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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 gene expression profiles of B cells transformed byΔ123 virus.(A) and (B) EBV latent gene transcription and translation in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus were examined by RT-qPCR (right panel) and western blot analysis (left panel) at day 11 (A) and day 36 (B) post-infection (dpi). Results from one representative experiment are presented. Intensity of western blot signals was measured using ImageJ software. Given below each western blot is the ratio between the intensity of the observed signal and the wt signal (wt set as 1). (C) BHRF1 gene transcription in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus as measured by RT-qPCR. Data are means from three independent infection experiments.
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ppat-1001294-g005: Latent gene expression profiles of B cells transformed byΔ123 virus.(A) and (B) EBV latent gene transcription and translation in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus were examined by RT-qPCR (right panel) and western blot analysis (left panel) at day 11 (A) and day 36 (B) post-infection (dpi). Results from one representative experiment are presented. Intensity of western blot signals was measured using ImageJ software. Given below each western blot is the ratio between the intensity of the observed signal and the wt signal (wt set as 1). (C) BHRF1 gene transcription in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus as measured by RT-qPCR. Data are means from three independent infection experiments.

Mentions: We then used RT-qPCR and western blots to gauge EBV latent gene expression. The results of one out of four experiments are shown in Figure 5. This set of experiments demonstrated that B cells generated with the Δ123 virus express all tested latent genes with some expressed at higher levels than in the controls at day 11 post-infection (Fig. 5A). Similar assays conducted at day 36 post-infection confirmed this trend; all latent genes were expressed at higher levels in Δ123-positive LCLs both at the mRNA and the protein level (Fig. 5B). This difference was particularly dramatic in the case of the EBNA-LP protein, which was five times more abundant in LCLs generated with Δ123 than in controls, but was also visible for EBNA1, EBNA2, EBNA3A, EBNA3B, and EBNA3C. LMP1 and LMP2-specific transcripts were also more abundant in LCLs infected by the triple mutant. Western blot analysis detected a marginal increase in LMP1 protein production in the same cells. Recent recognition that the BHRF1 protein is important for B cell transformation prompted us to assess expression of this gene [18]. Lymphoblastoid cell lines infected with wt, Δ123, or Δ123 Rev viruses evinced similar levels of BHRF1 transcripts (Fig. 5C).


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 gene expression profiles of B cells transformed byΔ123 virus.(A) and (B) EBV latent gene transcription and translation in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus were examined by RT-qPCR (right panel) and western blot analysis (left panel) at day 11 (A) and day 36 (B) post-infection (dpi). Results from one representative experiment are presented. Intensity of western blot signals was measured using ImageJ software. Given below each western blot is the ratio between the intensity of the observed signal and the wt signal (wt set as 1). (C) BHRF1 gene transcription in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus as measured by RT-qPCR. Data are means from three independent infection experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001294-g005: Latent gene expression profiles of B cells transformed byΔ123 virus.(A) and (B) EBV latent gene transcription and translation in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus were examined by RT-qPCR (right panel) and western blot analysis (left panel) at day 11 (A) and day 36 (B) post-infection (dpi). Results from one representative experiment are presented. Intensity of western blot signals was measured using ImageJ software. Given below each western blot is the ratio between the intensity of the observed signal and the wt signal (wt set as 1). (C) BHRF1 gene transcription in LCLs transformed by Δ123, Δ123 Rev, or EBV-wt virus as measured by RT-qPCR. Data are means from three independent infection experiments.
Mentions: We then used RT-qPCR and western blots to gauge EBV latent gene expression. The results of one out of four experiments are shown in Figure 5. This set of experiments demonstrated that B cells generated with the Δ123 virus express all tested latent genes with some expressed at higher levels than in the controls at day 11 post-infection (Fig. 5A). Similar assays conducted at day 36 post-infection confirmed this trend; all latent genes were expressed at higher levels in Δ123-positive LCLs both at the mRNA and the protein level (Fig. 5B). This difference was particularly dramatic in the case of the EBNA-LP protein, which was five times more abundant in LCLs generated with Δ123 than in controls, but was also visible for EBNA1, EBNA2, EBNA3A, EBNA3B, and EBNA3C. LMP1 and LMP2-specific transcripts were also more abundant in LCLs infected by the triple mutant. Western blot analysis detected a marginal increase in LMP1 protein production in the same cells. Recent recognition that the BHRF1 protein is important for B cell transformation prompted us to assess expression of this gene [18]. Lymphoblastoid cell lines infected with wt, Δ123, or Δ123 Rev viruses evinced similar levels of BHRF1 transcripts (Fig. 5C).

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