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

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.

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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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The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

Journal of Virology ◽

10.1128/jvi.72.11.8559-8567.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8559-8567 ◽

Cited By ~ 58

Author(s):

Zhigang Gao ◽

Anita Krithivas ◽

Jon E. Finan ◽

O. John Semmes ◽

Sifang Zhou ◽

...

Keyword(s):

Amino Acids ◽

Epstein Barr Virus ◽

Nuclear Translocation ◽

Intracellular Localization ◽

Activation Domain ◽

Replication Complex ◽

Barr Virus ◽

Amino Terminal ◽

Epstein Barr ◽

Primase Complex

ABSTRACT The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathioneS-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

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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

Journal of Virology ◽

10.1128/jvi.75.18.8556-8568.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8556-8568 ◽

Cited By ~ 10

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.

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Germinal Center B Cells Latently Infected with Epstein-Barr Virus Proliferate Extensively but Do Not Increase in Number

Journal of Virology ◽

10.1128/jvi.01780-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 1158-1168 ◽

Cited By ~ 42

Author(s):

Jill E. Roughan ◽

Charles Torgbor ◽

David A. Thorley-Lawson

Keyword(s):

B Cells ◽

Germinal Center ◽

Epstein Barr Virus ◽

Acute Infection ◽

Absolute Number ◽

Barr Virus ◽

Infected Cells ◽

The Absolute ◽

Epstein Barr

ABSTRACT In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo.

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Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

Journal of Virology ◽

10.1128/jvi.75.1.90-99.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 90-99 ◽

Cited By ~ 17

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.

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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

Journal of Virology ◽

10.1128/jvi.76.10.4699-4708.2002 ◽

2002 ◽

Vol 76 (10) ◽

pp. 4699-4708 ◽

Cited By ~ 69

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.

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An Epstein-Barr Virus That Expresses Only the First 231 LMP1 Amino Acids Efficiently Initiates Primary B-Lymphocyte Growth Transformation

Journal of Virology ◽

10.1128/jvi.73.12.10525-10530.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10525-10530 ◽

Cited By ~ 55

Author(s):

Kenneth M. Kaye ◽

Kenneth M. Izumi ◽

Hong Li ◽

Eric Johannsen ◽

David Davidson ◽

...

Keyword(s):

Amino Acids ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

B Lymphocyte ◽

Feeder Cells ◽

Barr Virus ◽

Infected Cells ◽

Carboxyl Terminal ◽

Epstein Barr

ABSTRACT An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675–683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-κB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.

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Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation

Journal of Virology ◽

10.1128/jvi.02682-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5261-5272 ◽

Cited By ~ 43

Author(s):

Kathy Shire ◽

Priya Kapoor ◽

Ke Jiang ◽

Margaret Ng Thow Hing ◽

Nirojini Sivachandran ◽

...

Keyword(s):

Amino Acids ◽

Transcriptional Activation ◽

Epstein Barr Virus ◽

Arginine Methylation ◽

Serine Phosphorylation ◽

Virus Protein ◽

Rich Region ◽

Barr Virus ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) EBNA1 protein is important for the replication and mitotic segregation of EBV genomes in latently infected cells and also activates the transcription of some of the viral latency genes. A Gly-Arg-rich region between amino acids 325 and 376 is required for both the segregation and transcriptional activation functions of EBNA1. Here we show that this region is modified by both arginine methylation and serine phosphorylation. Mutagenesis of the four potentially phosphorylated serines in this region indicated that phosphorylation of multiple serines contributes to the efficient segregation of EBV-based plasmids by EBNA1, at least in part by increasing EBNA1 binding to hEBP2. EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5. Multiple arginines in the 325-376 region were methylated in vitro by PRMT1 and PRMT5, as was an N-terminal Gly-Arg-rich region between amino acids 41 and 50. EBNA1 was also shown to be methylated in vivo, predominantly in the 325-376 region. Treatment of cells with a methylation inhibitor or down-regulation of PRMT1 altered EBNA1 localization, resulting in the formation of EBNA1 rings around the nucleoli. The results indicate that EBNA1 function is influenced by both serine phosphorylation and arginine methylation.

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Exosomes released in vitro from Epstein–Barr virus (EBV)-infected cells contain EBV-encoded latent phase mRNAs

Cancer Letters ◽

10.1016/j.canlet.2013.05.012 ◽

2013 ◽

Vol 337 (2) ◽

pp. 193-199 ◽

Cited By ~ 45

Author(s):

Andrea Canitano ◽

Giulietta Venturi ◽

Martina Borghi ◽

Maria Grazia Ammendolia ◽

Stefano Fais

Keyword(s):

Epstein Barr Virus ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr ◽

Latent Phase

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Epstein-Barr Virus Nuclear Antigen 3C Regulates Cyclin A/p27 Complexes and Enhances Cyclin A-Dependent Kinase Activity

Journal of Virology ◽

10.1128/jvi.78.4.1981-1991.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1981-1991 ◽

Cited By ~ 57

Author(s):

Jason S. Knight ◽

Erle S. Robertson

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Activity ◽

Cell Transformation ◽

Cyclin A ◽

Nuclear Antigen ◽

Cycle Progression ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.

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