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 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 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 MicroRNA-155 Is an Epstein-Barr Virus-Induced Gene That Modulates Epstein-Barr Virus-Regulated Gene Expression Pathways

2008 ◽  
Vol 82 (11) ◽  
pp. 5295-5306 ◽  
Author(s):  
Qinyan Yin ◽  
Jane McBride ◽  
Claire Fewell ◽  
Michelle Lacey ◽  
Xia Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Type I ◽  
Type Iii ◽  
Barr Virus ◽  
Transcriptional Regulatory ◽  
Infected Cells ◽  
Epstein Barr ◽  
Regulated Gene Expression ◽  
In Cells

ABSTRACT The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in Epstein-Barr virus (EBV)-infected cells displaying type III latency gene expression but not type I latency gene expression. We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested, and we show that expression in EBV-infected cells requires a conserved AP-1 element in the miR-155 promoter. Gene expression analysis was carried out in a type I latency cell line transduced with an miR-155-expressing retrovirus. This analysis identified both miR-155-suppressed and -induced cellular mRNAs and suggested that in addition to direct targeting of 3′ untranslated regions (UTRs), miR-155 alters gene expression in part through the alteration of signal transduction pathways. 3′ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes encoding BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets. Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus. Inspection of the promoters from genes regulated in EBV-infected cells and in cells infected with an miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3. Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV-mediated signaling in part through the modulation of transcriptional regulatory factors.

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 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 Changes in Expression Induced by Epstein-Barr Virus LMP1-CTAR1: Potential Role of bcl3

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Rachel Hood Edwards ◽  
Aron R. Marquitz ◽  
Nancy Raab-Traub
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Epstein Barr Virus ◽  
High Throughput Sequencing ◽  
Latent Membrane Protein 1 ◽  
Virus Protein ◽  
Barr Virus ◽  
Cellular Expression ◽  
Epstein Barr ◽  
Carboxy Terminal

ABSTRACTThe Epstein-Barr virus protein latent membrane protein 1 (LMP1) has two NF-κB activating domains within its intracellular carboxy terminus (carboxy-terminal activating region 1 [CTAR1] and CTAR2). LMP1-CTAR1 is required for B-lymphocyte transformation, is capable of transforming rodent fibroblasts, and uniquely activates phosphoinositol (PI3) kinase, the noncanonical NF-κB pathway, and expression of the epidermal growth factor receptor (EGFR). In this study, the effects of LMP1-CTAR1 on cellular gene expression were determined by high-throughput sequencing. Additionally, the binding of bcl3 was determined using chromatin immunoprecipitation (ChIP) and sequencing. LMP1-CTAR1 induced few changes in transcription with more genes showing decreased expression. Ingenuity pathway analysis indicated significant enrichment for genes involved in cancer and cellular movement, survival, growth, and proliferation pathways. ChIP in combination with high-throughput sequencing (ChIP-Seq) identified bcl3 binding for more than 2,000 genes in LMP1-CTAR1-expressing cells with more than 90% of the peaks at genes detected within the probable promoter region. Only a small subset of the genes with significant changes in expression had corresponding peaks in the bcl3 ChIP. However, both NFKB2 and PI3 kinase were identified in the bcl3 ChIP. Additionally, many of the predicted upstream regulators for the changes in expression were identified in the bcl3 ChIP. Analysis of the proteins in the NF-κB pathway revealed many changes identified by the high-throughput RNA sequencing (RNA-Seq) and bcl3 ChIP that would likely activate noncanonical NF-κB signaling and possibly inhibit canonical NF-κB signaling. These findings suggest that the two LMP1 signaling domains modulate their combined activity and that the bcl3 transcription factor is likely responsible for some of the unique effects of CTAR1 on cellular expression.IMPORTANCEThe Epstein-Barr virus protein latent membrane protein 1 (LMP1) has potent effects on cell growth. LMP1 has two regions, carboxy-terminal activating region 1 (CTAR1) and CTAR2, that distinctly activate NF-κB, a transcription factor complex involved in activation of important host genes. In this study, analysis of the effects on cellular gene expression revealed that CTAR1 significantly affected cellular expression in part through effects on a specific form of NF-κB. The data suggest that LMP1 can activate a distinct subset of host gene expression through its CTAR1 domain which in combination with other signaling effects induced by the CTAR2 domain likely affects cell movement, survival, and growth.

scholarly journals Interaction with the Epstein-Barr Virus Helicase Targets Zta to DNA Replication Compartments

2001 ◽  
Vol 75 (18) ◽  
pp. 8792-8802 ◽  
Author(s):  
Gangling Liao ◽  
Frederick Y. Wu ◽  
S. Diane Hayward
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Lytic Cycle ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Epstein Barr ◽  
Green Fluorescent

ABSTRACT Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.

scholarly journals Augmented Latent Membrane Protein 1 Expression from Epstein-Barr Virus Episomes with Minimal Terminal Repeats

2009 ◽  
Vol 84 (5) ◽  
pp. 2236-2244 ◽  
Author(s):  
Allison M. Repic ◽  
Mingxia Shi ◽  
Rona S. Scott ◽  
John W. Sixbey
Keyword(s):  
Membrane Protein ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Cell Clone ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
Mrna Levels ◽  
Barr Virus ◽  
Epstein Barr ◽  
Green Fluorescent

ABSTRACT The major oncogene of the Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), can be expressed from either of two promoters, ED-L1 or L1-TR, producing mRNAs of 2.8 kb or 3.5 kb, respectively. L1-TR, active in nasopharyngeal carcinoma and Hodgkin's lymphoma, is located within the first of a highly variable reiteration of terminal repeat (TR) sequences that are joined by random recombination upon circularization of the linear genome at entry into cells. To determine whether the resultant TR number affects LMP1 promoter activity, we isolated single-cell clones bearing episomes of distinct TR numbers (6TR to 12TR) from epithelial cells newly infected with EBV. LMP1 mRNA levels correlated directly with the quantity of LMP1 protein expressed but varied inversely to TR number. Unexpectedly, the 3.5-kb transcript predominated only at lower TR reiterations. Diminished L1-TR activity in the context of a higher TR count was confirmed with a green fluorescent protein (GFP) reporter construct driven by L1-TR. Various levels of LMP1, expressed from virus isogenic in all but TR number, produced divergent morphological and growth phenotypes in each cell clone. Abundant LMP1 in 6TR cells yielded a relatively cytostatic state compared to the proliferative one produced by intermediate and smaller amounts in 8TR and 12TR clones. These findings suggest that the diversification of TR number, inherent in a round of EBV reactivation and reinfection, may itself be a component of the oncogenic process. The replicative burst preceding onset of many EBV-linked cancers may increase the likelihood that LMP1 levels compatible with clonal outgrowth are achieved in a subset of infected cells.

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