scholarly journals Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

1997 ◽  
Vol 17 (1) ◽  
pp. 364-377 ◽  
Author(s):  
B C Schaefer ◽  
J L Strominger ◽  
S H Speck
Keyword(s):  
Host Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Gene Promoter ◽  
Cpg Methylation ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Barr Virus ◽  
Epstein Barr

Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs.

scholarly journals Regulation of Epstein-Barr Virus Latency Type by the Chromatin Boundary Factor CTCF

2006 ◽  
Vol 80 (12) ◽  
pp. 5723-5732 ◽  
Author(s):  
Charles M. Chau ◽  
Xiao-Yong Zhang ◽  
Steven B. McMahon ◽  
Paul M. Lieberman
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Type I ◽  
Mrna Levels ◽  
Type Iii ◽  
Barr Virus ◽  
Chromatin Boundary ◽  
Epstein Barr

ABSTRACT Epstein Barr virus (EBV) can establish distinct latency types with different growth-transforming properties. Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP). Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jκ response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA. Using DNA affinity, electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP), we found that CTCF binds both in vitro and in vivo to the EBV genome between OriP and Cp, with an ∼50-bp footprint at EBV coordinates 10515 to 10560. Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC. DNA affinity and ChIP showed more CTCF binding at this site in type I latency cell lines (MutuI and KemI) than in type III latency cell lines (LCL3456 and Raji). CTCF protein and mRNA expression levels were higher in type I than type III cell lines. Short interfering RNA depletion of CTCF in type I MutuI cells stimulated EBNA2 mRNA levels, while overexpression of CTCF in type III Raji cells inhibited EBNA2 mRNA levels. These results indicate that increased CTCF can repress EBNA2 transcription. We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination.

scholarly journals Interferon Regulatory Factor 2 Represses the Epstein-Barr Virus BamHI Q Latency Promoter in Type III Latency

1999 ◽  
Vol 19 (4) ◽  
pp. 3216-3223 ◽  
Author(s):  
Luwen Zhang ◽  
Joseph S. Pagano
Keyword(s):  
Epstein Barr Virus ◽  
Mrna Level ◽  
Negative Regulator ◽  
Nuclear Antigen ◽  
Major Protein ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is the essential protein for maintenance of the EBV episome and establishment of latency. The BamHI Q promoter (Qp) is used for the transcription of EBNA-1 mRNA in type I and type II latency, which are EBV infection states exemplified by Burkitt’s lymphoma and nasopharyngeal carcinoma. However, Qp is inactive in type III latency, and other promoters (the BamHI C promoter and/or theBamHI W promoter) are used for EBNA-1. The involvement of interferon regulatory factors (IRFs) in the regulation of Qp is suggested by the presence of an essential interferon-stimulated response element (ISRE) in the promoter. In this work, expression of IRF-2 is shown to be inversely associated with Qp status, i.e., IRF-2 levels are high in type III latency (when Qp is inactive) and low in type I latency (when Qp is active). Also, IRF-2 is identified by electrophoretic mobility shift assay as the major protein binding to the Qp ISRE in type III latency. In transient transfection assays, IRF-2 represses the activity of Qp-reporter constructs. Overexpression of IRF-2 in a type I latency cell line did not activate the endogenous Qp but marginally reduced the EBNA-1 mRNA level. Switching from type III latency (Qp inactive) to type II latency (Qp active), as produced by cell fusion, is directly associated with greatly reduced expression of IRF-2. These data strongly suggest that IRF-2 is a negative regulator of Qp and may contribute to the silencing of Qp in type III latency.

scholarly journals Visualization of Alternative Epstein-Barr Virus Expression Programs by Fluorescent In Situ Hybridization at the Cell Level

1999 ◽  
Vol 73 (6) ◽  
pp. 5064-5069 ◽  
Author(s):  
Anna Szeles ◽  
Kerstin I. Falk ◽  
Stephan Imreh ◽  
George Klein
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Fluorescent In Situ Hybridization ◽  
Epstein Barr Virus ◽  
Type I ◽  
Type Iii ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). They regularly express six virally encoded nuclear proteins (EBNA1 to EBNA6) and three membrane proteins (LMP1, LMP2A, and LMP2B). In contrast, EBV-carrying Burkitt lymphoma (BL) cells in vivo and derived type I cell lines that maintain the BL phenotype express only EBNA1. During prolonged in vitro culturing, most EBV-carrying BL lines drift toward a more immunoblastic (type II or III) phenotype. Their viral antigen expression is upregulated in parallel. We have used fluorescent in situ hybridization to visualize viral transcripts in type I and III BL lines and LCLs. In type I cells, EBNA1 is encoded by a monocistronic message that originates from the Qp promoter. In type III cells, the EBNA1 transcript is spliced from a giant polycistronic message that originates from one of several alternative Wp or Cp promoters and encodes all six EBNAs. We have obtained a “track” signal with aBamHI W DNA probe that could hybridize with the polycistronic but not with the monocistronic message in two type III BL lines (Namalwa-Cl8 and MUTU III) and three LCLs (LCL IB4-D, LCL-970402, and IARC-171). A BamHI K probe that can hybridize to both the monocistronic and the polycistronic message visualized the same pattern in the type III BLs and the LCLs as the BamHI W probe. A positive signal was obtained with the BamHI K but not the BamHI W probe in the type I BL lines MUTU I and Rael. The RNA track method can thus distinguish between cells that use a type III and those that use a type I program. The former cells hybridize with both the W and the K probes, but the latter cells hybridize with only the K probe. Our findings may open the way for studies of the important but still unanswered question of whether cells with type I latency arise from immunoblasts with a full type III program or are generated by a separate pathway during primary infection.

