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 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 Epstein–Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular

2002 ◽  
Vol 83 (10) ◽  
pp. 2377-2383 ◽  
Author(s):  
Sayuri Ito ◽  
Eisuke Gotoh ◽  
Shigeru Ozawa ◽  
Kazuo Yanagi
Keyword(s):  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Chromosomal Localization ◽  
Nuclear Antigen ◽  
Interphase Chromatin ◽  
Premature Chromosome Condensation ◽  
Metaphase Chromosomes ◽  
Barr Virus ◽  
Epstein Barr ◽  
Green Fluorescent

Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP–EBNA-1) is examined by confocal microscopy combined with a ‘premature chromosome condensation’ (PCC) procedure. Analyses show that GFP–EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP–EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.

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 High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (4) ◽  
pp. 1657-1664 ◽  
Author(s):  
Ngan Lam ◽  
Mark L. Sandberg ◽  
Bill Sugden
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Barr Virus ◽  
Signaling Domain ◽  
Infected Cells ◽  
Epstein Barr ◽  
Green Fluorescent ◽  
Carboxy Terminal

ABSTRACT LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

scholarly journals Identification of the nuclear localization signals within the Epstein–Barr virus EBNA-6 protein

2004 ◽  
Vol 85 (1) ◽  
pp. 165-172 ◽  
Author(s):  
Kenia Krauer ◽  
Marion Buck ◽  
James Flanagan ◽  
Deanna Belzer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Epstein Barr

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.

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 Epstein-Barr Virus Encodes a Novel Homolog of the bcl-2 Oncogene That Inhibits Apoptosis and Associates with Bax and Bak

1999 ◽  
Vol 73 (6) ◽  
pp. 5181-5185 ◽  
Author(s):  
William L. Marshall ◽  
Ching Yim ◽  
Erik Gustafson ◽  
Thomas Graf ◽  
David R. Sage ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Apoptosis Resistance ◽  
Significant Sequence Similarity ◽  
Reading Frame ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr ◽  
Green Fluorescent

ABSTRACT The sequenced gammaherpesviruses each contain a single viralbcl-2 homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2 homologs and cellular bcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2.

scholarly journals Epstein-Barr Virus Entry Utilizing HLA-DP or HLA-DQ as a Coreceptor

2000 ◽  
Vol 74 (5) ◽  
pp. 2451-2454 ◽  
Author(s):  
Keith M. Haan ◽  
William W. Kwok ◽  
Richard Longnecker ◽  
Peter Speck
Keyword(s):  
Membrane Fusion ◽  
Epstein Barr Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Surface Expression ◽  
Barr Virus ◽  
Hla Dr ◽  
Hla Dq ◽  
Epstein Barr ◽  
Green Fluorescent

ABSTRACT Epstein-Barr virus (EBV) glycoprotein gp350/gp220 association with cellular CD21 facilitates virion attachment to B lymphocytes. Membrane fusion requires the additional interaction between virion gp42 and cellular HLA-DR. This binding is thought to catalyze membrane fusion through a further association with the gp85-gp25 (gH-gL) complex. Cell lines expressing CD21 but lacking expression of HLA class II molecules are resistant to infection by a recombinant EBV expressing enhanced green fluorescent protein. Surface expression of HLA-DR, HLA-DP, or HLA-DQ confers susceptibility to EBV infection on resistant cells that express CD21. Therefore, HLA-DP or HLA-DQ can substitute for HLA-DR and serve as a coreceptor in EBV entry.

scholarly journals Key motifs in EBV (Epstein–Barr virus)-encoded protein kinase for phosphorylation activity and nuclear localization

2010 ◽  
Vol 431 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Svetlana Gershburg ◽  
Leann Murphy ◽  
Manfred Marschall ◽  
Edward Gershburg
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Nuclear Translocation ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Barr Virus ◽  
Antiviral Compounds ◽  
Epstein Barr

A sole EBV (Epstein–Barr virus)-encoded protein kinase (EBV-PK) (the BGLF4 gene product) plays important roles in viral infection. Although a number of targets of this protein have been identified, the kinase itself remains largely unstudied with regard to its enzymology and structure. In the present study, site-directed mutagenesis has been employed to generate mutations targeting residues involved in nuclear localization of the EBV-PK, core residues in subdomain III of the protein kinase domain conserved in most protein kinases or residues in subdomain VIa conserved only within the HPK (herpesvirus-encoded protein kinase) group. Deletion of amino acids 389–391 resulted in exclusive cytoplasmic localization of the protein, indicating the involvement of this region in nuclear translocation of the EBV-PK. Mutations at the amino acids Glu113 (core component), Phe175, Leu178, Phe184, Leu185 and Asn186 (conserved in HPKs) resulted in loss of EBV-PK autophosphorylation, protein substrate [EBV EA-D (early antigen diffused)] phosphorylation, and ability to facilitate ganciclovir phosphorylation. These results reiterate the unique features of this group of kinases and present an opportunity for designing more specific antiviral compounds.

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