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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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B cells transformed by Δ123 virus show defective cell growth relative to its wild type counterparts.(A) B cell transformation assays were carried out at low B cell concentration (102/well) with an MOI of 1 gru per B cell. An average of the results of three independent infection experiments is presented. (B) Growth curve of EBV-infected B cells in the first weeks post-infection. Mean values of three independent B cell infection experiments are shown. (C) Cell cycle analysis of BrdU-7AAD-stained LCLs generated with Δ123, Δ123 Rev, and EBV-wt viruses. The percentage of cells present in S or G2/M phase of the cell cycle is given. (D) BHRF1 miRNA expression profile. Shown are RT-PCR amplification products from one LCL set generated with Δ123, Δ123 Rev, or EBV-wt viruses using primers specific for miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3. Amplification of RNU48 served as an internal reference, and BJAB cells were used as a miRNA negative control. (E) Three unrelated B cell samples (a, b, and c) were infected with Δ123, Δ123 Rev, or EBV-wt viruses and BHRF1 miRNA expression was evaluated at day 11 p.i. Results are presented relative to the values obtained with one LCL (a) exposed to EBV-wt. The mean value of three independent analyses are shown.
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ppat-1001294-g003: B cells transformed by Δ123 virus show defective cell growth relative to its wild type counterparts.(A) B cell transformation assays were carried out at low B cell concentration (102/well) with an MOI of 1 gru per B cell. An average of the results of three independent infection experiments is presented. (B) Growth curve of EBV-infected B cells in the first weeks post-infection. Mean values of three independent B cell infection experiments are shown. (C) Cell cycle analysis of BrdU-7AAD-stained LCLs generated with Δ123, Δ123 Rev, and EBV-wt viruses. The percentage of cells present in S or G2/M phase of the cell cycle is given. (D) BHRF1 miRNA expression profile. Shown are RT-PCR amplification products from one LCL set generated with Δ123, Δ123 Rev, or EBV-wt viruses using primers specific for miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3. Amplification of RNU48 served as an internal reference, and BJAB cells were used as a miRNA negative control. (E) Three unrelated B cell samples (a, b, and c) were infected with Δ123, Δ123 Rev, or EBV-wt viruses and BHRF1 miRNA expression was evaluated at day 11 p.i. Results are presented relative to the values obtained with one LCL (a) exposed to EBV-wt. The mean value of three independent analyses are shown.

Mentions: To assess the contribution of the BHRF1 miRNA cluster to EBV's transforming properties, we exposed resting primary B cells to wild type, Δ123, and Δ123 Rev viruses. Infections were carried out at an MOI of 1 infectious particle per B cell (i.e. one gru/B cell), and cell outgrowth was monitored. Infected B cells were either seeded at low concentration, i.e. 2×103 per ml in a 96-well plate containing feeder cells or at high concentration i.e. 2×106 cells per ml. EBV-infected cells grow much more easily when infected at high concentration. Therefore, the first culture conditions are very stringent and allow detection of differences in terms of transformation efficiency but they do not allow monitoring of the infected B cells at the early stages of transformation. The percentage of wells containing outgrowing cell clones was assessed 8 weeks after infection. The results of three independent infection experiments is presented in Figure 3A. On average, wild type and revertant viruses respectively induced 82 and 75% cell outgrowth at an MOI of 1. In contrast, only 3% of the wells containing B cells infected with Δ123 virus showed outgrowth (Fig. 3A). Note that the standard variation between the different transformation assays was high. This reflects the fact that B cells from different individuals differ in their ability to form continuously growing cell lines. We conclude from these data that the BHRF1 miRNA cluster markedly increases the efficiency of transformation at low B cell concentration. The results of the bulk infection revealed similar though less pronounced effects. Monitoring of cell growth in B cells exposed to EBV-wt, Δ123, and Δ123 Rev virus evidenced slower growth in samples infected with Δ123. After 29 days in culture, B cells exposed to wild type or Δ123 Rev viruses expanded from 2×106 to 3.6×108 and 3.3×108 cells, respectively. This compares with 1.3×108 for B cells transformed by Δ123 (Fig. 3B). This prompted us to study the cell cycle parameters of B cell populations, by performing a BrdU incorporation assay (Fig. 3C). Whilst between 22 and 23% of B cells infected with both wild type controls entered S phase within 30 minutes, this percentage fell to 9.4% in B cells infected with Δ123 virus. We also noted that the ratio between cells still present in G2/M phase at the time of analysis and those that had entered S phase was ∼0.3 for the controls and ∼0.9 for B cells infected Δ123 virus. From this set of data, we conclude that the absence of BHRF1 miRNAs does not suppress EBV's transforming properties, but instead markedly slows down the growth rate of infected target cells. The permanently growing cell lines obtained after Δ123 infection provided material to quantify BHRF1 miRNA expression by RT-qPCR using the RNU48 snoRNA as an internal reference. The results of this analysis are given in Fig. 3D and show expression of the three BHRF1 miRNAs in all control lines tested, but not in those generated with Δ123, as expected. Of note, the expression level of the BHRF1 miRNAs varies within the control LCLs established from three different B cell infections (1 to 5 range for miR-BHRF1-1, 1 to 4 range for miR-BHRF1-2, 1 to 2 range for miR-BHRF1-3). This variation most likely reflects different activity levels of the Cp/Wp promoter among different LCLs [15].