scholarly journals Infection of Epstein–Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines

Cancers ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 237 ◽  
Author(s):  
Asuka Nanbo ◽  
Harutaka Katano ◽  
Michiyo Kataoka ◽  
Shiho Hoshina ◽  
Tsuyoshi Sekizuka ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Burkitt Lymphoma ◽  
Epstein Barr Virus ◽  
Type I ◽  
Type Iii ◽  
Barr Virus ◽  
Ebv Infection ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

scholarly journals IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency.

1997 ◽  
Vol 17 (10) ◽  
pp. 5748-5757 ◽  
Author(s):  
L Zhang ◽  
J S Pagano
Keyword(s):  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Type I ◽  
Blood Leukocytes ◽  
Type Iii ◽  
Barr Virus ◽  
Virus Latency ◽  
Restricted Type ◽  
Epstein Barr

The Epstein-Barr virus (EBV) BamHI Q promoter (Qp) is the only promoter used for the transcription of Epstein-Barr virus nuclear antigen 1 (EBNA-1) mRNA in cells in the most restricted (type I) latent infection state. However, Qp is inactive in type III latency. With the use of the yeast one-hybrid system, a new cellular gene has been identified that encodes proteins which bind to sequence in Qp. The deduced amino acid sequence of the gene has significant homology to the interferon regulatory factors (IRFs). This new gene and products including two splicing variants are designated IRF-7A, IRF-7B, and IRF-7C. The expression of IRF-7 is predominantly in spleen, thymus, and peripheral blood leukocytes (PBL). IRF-7 proteins were identified in primary PBL with specific antiserum against IRF-7B protein. IRF-7s can bind to interferon-stimulated response element (ISRE) sequence and repress transcriptional activation by both interferon and IRF-1. Additionally, a functional viral ISRE sequence, 5'-GCGAAAACGAAAGT-3', has been identified in Qp. Finally, the expression of IRF-7 is consistently high in type III latency cells and almost undetectable in type I latency, corresponding to the activity of endogenous Qp in these latency states and the ability of the IRF-7 proteins to repress Qp-reporter constructs. The identification of a functional viral ISRE and association of IRF-7 with type III latency may be relevant to the mechanism of regulation of Qp.

A Novel Epstein-Barr Virus-Like Virus, HVMNE, in aMacaca Nemestrina With Mycosis Fungoides

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2090-2101
Author(s):  
E.D. Rivadeneira ◽  
M.G. Ferrari ◽  
R.F. Jarrett ◽  
A.A. Armstrong ◽  
P. Markham ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cd8 T Cell ◽  
Macaca Nemestrina ◽  
Type I ◽  
Polymerase Gene ◽  
Barr Virus ◽  
Epstein Barr ◽  
T Cell Lines

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HVMNE) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3+/CD8+ phenotype. Two primary transformed CD8+ T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2–independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell lines as well as in the infiltrating CD8+ T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HVMNE placed this virus within theLymphocryptovirus genus and demonstrated that HVMNEis a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.

scholarly journals Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein–Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

2009 ◽  
Vol 90 (5) ◽  
pp. 1183-1189 ◽  
Author(s):  
Daniel Salamon ◽  
Ferenc Banati ◽  
Anita Koroknai ◽  
Mate Ravasz ◽  
Kalman Szenthe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cpg Methylation ◽  
Ctcf Binding ◽  
Mobility Shift ◽  
Barr Virus ◽  
Binding Factor ◽  
Epstein Barr ◽  
Mobility Shift Assay

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.

scholarly journals Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways

2007 ◽  
Vol 82 (4) ◽  
pp. 1946-1958 ◽  
Author(s):  
Jennifer E. Cameron ◽  
Qinyan Yin ◽  
Claire Fewell ◽  
Michelle Lacey ◽  
Jane McBride ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Interferon Response ◽  
Latent Membrane Protein 1 ◽  
Type I ◽  
Induced Expression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor κB (NF-κB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-κB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

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.

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