scholarly journals Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6556-6566 ◽  
Author(s):  
Oliver Dorigo ◽  
Jose S. Gil ◽  
Sean D. Gallaher ◽  
Brenton T. Tan ◽  
Maria G. Castro ◽  
...  
Keyword(s):  
Hybrid System ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Genetic Defects ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

scholarly journals Latent Gene Sequencing Reveals Familial Relationships among Chinese Epstein-Barr Virus Strains and Evidence for Positive Selection of A11 Epitope Changes

2003 ◽  
Vol 77 (21) ◽  
pp. 11517-11530 ◽  
Author(s):  
R. S. Midgley ◽  
A. I. Bell ◽  
D. J. McGeoch ◽  
A. B. Rickinson
Keyword(s):  
Epstein Barr Virus ◽  
Sequence Divergence ◽  
Nuclear Antigen ◽  
Coding Region ◽  
Immune Selection ◽  
Genomic Context ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) strains from the highly HLA-A11-positive Chinese population are predominantly type 1 and show a variety of sequence changes (relative to the contemporary Caucasian prototype strain B95.8) in the nuclear antigen EBNA3B sequences encoding two immunodominant HLA-A11 epitopes, here called IVT and AVF. This has been interpreted by some as evidence of immune selection and by others as random genetic drift. To study epitope variation in a broader genomic context, we sequenced the whole of EBNA3B and parts of the EBNA2, 3A, and 3C genes from each of 31 Chinese EBV isolates. At each locus, type 1 viruses showed <2% nucleotide divergence from the B95.8 prototype while type 2 sequences remained even closer to the contemporary African prototype Ag876. However, type 1 isolates could clearly be divided into families based on linked patterns of sequence divergence from B95.8 across all four EBNA loci. Different patterns of IVT and AVF variation were associated with the different type 1 families, and there was additional epitope diversity within families. When the EBNA3 gene sequences of type 1 Chinese strains were subject to computer-based analysis, particular codons within the A11-epitope-coding region were among the few identified as being under positive or diversifying selection pressure. From these results, and the observation that mutant epitopes are consistently nonimmunogenic in vivo, we conclude that the immune selection hypothesis remains viable and worthy of further investigation.

scholarly journals Epstein–Barr virus-based plasmid enables inheritable transgene expression in mouse cerebral cortex

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0258026
Author(s):  
Tomoko Satake
Keyword(s):  
Cerebral Cortex ◽  
Glial Cells ◽  
Transgene Expression ◽  
Epstein Barr Virus ◽  
Continuous Development ◽  
Shrna Expression ◽  
Embryonic Mouse ◽  
Barr Virus ◽  
Epstein Barr

Continuous development of the cerebral cortex from the prenatal to postnatal period depends on neurons and glial cells, both of which are generated from neural progenitor cells (NPCs). Owing to technical limitations regarding the transfer of genes into mouse brain, the mechanisms behind the long-term development of the cerebral cortex have not been well studied. Plasmid transfection into NPCs in embryonic mouse brains by in utero electroporation (IUE) is a widely used technique aimed at expressing transgenes in NPCs and their recent progeny neurons. Because the plasmids in NPCs are attenuated with each cell division, the transgene is not expressed in their descendants, including glial cells. The present study shows that an Epstein–Barr virus-based plasmid (EB-oriP plasmid) is helpful for studying long-term cerebral cortex development. The use of the EB-oriP plasmid for IUE allowed transgene expression even in the descendant progeny cells of adult mouse brains. Combining the EB-oriP plasmid with the shRNA expression cassette allowed examination of the genes of interest in the continuous development of the cerebral cortex. Furthermore, preferential transgene expression was achieved in combination with cell type-specific promoter-driven transgene expression. Meanwhile, introducing the EB-oriP plasmid twice into the same individual embryos during separate embryonic development stages suggested heterogeneity of NPCs. In summary, IUE using the EB-oriP plasmid is a novel option to study the long-term development of the cerebral cortex in mice.

scholarly journals Differential Hyperacetylation of Histones H3 and H4 upon Promoter-Specific Recruitment of EBNA2 in Epstein-Barr Virus Chromatin

2003 ◽  
Vol 77 (14) ◽  
pp. 8166-8172 ◽  
Author(s):  
Nathalie Alazard ◽  
Henri Gruffat ◽  
Edwige Hiriart ◽  
Alain Sergeant ◽  
Evelyne Manet
Keyword(s):  
B Lymphocytes ◽  
Chromatin Immunoprecipitation ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Viral Promoters ◽  
Epstein Barr ◽  
Cellular Dna ◽  
First Time

ABSTRACT Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transcriptional activator involved in the immortalization of B lymphocytes by the virus. EBNA2 is targeted to the promoters of its responsive genes, via interaction with cellular DNA-binding proteins. Using chromatin immunoprecipitation assays, we show for the first time the conditional recruitment of EBNA2 on two specific viral promoters in vivo and demonstrate a correlation between this recruitment and a local change in the acetylation of histones H3 and H4, which is promoter dependent.

scholarly journals The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE.

