scholarly journals The Conserved Domain CR2 of Epstein–Barr Virus Nuclear Antigen Leader Protein Is Responsible Not Only for Nuclear Matrix Association but Also for Nuclear Localization

Virology ◽  
2001 ◽  
Vol 279 (2) ◽  
pp. 401-413 ◽  
Author(s):  
Akihiko Yokoyama ◽  
Yasushi Kawaguchi ◽  
Issay Kitabayashi ◽  
Misao Ohki ◽  
Kanji Hirai
Keyword(s):  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Conserved Domain ◽  
Leader Protein ◽  
Epstein Barr

scholarly journals 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

2002 ◽  
Vol 76 (3) ◽  
pp. 1025-1032 ◽  
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.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
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.

scholarly journals Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Chong Wang ◽  
Hufeng Zhou ◽  
Yong Xue ◽  
Jun Liang ◽  
Yohei Narita ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Transcription Activator ◽  
Lymphoproliferative Diseases ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCEEpstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

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 Identification of protein kinases responsible for phosphorylation of Epstein–Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function

2003 ◽  
Vol 84 (12) ◽  
pp. 3381-3392 ◽  
Author(s):  
Kentaro Kato ◽  
Akihiko Yokoyama ◽  
Yukinobu Tohya ◽  
Hiroomi Akashi ◽  
Yukihiro Nishiyama ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

scholarly journals Genetic analysis of the Epstein–Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function

2001 ◽  
Vol 82 (12) ◽  
pp. 3067-3079 ◽  
Author(s):  
Eamon M. McCann ◽  
Gemma L. Kelly ◽  
Alan B. Rickinson ◽  
Andrew I. Bell
Keyword(s):  
Genetic Analysis ◽  
Epstein Barr Virus ◽  
Cell Transformation ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Lmp1 Expression ◽  
Leader Protein ◽  
Epstein Barr ◽  
The Individual ◽  
Unique Domain

Co-operation between the Epstein–Barr virus (EBV)-coded leader protein EBNA-LP and the nuclear antigen EBNA2 appears to be critical for efficient virus-induced B cell transformation. Here we report the genetic analysis of EBNA-LP function using two transient co-transfection assays of co-operativity, activation of latent membrane protein 1 (LMP1) expression from a resident EBV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regions (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within all three CRs in the EBNA-LP W1W2 repeat domain completely abrogated function, through inhibition of nuclear localization in the cases of CR1 and CR2 but not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had relatively little effect, yet loss of the whole Y2 sequence blocked activity. Alanine substitution of serine residues within potential phosphorylation sites identified two mutants of particular interest. Substitution of three such residues (S34,36,63) within W1W2 not only abrogated EBNA-LP activity but was associated with a complete loss of EBNA2 detectability in co-transfected cells, implying possible destabilization of the co-expressed EBNA2 protein. More importantly the individual substitution of S36 completely blocked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We infer critical roles for the CR3 domain and for the S36 residue in EBNA-LP’s co-operative function.

scholarly journals Sequences Adjacent to oriP Improve the Persistence of Epstein-Barr Virus-Based Episomes in B Cells

2001 ◽  
Vol 75 (22) ◽  
pp. 11249-11252 ◽  
Author(s):  
Robert E. White ◽  
Richard Wade-Martins ◽  
Michael R. James
Keyword(s):  
B Cells ◽  
Nuclear Matrix ◽  
Epstein Barr Virus ◽  
Matrix Attachment Region ◽  
Nuclear Antigen ◽  
Adjacent Region ◽  
Barr Virus ◽  
Attachment Region ◽  
Nuclear Matrix Attachment Region ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spansoriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriPalone.

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.

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