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)

B cells transformed by Δ123 virus show defective cell growth relative to its wild type counterparts.(A) B cell transformation assays were carried out at low B cell concentration (102/well) with an MOI of 1 gru per B cell. An average of the results of three independent infection experiments is presented. (B) Growth curve of EBV-infected B cells in the first weeks post-infection. Mean values of three independent B cell infection experiments are shown. (C) Cell cycle analysis of BrdU-7AAD-stained LCLs generated with Δ123, Δ123 Rev, and EBV-wt viruses. The percentage of cells present in S or G2/M phase of the cell cycle is given. (D) BHRF1 miRNA expression profile. Shown are RT-PCR amplification products from one LCL set generated with Δ123, Δ123 Rev, or EBV-wt viruses using primers specific for miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3. Amplification of RNU48 served as an internal reference, and BJAB cells were used as a miRNA negative control. (E) Three unrelated B cell samples (a, b, and c) were infected with Δ123, Δ123 Rev, or EBV-wt viruses and BHRF1 miRNA expression was evaluated at day 11 p.i. Results are presented relative to the values obtained with one LCL (a) exposed to EBV-wt. The mean value of three independent analyses are shown.
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Related In: Results  -  Collection

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

ppat-1001294-g003: B cells transformed by Δ123 virus show defective cell growth relative to its wild type counterparts.(A) B cell transformation assays were carried out at low B cell concentration (102/well) with an MOI of 1 gru per B cell. An average of the results of three independent infection experiments is presented. (B) Growth curve of EBV-infected B cells in the first weeks post-infection. Mean values of three independent B cell infection experiments are shown. (C) Cell cycle analysis of BrdU-7AAD-stained LCLs generated with Δ123, Δ123 Rev, and EBV-wt viruses. The percentage of cells present in S or G2/M phase of the cell cycle is given. (D) BHRF1 miRNA expression profile. Shown are RT-PCR amplification products from one LCL set generated with Δ123, Δ123 Rev, or EBV-wt viruses using primers specific for miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3. Amplification of RNU48 served as an internal reference, and BJAB cells were used as a miRNA negative control. (E) Three unrelated B cell samples (a, b, and c) were infected with Δ123, Δ123 Rev, or EBV-wt viruses and BHRF1 miRNA expression was evaluated at day 11 p.i. Results are presented relative to the values obtained with one LCL (a) exposed to EBV-wt. The mean value of three independent analyses are shown.
Mentions: To assess the contribution of the BHRF1 miRNA cluster to EBV's transforming properties, we exposed resting primary B cells to wild type, Δ123, and Δ123 Rev viruses. Infections were carried out at an MOI of 1 infectious particle per B cell (i.e. one gru/B cell), and cell outgrowth was monitored. Infected B cells were either seeded at low concentration, i.e. 2×103 per ml in a 96-well plate containing feeder cells or at high concentration i.e. 2×106 cells per ml. EBV-infected cells grow much more easily when infected at high concentration. Therefore, the first culture conditions are very stringent and allow detection of differences in terms of transformation efficiency but they do not allow monitoring of the infected B cells at the early stages of transformation. The percentage of wells containing outgrowing cell clones was assessed 8 weeks after infection. The results of three independent infection experiments is presented in Figure 3A. On average, wild type and revertant viruses respectively induced 82 and 75% cell outgrowth at an MOI of 1. In contrast, only 3% of the wells containing B cells infected with Δ123 virus showed outgrowth (Fig. 3A). Note that the standard variation between the different transformation assays was high. This reflects the fact that B cells from different individuals differ in their ability to form continuously growing cell lines. We conclude from these data that the BHRF1 miRNA cluster markedly increases the efficiency of transformation at low B cell concentration. The results of the bulk infection revealed similar though less pronounced effects. Monitoring of cell growth in B cells exposed to EBV-wt, Δ123, and Δ123 Rev virus evidenced slower growth in samples infected with Δ123. After 29 days in culture, B cells exposed to wild type or Δ123 Rev viruses expanded from 2×106 to 3.6×108 and 3.3×108 cells, respectively. This compares with 1.3×108 for B cells transformed by Δ123 (Fig. 3B). This prompted us to study the cell cycle parameters of B cell populations, by performing a BrdU incorporation assay (Fig. 3C). Whilst between 22 and 23% of B cells infected with both wild type controls entered S phase within 30 minutes, this percentage fell to 9.4% in B cells infected with Δ123 virus. We also noted that the ratio between cells still present in G2/M phase at the time of analysis and those that had entered S phase was ∼0.3 for the controls and ∼0.9 for B cells infected Δ123 virus. From this set of data, we conclude that the absence of BHRF1 miRNAs does not suppress EBV's transforming properties, but instead markedly slows down the growth rate of infected target cells. The permanently growing cell lines obtained after Δ123 infection provided material to quantify BHRF1 miRNA expression by RT-qPCR using the RNU48 snoRNA as an internal reference. The results of this analysis are given in Fig. 3D and show expression of the three BHRF1 miRNAs in all control lines tested, but not in those generated with Δ123, as expected. Of note, the expression level of the BHRF1 miRNAs varies within the control LCLs established from three different B cell infections (1 to 5 range for miR-BHRF1-1, 1 to 4 range for miR-BHRF1-2, 1 to 2 range for miR-BHRF1-3). This variation most likely reflects different activity levels of the Cp/Wp promoter among different LCLs [15].

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