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

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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Nuclear localization of the Epstein–Barr virus EBNA3B protein

Journal of General Virology ◽

10.1099/vir.0.81640-0 ◽

2006 ◽

Vol 87 (4) ◽

pp. 789-793 ◽

Cited By ~ 7

Author(s):

Anita Burgess ◽

Marion Buck ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Computer Analyses ◽

Epstein Barr ◽

Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

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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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Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association

Journal of Virology ◽

10.1128/jvi.76.3.1025-1032.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1025-1032 ◽

Cited By ~ 13

Author(s):

Michiko Tanaka ◽

Akihiko Yokoyama ◽

Mie Igarashi ◽

Go Matsuda ◽

Kentaro Kato ◽

...

Keyword(s):

Amino Acid ◽

Nuclear Localization ◽

Nuclear Matrix ◽

Epstein Barr Virus ◽

Cellular Proteins ◽

Nuclear Antigen ◽

Barr Virus ◽

Leader Protein ◽

Epstein Barr ◽

Self Association

ABSTRACT Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate γ-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401–413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

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

Journal of Virology ◽

10.1128/jvi.78.7.3542-3552.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3542-3552 ◽

Cited By ~ 15

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.

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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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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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