Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro differentiation of EBV-transduced mESC lines.(A) Schematic protocol of the different steps, which lead to in vitro differentiated murine B cells. Cobblestone indicates the appearance of cell areas in which hematopoietic precursors develop. (B) Flow cytometry analysis of selected cell cultures gated on lymphocytes of EB5 mESCs, the Bruce4 EBNA1 clone C1 and two derivatives transduced with the recombinant EBV genomes p3053 and p3314 as indicated. Numbers indicate the percentage of B cells (B220+/CD19+), IgM+ B cells and IgM+/IgD+ B cells within the lymphocyte gate. Cells were analyzed between day 20 and day 23 of the differentiation protocol.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2289878&req=5

pone-0001996-g005: In vitro differentiation of EBV-transduced mESC lines.(A) Schematic protocol of the different steps, which lead to in vitro differentiated murine B cells. Cobblestone indicates the appearance of cell areas in which hematopoietic precursors develop. (B) Flow cytometry analysis of selected cell cultures gated on lymphocytes of EB5 mESCs, the Bruce4 EBNA1 clone C1 and two derivatives transduced with the recombinant EBV genomes p3053 and p3314 as indicated. Numbers indicate the percentage of B cells (B220+/CD19+), IgM+ B cells and IgM+/IgD+ B cells within the lymphocyte gate. Cells were analyzed between day 20 and day 23 of the differentiation protocol.

Mentions: Our final goal was to study EBV's phenotype in mouse B cells in vitro. We also expected that viral genes like EBNA2, which were not expressed in mESCs (Fig. 3C), might be expressed upon differentiation to cells of the B-cell lineage. mESCs can be differentiated to mature B cells in vitro [20], [21]. We optimized the published protocols to improve the rate of B cell differentiation (Fig. 5A) to obtain sufficient numbers of B cells from the originally described EB5 mESC line, EBNA1-expressing Bruce4 mESCs and derivatives carrying the recombinant maxi- and mini-EBV genomes. Drug selection for the maintenance of the recombinant EBV genomes could not be applied during the differentiation process because OP9 feeder cells are sensitive to G418. The recombinant EBV genomes were stably maintained in mESCs for several weeks without selection (data not shown) and under conditions of mESC differentiation (below).


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)

In vitro differentiation of EBV-transduced mESC lines.(A) Schematic protocol of the different steps, which lead to in vitro differentiated murine B cells. Cobblestone indicates the appearance of cell areas in which hematopoietic precursors develop. (B) Flow cytometry analysis of selected cell cultures gated on lymphocytes of EB5 mESCs, the Bruce4 EBNA1 clone C1 and two derivatives transduced with the recombinant EBV genomes p3053 and p3314 as indicated. Numbers indicate the percentage of B cells (B220+/CD19+), IgM+ B cells and IgM+/IgD+ B cells within the lymphocyte gate. Cells were analyzed between day 20 and day 23 of the differentiation protocol.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001996-g005: In vitro differentiation of EBV-transduced mESC lines.(A) Schematic protocol of the different steps, which lead to in vitro differentiated murine B cells. Cobblestone indicates the appearance of cell areas in which hematopoietic precursors develop. (B) Flow cytometry analysis of selected cell cultures gated on lymphocytes of EB5 mESCs, the Bruce4 EBNA1 clone C1 and two derivatives transduced with the recombinant EBV genomes p3053 and p3314 as indicated. Numbers indicate the percentage of B cells (B220+/CD19+), IgM+ B cells and IgM+/IgD+ B cells within the lymphocyte gate. Cells were analyzed between day 20 and day 23 of the differentiation protocol.
Mentions: Our final goal was to study EBV's phenotype in mouse B cells in vitro. We also expected that viral genes like EBNA2, which were not expressed in mESCs (Fig. 3C), might be expressed upon differentiation to cells of the B-cell lineage. mESCs can be differentiated to mature B cells in vitro [20], [21]. We optimized the published protocols to improve the rate of B cell differentiation (Fig. 5A) to obtain sufficient numbers of B cells from the originally described EB5 mESC line, EBNA1-expressing Bruce4 mESCs and derivatives carrying the recombinant maxi- and mini-EBV genomes. Drug selection for the maintenance of the recombinant EBV genomes could not be applied during the differentiation process because OP9 feeder cells are sensitive to G418. The recombinant EBV genomes were stably maintained in mESCs for several weeks without selection (data not shown) and under conditions of mESC differentiation (below).

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