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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

RT-PCR analysis of the Cp and Wp promoters indicated incorrect splicing and premature termination of viral EBNA transcripts in mESCs.RT-PCR analyses of EBNA-specific transcripts with primer pairs, which selectively amplify Cp or Wp originating transcripts. The expected patterns of multiply spliced exon combinations are schematically depicted. (A) C1/W2 primer pairs amplify transcripts, which originate from Cp. (B) W0W1'B/W2 primer pairs detect transcripts from Wp. The agarose gels show the RT-PCR patterns of different EBV-transduced Bruce4 EBNA1 clones together with the positive controls derived from B95.8 cells. (C) Off-size PCR products were isolated from gels as shown in B and directly sequenced. Their composition is schematically shown.
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pone-0001996-g004: RT-PCR analysis of the Cp and Wp promoters indicated incorrect splicing and premature termination of viral EBNA transcripts in mESCs.RT-PCR analyses of EBNA-specific transcripts with primer pairs, which selectively amplify Cp or Wp originating transcripts. The expected patterns of multiply spliced exon combinations are schematically depicted. (A) C1/W2 primer pairs amplify transcripts, which originate from Cp. (B) W0W1'B/W2 primer pairs detect transcripts from Wp. The agarose gels show the RT-PCR patterns of different EBV-transduced Bruce4 EBNA1 clones together with the positive controls derived from B95.8 cells. (C) Off-size PCR products were isolated from gels as shown in B and directly sequenced. Their composition is schematically shown.

Mentions: EBNA2 and other members of the EBNA gene family (EBNA-LP, EBNA3A, -B, -C, and EBNA1) are expressed from multiply spliced transcripts, which originate from the Cp and/or Wp promoters (Fig. 2). By RT-PCR, we analyzed the activities of the Cp and Wp promoters with appropriate primer pairs (Fig. 4A, B and Table. S1). The positive control with cDNA derived from the B95.8 cell line gave the characteristic ladder of orderly spliced exon repeats (Fig. 4A, B). Cp- and Wp-specific cDNAs could be readily detected in EBV-transduced mESCs but the amplified cDNAs revealed only two of the expected PCR products (312 bp and 510 bp with Cp-specific primers; 110 bp and 308 bp with Wp-specific primers; Fig. 4A, B) in contrast to B95.8. Moreover, the PCR products indicative of longer, multiply spliced adducts were absent in EBV-positive mESCs while additional, smaller and prominent bands appeared which were not present in B95.8 cells (Fig. 4A, B). The unexpected bands were isolated from the gels and directly sequenced. The three aberrant bands amplified with the Wp-specific primer pair were 192, 390, and 588 bp in size and contained intron sequences as schematically shown in Fig. 4C; similar to the unexpected Cp-specific fragments (Fig. 4B and data not shown). It thus appears that alternative RNA splicing in mESCs resulted in retained introns in common EBNA transcripts. In addition, premature termination of aberrantly spliced mRNAs was evident. It is likely that both processes contributed to the failure of these cells to express EBNA2 and other members of the EBNA gene family, including EBNA1.


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)

RT-PCR analysis of the Cp and Wp promoters indicated incorrect splicing and premature termination of viral EBNA transcripts in mESCs.RT-PCR analyses of EBNA-specific transcripts with primer pairs, which selectively amplify Cp or Wp originating transcripts. The expected patterns of multiply spliced exon combinations are schematically depicted. (A) C1/W2 primer pairs amplify transcripts, which originate from Cp. (B) W0W1'B/W2 primer pairs detect transcripts from Wp. The agarose gels show the RT-PCR patterns of different EBV-transduced Bruce4 EBNA1 clones together with the positive controls derived from B95.8 cells. (C) Off-size PCR products were isolated from gels as shown in B and directly sequenced. Their composition is schematically shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001996-g004: RT-PCR analysis of the Cp and Wp promoters indicated incorrect splicing and premature termination of viral EBNA transcripts in mESCs.RT-PCR analyses of EBNA-specific transcripts with primer pairs, which selectively amplify Cp or Wp originating transcripts. The expected patterns of multiply spliced exon combinations are schematically depicted. (A) C1/W2 primer pairs amplify transcripts, which originate from Cp. (B) W0W1'B/W2 primer pairs detect transcripts from Wp. The agarose gels show the RT-PCR patterns of different EBV-transduced Bruce4 EBNA1 clones together with the positive controls derived from B95.8 cells. (C) Off-size PCR products were isolated from gels as shown in B and directly sequenced. Their composition is schematically shown.
Mentions: EBNA2 and other members of the EBNA gene family (EBNA-LP, EBNA3A, -B, -C, and EBNA1) are expressed from multiply spliced transcripts, which originate from the Cp and/or Wp promoters (Fig. 2). By RT-PCR, we analyzed the activities of the Cp and Wp promoters with appropriate primer pairs (Fig. 4A, B and Table. S1). The positive control with cDNA derived from the B95.8 cell line gave the characteristic ladder of orderly spliced exon repeats (Fig. 4A, B). Cp- and Wp-specific cDNAs could be readily detected in EBV-transduced mESCs but the amplified cDNAs revealed only two of the expected PCR products (312 bp and 510 bp with Cp-specific primers; 110 bp and 308 bp with Wp-specific primers; Fig. 4A, B) in contrast to B95.8. Moreover, the PCR products indicative of longer, multiply spliced adducts were absent in EBV-positive mESCs while additional, smaller and prominent bands appeared which were not present in B95.8 cells (Fig. 4A, B). The unexpected bands were isolated from the gels and directly sequenced. The three aberrant bands amplified with the Wp-specific primer pair were 192, 390, and 588 bp in size and contained intron sequences as schematically shown in Fig. 4C; similar to the unexpected Cp-specific fragments (Fig. 4B and data not shown). It thus appears that alternative RNA splicing in mESCs resulted in retained introns in common EBNA transcripts. In addition, premature termination of aberrantly spliced mRNAs was evident. It is likely that both processes contributed to the failure of these cells to express EBNA2 and other members of the EBNA gene family, including EBNA1.

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