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Restricted expression of Epstein-Barr virus latent genes in murine B cells derived from embryonic stem cells.

Zychlinska M, Herrmann H, Zimber-Strobl U, Hammerschmidt W - PLoS ONE (2008)

Bottom Line: In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs).In contrast to human B cells, EBV's nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro.Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environment and Health, Munich, Germany.

ABSTRACT

Background: Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95% of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV's pathogenesis and latency in a suitable and amenable host.

Methodology/principal findings: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV's nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation.

Conclusions/significance: Our findings indicate that fundamental differences in gene regulation between mouse and man might block the route towards a tractable murine model for EBV.

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Related in: MedlinePlus

Targeting of EBNA1 into the rosa26 locus.(A) Molecular cloning strategy. A loxP flanked transcriptional stop cassette (stop) and the selectable maker gene for G418 resistance (neor) precede the EBNA1 gene in the rosa26 intron bracketed by exon 1 (e1) and exon 2 (e2) (top). After Cre-mediated deletion EBNA1 is transcribed from the ubiquitously expressed rosa26 locus in Bruce4 EBNA1 cells (bottom). (B) Southern blot analysis of several Bruce4 EBNA1 cell lines after Cre-mediated deletion of the stop cassette. The radioactive probe, depicted in (A), distinguishes between the initial targeted situation (8.7 kb) and deletion of the stop cassette (4.8 kb). (C) Expression of EBNA1 in different Bruce4 EBNA1 cell lines as confirmed by immunodetection (arrowhead). EBNA1-positive 293 cells serve as positive control, negative controls are mouse embryonic fibroblasts and parental mESCs (Bruce4).
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pone-0001996-g001: Targeting of EBNA1 into the rosa26 locus.(A) Molecular cloning strategy. A loxP flanked transcriptional stop cassette (stop) and the selectable maker gene for G418 resistance (neor) precede the EBNA1 gene in the rosa26 intron bracketed by exon 1 (e1) and exon 2 (e2) (top). After Cre-mediated deletion EBNA1 is transcribed from the ubiquitously expressed rosa26 locus in Bruce4 EBNA1 cells (bottom). (B) Southern blot analysis of several Bruce4 EBNA1 cell lines after Cre-mediated deletion of the stop cassette. The radioactive probe, depicted in (A), distinguishes between the initial targeted situation (8.7 kb) and deletion of the stop cassette (4.8 kb). (C) Expression of EBNA1 in different Bruce4 EBNA1 cell lines as confirmed by immunodetection (arrowhead). EBNA1-positive 293 cells serve as positive control, negative controls are mouse embryonic fibroblasts and parental mESCs (Bruce4).

Mentions: In preliminary experiments (discussed below), we noticed that mESCs did not survive selection when transfected with recombinant EBV genomes encoding the G418 resistance gene neo. Because nuclear retention and DNA replication of extrachromosomal EBV genomes depend on expression of the viral EBNA1 gene we considered that it might be expressed at insufficient levels in mESCs. Therefore, we stably introduced EBNA1 into the mouse rosa26 locus. This locus is known to assure stable expression of inserted genes in all cell types [15]. Our genetic strategy is outlined in Fig. 1A. EBNA1 was integrated into the first intron of the rosa26 gene in Bruce4 mESCs in two steps. First, the locus was targeted with the linearized p3032 plasmid fragment carrying EBNA1 with a preceding transcriptional stop cassette encompassing the neo gene. The stop cassette was flanked by two loxP sites oriented in the same direction. After successful transfection and selection, 400 G418-resistant single cell clones were obtained and analyzed for correct homologous recombination by Southern blot hybridization. Several correct clones were identified. In the second step, Cre recombinase was transiently expressed in one of the mESC clones, which led to the deletion of the stop cassette in 17 out of 20 clones analyzed (Fig. 1A). Southern blot hybridizations confirmed this genetic manipulation and concomitant removal of the neo gene (Fig. 1B). Expression of EBNA1 was assessed by Western blots in nine clones, four of which are shown in Fig 1C.