1995 ◽  
Vol 15 (9) ◽  
pp. 4735-4744 ◽  
Author(s):  
X Tong ◽  
R Drapkin ◽  
R Yalamanchili ◽  
G Mosialos ◽  
E Kieff
Keyword(s):  
Epstein Barr Virus ◽  
Nuclear Protein ◽  
Lymphocyte Transformation ◽  
Nuclear Antigen ◽  
Cdna Clones ◽  
Barr Virus ◽  
Acidic Domain ◽  
Epstein Barr

Epstein-Barr virus nuclear antigen 2 (EBNA 2) activates transcription of specific genes and is essential for B-lymphocyte transformation. EBNA 2 has an acidic activation domain which interacts with general transcription factors TFIIB, TFIIH, and TAF40. We now show that EBNA 2 is specifically bound to a novel nuclear protein, p100, and that p100 can coactivate gene expression mediated by the EBNA 2 acidic domain. The EBNA 2 acidic domain was used to affinity purify p100. cDNA clones encoding the p100 open reading frame were identified on the basis of peptide sequences of the purified protein. Antibody against p100 coimmunoprecipitated p100 and EBNA 2 from Epstein-Barr virus-transformed lymphocyte extracts, indicating that EBNA 2 and p100 are complexed in vivo. p100 overexpression in cells specifically augmented EBNA 2 acidic domain-mediated activation. The coactivating effect is probably mediated by p100 interaction with TFIIE. Bacterially expressed p100 specifically adsorbs TFIIE from nuclear extracts, and in vitro-translated p56 or p34 TFIIE subunit can independently bind to p100. p100 also appears to be essential for normal cell growth, since cell viability was reduced by antisense p100 RNA and restored by sense p100 RNA expression.

scholarly journals Physical and Functional Interactions between the Corepressor CtBP and the Epstein-Barr Virus Nuclear Antigen EBNA3C

2001 ◽  
Vol 75 (16) ◽  
pp. 7749-7755 ◽  
Author(s):  
Robert Touitou ◽  
Mark Hickabottom ◽  
Gillian Parker ◽  
Tim Crook ◽  
Martin J. Allday
Keyword(s):  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Rat Embryo ◽  
Barr Virus ◽  
Functional Interactions ◽  
C Terminus ◽  
Epstein Barr ◽  
Repressor Activity

ABSTRACT CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). Here we show that EBNA3C binds to CtBP both in vitro and in vivo and that the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest trans-repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif had little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts was also revealed.

scholarly journals Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

1995 ◽  
Vol 15 (5) ◽  
pp. 2893-2903 ◽  
Author(s):  
R D Little ◽  
C L Schildkraut
Keyword(s):  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Virus Genome ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Small Plasmid ◽  
Barr Virus ◽  
Latently Infected ◽  
Epstein Barr

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.

Hypovitaminosis-D and EBV: no interdependence between two MS risk factors in a healthy young UK autumn cohort

2013 ◽  
Vol 20 (6) ◽  
pp. 751-753 ◽  
Author(s):  
Caren Ramien ◽  
Annette Pachnio ◽  
Sofia Sisay ◽  
Jusnara Begum ◽  
Alison Leese ◽  
...  
Keyword(s):  
Risk Factors ◽  
Multiple Sclerosis ◽  
Epstein Barr Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Hypovitaminosis D ◽  
Nuclear Antigen ◽  
Epstein Barr Virus Infection ◽  
Barr Virus ◽  
Epstein Barr

Late Epstein-Barr virus infection and hypovitaminosis-D as environmental risk factors in the pathogenesis of multiple sclerosis are gaining great interest. We, therefore, tested for in-vivo interdependence between Epstein-Barr-virus (EBV)-status and 25-hydroxyvitamin D3 (25(OH)D3) -level in healthy young individuals from a United Kingdom (UK) autumn cohort. EBV-load was measured by quantitative polymerase chain reaction and 25(OH)D3 levels by isotope-dilution liquid chromatography-tandem mass spectrometry. This young, healthy UK autumn cohort showed surprisingly low levels of 25(OH)D3 (mean value: 40.5 nmol/L ± 5.02). Furthermore, we found that low 25(OH)D3 levels did not impact on EBV load and anti-EBV nuclear antigen-1 (EBNA-1) titers. However, we observed a correlation between EBV load and EBNA-1 titers. These observations should be of value in the study of the potential relationship between hypovitaminosis-D and EBV-status in the pathophysiology of multiple sclerosis.

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