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A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus.

Feederle R, Linnstaedt SD, Bannert H, Lips H, Bencun M, Cullen BR, Delecluse HJ - PLoS Pathog. (2011)

Bottom Line: EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas.Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels.Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Virus Associated Tumours, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

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Latent promoter usage and clonality studies of transformed B cells.(A) Wp and Cp-initiated transcripts were monitored by RT-qPCR in B cells transformed with Δ123, Δ123 Rev, or EBV-wt virus at multiple time points post-infection. Results obtained at a given time point are grouped and presented relative to EBV-wt at the same time point. Results represent mean values obtained from three independent analyses. (B) The same data as in (A) presented relative to the value observed in B cells infected by EBV-wt five days after infection. This illustrates Wp and Cp activities over time in B cells infected by Δ123 mutant or wild type counterparts. (C) Clonality of LCLs generated with Δ123, Δ123 Rev, or EBV-wt virus was determined by a PCR-based assay using primers specific to the IgVH sequences. Cell populations were analyzed at different time points post-infection. Akata is a monoclonal B cell line; non-infected primary B cells (day 0) are polyclonal in nature. NTC: no template control.
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ppat-1001294-g004: Latent promoter usage and clonality studies of transformed B cells.(A) Wp and Cp-initiated transcripts were monitored by RT-qPCR in B cells transformed with Δ123, Δ123 Rev, or EBV-wt virus at multiple time points post-infection. Results obtained at a given time point are grouped and presented relative to EBV-wt at the same time point. Results represent mean values obtained from three independent analyses. (B) The same data as in (A) presented relative to the value observed in B cells infected by EBV-wt five days after infection. This illustrates Wp and Cp activities over time in B cells infected by Δ123 mutant or wild type counterparts. (C) Clonality of LCLs generated with Δ123, Δ123 Rev, or EBV-wt virus was determined by a PCR-based assay using primers specific to the IgVH sequences. Cell populations were analyzed at different time points post-infection. Akata is a monoclonal B cell line; non-infected primary B cells (day 0) are polyclonal in nature. NTC: no template control.

Mentions: The latent viral proteins have been recognized as the principal mediators of EBV's transforming properties. It was therefore important to assess latent gene expression in B cells infected with the EBV Δ123 mutant. To this aim, we carried out RT-qPCR analysis of the viral latent transcripts produced from the Wp and Cp promoters in transformed Bcell lines at 5, 11, 25, 36, and 73 days after infection (Fig. 4A). At day 5, the level of transcription from the Cp and Wp promoters was very similar in cells infected with Δ123 and in wild type controls. However, from day 11 on, the activity of the Wp promoter, and of the Cp promoter gradually increased in B cells transformed with the triple mutant relative to their control counterparts (Fig. 4A). Rather than an absolute increase of Wp transcriptional activity in Δ123-positive B cell lines, the observed differences could be ascribed to a stronger down-regulation of Wp in the controls (Fig. 4B).


A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus.

Feederle R, Linnstaedt SD, Bannert H, Lips H, Bencun M, Cullen BR, Delecluse HJ - PLoS Pathog. (2011)

Latent promoter usage and clonality studies of transformed B cells.(A) Wp and Cp-initiated transcripts were monitored by RT-qPCR in B cells transformed with Δ123, Δ123 Rev, or EBV-wt virus at multiple time points post-infection. Results obtained at a given time point are grouped and presented relative to EBV-wt at the same time point. Results represent mean values obtained from three independent analyses. (B) The same data as in (A) presented relative to the value observed in B cells infected by EBV-wt five days after infection. This illustrates Wp and Cp activities over time in B cells infected by Δ123 mutant or wild type counterparts. (C) Clonality of LCLs generated with Δ123, Δ123 Rev, or EBV-wt virus was determined by a PCR-based assay using primers specific to the IgVH sequences. Cell populations were analyzed at different time points post-infection. Akata is a monoclonal B cell line; non-infected primary B cells (day 0) are polyclonal in nature. NTC: no template control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040666&req=5

ppat-1001294-g004: Latent promoter usage and clonality studies of transformed B cells.(A) Wp and Cp-initiated transcripts were monitored by RT-qPCR in B cells transformed with Δ123, Δ123 Rev, or EBV-wt virus at multiple time points post-infection. Results obtained at a given time point are grouped and presented relative to EBV-wt at the same time point. Results represent mean values obtained from three independent analyses. (B) The same data as in (A) presented relative to the value observed in B cells infected by EBV-wt five days after infection. This illustrates Wp and Cp activities over time in B cells infected by Δ123 mutant or wild type counterparts. (C) Clonality of LCLs generated with Δ123, Δ123 Rev, or EBV-wt virus was determined by a PCR-based assay using primers specific to the IgVH sequences. Cell populations were analyzed at different time points post-infection. Akata is a monoclonal B cell line; non-infected primary B cells (day 0) are polyclonal in nature. NTC: no template control.
Mentions: The latent viral proteins have been recognized as the principal mediators of EBV's transforming properties. It was therefore important to assess latent gene expression in B cells infected with the EBV Δ123 mutant. To this aim, we carried out RT-qPCR analysis of the viral latent transcripts produced from the Wp and Cp promoters in transformed Bcell lines at 5, 11, 25, 36, and 73 days after infection (Fig. 4A). At day 5, the level of transcription from the Cp and Wp promoters was very similar in cells infected with Δ123 and in wild type controls. However, from day 11 on, the activity of the Wp promoter, and of the Cp promoter gradually increased in B cells transformed with the triple mutant relative to their control counterparts (Fig. 4A). Rather than an absolute increase of Wp transcriptional activity in Δ123-positive B cell lines, the observed differences could be ascribed to a stronger down-regulation of Wp in the controls (Fig. 4B).

Bottom Line: EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas.Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels.Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Virus Associated Tumours, German Cancer Research Center, Heidelberg, Germany.

ABSTRACT
Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

Show MeSH
Related in: MedlinePlus