Restricted expression of Epstein-Barr virus latent genes in murine B cells derived from embryonic stem cells.

Zychlinska M, Herrmann H, Zimber-Strobl U, Hammerschmidt W - PLoS ONE (2008)

Targeting of EBNA1 into the rosa26 locus.(A) Molecular cloning strategy. A loxP flanked transcriptional stop cassette (stop) and the selectable maker gene for G418 resistance (neor) precede the EBNA1 gene in the rosa26 intron bracketed by exon 1 (e1) and exon 2 (e2) (top). After Cre-mediated deletion EBNA1 is transcribed from the ubiquitously expressed rosa26 locus in Bruce4 EBNA1 cells (bottom). (B) Southern blot analysis of several Bruce4 EBNA1 cell lines after Cre-mediated deletion of the stop cassette. The radioactive probe, depicted in (A), distinguishes between the initial targeted situation (8.7 kb) and deletion of the stop cassette (4.8 kb). (C) Expression of EBNA1 in different Bruce4 EBNA1 cell lines as confirmed by immunodetection (arrowhead). EBNA1-positive 293 cells serve as positive control, negative controls are mouse embryonic fibroblasts and parental mESCs (Bruce4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289878&req=5

pone-0001996-g001: Targeting of EBNA1 into the rosa26 locus.(A) Molecular cloning strategy. A loxP flanked transcriptional stop cassette (stop) and the selectable maker gene for G418 resistance (neor) precede the EBNA1 gene in the rosa26 intron bracketed by exon 1 (e1) and exon 2 (e2) (top). After Cre-mediated deletion EBNA1 is transcribed from the ubiquitously expressed rosa26 locus in Bruce4 EBNA1 cells (bottom). (B) Southern blot analysis of several Bruce4 EBNA1 cell lines after Cre-mediated deletion of the stop cassette. The radioactive probe, depicted in (A), distinguishes between the initial targeted situation (8.7 kb) and deletion of the stop cassette (4.8 kb). (C) Expression of EBNA1 in different Bruce4 EBNA1 cell lines as confirmed by immunodetection (arrowhead). EBNA1-positive 293 cells serve as positive control, negative controls are mouse embryonic fibroblasts and parental mESCs (Bruce4).
Mentions: In preliminary experiments (discussed below), we noticed that mESCs did not survive selection when transfected with recombinant EBV genomes encoding the G418 resistance gene neo. Because nuclear retention and DNA replication of extrachromosomal EBV genomes depend on expression of the viral EBNA1 gene we considered that it might be expressed at insufficient levels in mESCs. Therefore, we stably introduced EBNA1 into the mouse rosa26 locus. This locus is known to assure stable expression of inserted genes in all cell types [15]. Our genetic strategy is outlined in Fig. 1A. EBNA1 was integrated into the first intron of the rosa26 gene in Bruce4 mESCs in two steps. First, the locus was targeted with the linearized p3032 plasmid fragment carrying EBNA1 with a preceding transcriptional stop cassette encompassing the neo gene. The stop cassette was flanked by two loxP sites oriented in the same direction. After successful transfection and selection, 400 G418-resistant single cell clones were obtained and analyzed for correct homologous recombination by Southern blot hybridization. Several correct clones were identified. In the second step, Cre recombinase was transiently expressed in one of the mESC clones, which led to the deletion of the stop cassette in 17 out of 20 clones analyzed (Fig. 1A). Southern blot hybridizations confirmed this genetic manipulation and concomitant removal of the neo gene (Fig. 1B). Expression of EBNA1 was assessed by Western blots in nine clones, four of which are shown in Fig 1C.

Bottom Line: In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs).In contrast to human B cells, EBV's nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro.Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environment and Health, Munich, Germany.

ABSTRACT

Background: Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95% of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV's pathogenesis and latency in a suitable and amenable host.

Methodology/principal findings: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV's nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation.

Conclusions/significance: Our findings indicate that fundamental differences in gene regulation between mouse and man might block the route towards a tractable murine model for EBV.

Show MeSH
Related in: MedlinePlus