during IM. IgA and IgG were the most frequent and IgM the least frequent immunoglobulin isotypes detected in EBNA-positive cells. In acute disease EBNA was present in 5.5--20% of T cell-depleted blood lymphocytes but in the 2nd or 3rd wk of illness the number of EBNA-positive cells sharply decreased to 0.4--1.4%. At the same time the fraction of antigen-positive cells containing cytoplasmic immunoglobulins also diminished, suggesting either that differentiation of infected cells was altered during the disease or that nondifferentiated antigen-positive cells had a survival advantage. Both the high proportion of plasmacytic EBNA-positive cells seen during acute disease and the apparent loss of differentiation by these cells later in disease may be regulated by host immunologic factors. Immunoglobulin-producing EBNA-positive cells may be the source of heterophile antibodies and other seemingly inappropriate antibodies usually found in serum during IM; however, increased numbers of noninfected plasma cells were present in some patients and may also be a potential source of these unusual antibodies.

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