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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

Recombinant EBV genomes.The maxi-EBV construct p3053 encompasses the prototypic EBV genome of the B95.8 strain [16], while the mini-EBV constructs p3314 lacks most lytic genes but encodes all latent viral genes [17]. Both plasmids can be selectively propagated in bacteria (F-factor replicon) under chloramphenicol selection (camr in p3314, not marked in p3053) and in eukaryotic cells under G418 selection (neor). p3053 also encodes gfp. Only latent viral genes are depicted, blue stands for EBNA genes and orange for LMP genes. Selected promoters are shown with arrowheads. ‘W’ stands for BamHI-W-repeats; oriP stands for the plasmid origin of replication, oriLyt is the active replication origin during virus synthesis. Terminal repeats (TR) are indispensable for encapsidation of virion DNA.
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pone-0001996-g002: Recombinant EBV genomes.The maxi-EBV construct p3053 encompasses the prototypic EBV genome of the B95.8 strain [16], while the mini-EBV constructs p3314 lacks most lytic genes but encodes all latent viral genes [17]. Both plasmids can be selectively propagated in bacteria (F-factor replicon) under chloramphenicol selection (camr in p3314, not marked in p3053) and in eukaryotic cells under G418 selection (neor). p3053 also encodes gfp. Only latent viral genes are depicted, blue stands for EBNA genes and orange for LMP genes. Selected promoters are shown with arrowheads. ‘W’ stands for BamHI-W-repeats; oriP stands for the plasmid origin of replication, oriLyt is the active replication origin during virus synthesis. Terminal repeats (TR) are indispensable for encapsidation of virion DNA.

Mentions: One EBNA1+ mESC clone, Bruce4 EBNA1 C1 (Fig. 1B, C), was electroporated with three different recombinant genomic EBV plasmids. Plasmid p3053 contains the complete EBV genome of the prototypic EBV strain B95.8, which was cloned onto a mini-F-plasmid in E.coli. Like its parental version p2089 [16], p3053 encodes gfp but in addition carries the G418 resistance gene neo located in the prokaryotic backbone (Fig 2A). This maxi-EBV plasmid is fully competent to give rise to progeny virus when propagated in appropriate cells or can directly mediate growth transformation of human B cells upon DNA transfection [16], [17]. p3298 is a variant of p3053 that is incapable of expressing EBV's latent genes other than LMP2B and EBERs and was used as a negative control (not shown). The mini-EBV plasmid p3314 is approximately half the size of a wild-type EBV genome, lacks the majority of EBV's lytic genes required for de novo virus synthesis (Fig. 2B) but carries all known latent EBV genes. This recombinant EBV genome is capable of transforming primary human B cells as efficiently as wild-type EBV [17], [18].


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)

Recombinant EBV genomes.The maxi-EBV construct p3053 encompasses the prototypic EBV genome of the B95.8 strain [16], while the mini-EBV constructs p3314 lacks most lytic genes but encodes all latent viral genes [17]. Both plasmids can be selectively propagated in bacteria (F-factor replicon) under chloramphenicol selection (camr in p3314, not marked in p3053) and in eukaryotic cells under G418 selection (neor). p3053 also encodes gfp. Only latent viral genes are depicted, blue stands for EBNA genes and orange for LMP genes. Selected promoters are shown with arrowheads. ‘W’ stands for BamHI-W-repeats; oriP stands for the plasmid origin of replication, oriLyt is the active replication origin during virus synthesis. Terminal repeats (TR) are indispensable for encapsidation of virion DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001996-g002: Recombinant EBV genomes.The maxi-EBV construct p3053 encompasses the prototypic EBV genome of the B95.8 strain [16], while the mini-EBV constructs p3314 lacks most lytic genes but encodes all latent viral genes [17]. Both plasmids can be selectively propagated in bacteria (F-factor replicon) under chloramphenicol selection (camr in p3314, not marked in p3053) and in eukaryotic cells under G418 selection (neor). p3053 also encodes gfp. Only latent viral genes are depicted, blue stands for EBNA genes and orange for LMP genes. Selected promoters are shown with arrowheads. ‘W’ stands for BamHI-W-repeats; oriP stands for the plasmid origin of replication, oriLyt is the active replication origin during virus synthesis. Terminal repeats (TR) are indispensable for encapsidation of virion DNA.
Mentions: One EBNA1+ mESC clone, Bruce4 EBNA1 C1 (Fig. 1B, C), was electroporated with three different recombinant genomic EBV plasmids. Plasmid p3053 contains the complete EBV genome of the prototypic EBV strain B95.8, which was cloned onto a mini-F-plasmid in E.coli. Like its parental version p2089 [16], p3053 encodes gfp but in addition carries the G418 resistance gene neo located in the prokaryotic backbone (Fig 2A). This maxi-EBV plasmid is fully competent to give rise to progeny virus when propagated in appropriate cells or can directly mediate growth transformation of human B cells upon DNA transfection [16], [17]. p3298 is a variant of p3053 that is incapable of expressing EBV's latent genes other than LMP2B and EBERs and was used as a negative control (not shown). The mini-EBV plasmid p3314 is approximately half the size of a wild-type EBV genome, lacks the majority of EBV's lytic genes required for de novo virus synthesis (Fig. 2B) but carries all known latent EBV genes. This recombinant EBV genome is capable of transforming primary human B cells as efficiently as wild-type EBV [17], [18].

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