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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

mini- and maxi-EBVs in Bruce4 EBNA1 mESCs.(A) GFP expression of the stably introduced maxi-EBV constructs p3053 and p3298 in Bruce4 EBNA1 cells. (B) Gardella gel hybridizations reveal the extrachromosomal status of the recombinant EBV genomes. Raji cells, which contain about 50 EBV genomes per cell served as a positive control. In each single lane, 5×106 Bruce4 EBNA1 cells carrying different recombinant EBVs as indicated were loaded. (C) Detection of cellular transcripts (actin, Oct4, Nanog) and viral transcripts (EBNA2, LMP1, LMP2) by RT-PCR amplification. Selected Bruce4 EBNA1 clones stably transduced with the indicated recombinant EBV constructs are shown.
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pone-0001996-g003: mini- and maxi-EBVs in Bruce4 EBNA1 mESCs.(A) GFP expression of the stably introduced maxi-EBV constructs p3053 and p3298 in Bruce4 EBNA1 cells. (B) Gardella gel hybridizations reveal the extrachromosomal status of the recombinant EBV genomes. Raji cells, which contain about 50 EBV genomes per cell served as a positive control. In each single lane, 5×106 Bruce4 EBNA1 cells carrying different recombinant EBVs as indicated were loaded. (C) Detection of cellular transcripts (actin, Oct4, Nanog) and viral transcripts (EBNA2, LMP1, LMP2) by RT-PCR amplification. Selected Bruce4 EBNA1 clones stably transduced with the indicated recombinant EBV constructs are shown.

Mentions: The recombinant maxi- and mini-EBV plasmids were separately introduced into the mESC clone Bruce4 EBNA1 C1 and selected with G418. Cells derived from several independent experiments were phenotypically and genotypically analyzed. GFP expression could be detected in mESCs electroporated with p3053 or p3298 (Fig. 3A) and the extrachromosomal status of the recombinant EBV genomes in all cell lines was confirmed by Gardella gel analysis (Fig. 3B). With this technique, intact large extrachromosomal EBV plasmids can be separated from genomic DNA and detected by Southern blot hybridization. Clearly, this technique does not exclude the possibility of integrated EBV DNA also present in the selected mESCs. In summary, the Bruce4 EBNA1 C1 mESC clone readily supported the maintenance of all different EBV plasmids, whereas the parental EBNA1-negative Bruce4 cells did not (data not shown).


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)

mini- and maxi-EBVs in Bruce4 EBNA1 mESCs.(A) GFP expression of the stably introduced maxi-EBV constructs p3053 and p3298 in Bruce4 EBNA1 cells. (B) Gardella gel hybridizations reveal the extrachromosomal status of the recombinant EBV genomes. Raji cells, which contain about 50 EBV genomes per cell served as a positive control. In each single lane, 5×106 Bruce4 EBNA1 cells carrying different recombinant EBVs as indicated were loaded. (C) Detection of cellular transcripts (actin, Oct4, Nanog) and viral transcripts (EBNA2, LMP1, LMP2) by RT-PCR amplification. Selected Bruce4 EBNA1 clones stably transduced with the indicated recombinant EBV constructs are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001996-g003: mini- and maxi-EBVs in Bruce4 EBNA1 mESCs.(A) GFP expression of the stably introduced maxi-EBV constructs p3053 and p3298 in Bruce4 EBNA1 cells. (B) Gardella gel hybridizations reveal the extrachromosomal status of the recombinant EBV genomes. Raji cells, which contain about 50 EBV genomes per cell served as a positive control. In each single lane, 5×106 Bruce4 EBNA1 cells carrying different recombinant EBVs as indicated were loaded. (C) Detection of cellular transcripts (actin, Oct4, Nanog) and viral transcripts (EBNA2, LMP1, LMP2) by RT-PCR amplification. Selected Bruce4 EBNA1 clones stably transduced with the indicated recombinant EBV constructs are shown.
Mentions: The recombinant maxi- and mini-EBV plasmids were separately introduced into the mESC clone Bruce4 EBNA1 C1 and selected with G418. Cells derived from several independent experiments were phenotypically and genotypically analyzed. GFP expression could be detected in mESCs electroporated with p3053 or p3298 (Fig. 3A) and the extrachromosomal status of the recombinant EBV genomes in all cell lines was confirmed by Gardella gel analysis (Fig. 3B). With this technique, intact large extrachromosomal EBV plasmids can be separated from genomic DNA and detected by Southern blot hybridization. Clearly, this technique does not exclude the possibility of integrated EBV DNA also present in the selected mESCs. In summary, the Bruce4 EBNA1 C1 mESC clone readily supported the maintenance of all different EBV plasmids, whereas the parental EBNA1-negative Bruce4 cells did not (data not shown).

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