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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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Characterization of viral recombinants.(A) EBV-wt, Δ123, and Δ123 Rev restriction enzyme analysis. A schematic map of the EBV-wt genome segment encompassing the BHRF1 region with cleavage sites for AclI and HindIII and the expected fragment sizes are shown on the top panel. The recombinant viral genomes isolated either from E.coli cells or rescued from stably transfected HEK293 cells were cleaved with AclI (left) or HindIII (right). While the exchange of miR-BHRF1-2 and miR-BHRF1-3 against a frt-site introduced a HindIII restriction site, the mutation of miR-BHRF1-1 introduced an AclI restriction site. The fragment size predictions for each enzyme are given and the resulting fragment changes observed after restriction analysis are indicated with arrows (lower panel). H3: HindIII; M: molecular weight marker; frt: flp recombinase target site. (B) Viral genome DNA equivalents (geq) per ml of supernatant were quantified by qPCR amplification of the viral BALF5 gene. Concentration of infectious particles per ml of supernatant were calculated by counting gfp-positive Raji units (gru). Mean values of 3 different supernatants are presented.
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ppat-1001294-g002: Characterization of viral recombinants.(A) EBV-wt, Δ123, and Δ123 Rev restriction enzyme analysis. A schematic map of the EBV-wt genome segment encompassing the BHRF1 region with cleavage sites for AclI and HindIII and the expected fragment sizes are shown on the top panel. The recombinant viral genomes isolated either from E.coli cells or rescued from stably transfected HEK293 cells were cleaved with AclI (left) or HindIII (right). While the exchange of miR-BHRF1-2 and miR-BHRF1-3 against a frt-site introduced a HindIII restriction site, the mutation of miR-BHRF1-1 introduced an AclI restriction site. The fragment size predictions for each enzyme are given and the resulting fragment changes observed after restriction analysis are indicated with arrows (lower panel). H3: HindIII; M: molecular weight marker; frt: flp recombinase target site. (B) Viral genome DNA equivalents (geq) per ml of supernatant were quantified by qPCR amplification of the viral BALF5 gene. Concentration of infectious particles per ml of supernatant were calculated by counting gfp-positive Raji units (gru). Mean values of 3 different supernatants are presented.

Mentions: We deleted the BHRF1 miRNA cluster from the B95.8 genome cloned in E.coli in a sequential manner (Fig. 1). We first exchanged the miR-BHRF1-1 seed region for an unrelated sequence by chromosomal building using a shuttle plasmid that carries an ampicillin resistance cassette [16]. The modified miR-BHRF1-1 seed region introduces an AclI restriction site that allows unequivocal identification of the properly recombined mutant. In a second step, we replaced the DNA region that spans the miR-BHRF1-2 and miR-BHRF1-3 mature miRNAs by a kanamycin resistance cassette flanked by Flp recombinase target sites using recA-mediated homologous recombination. The last step consisted in excising the kanamycin resistance cassette by transient expression of the Flp recombinase. As a result, the miR-BHRF1-2 and miR-BHRF1-3 miRNAs were exchanged against one Flp recombinase target site (Fig. 1 and Fig. S1). The modified viral DNA, which carries a hygromycin resistance cassette, hereinafter referred to as Δ123, was then transfected into 293 cells. Clones from these stably transfected 293 cells (293/Δ123) were obtained by hygromycin selection and the viral mutant genomes present in these producing cell lines were transferred back into E.coli and their global integrity was confirmed by restriction enzyme analysis (Fig. 2A). Furthermore, sequencing the DNA fragments that were modified during virus construction confirmed the exactitude of the introduced alterations (Fig. S2) and the complete identity of sequences outside the miRNAs with wild type genome. Next, the producer cell clones were tested for their ability to sustain viral lytic replication. The viral structural titers were detected by quantitative PCR and found to be similar to those observed with wild type producer cell lines. The mean values ranged between 2.2×107 and 2.9×107 genome equivalents per ml of supernatant for Δ123 and wt, respectively, showing that the BHRF1 miRNAs are not required for virus production (Fig. 2B). We then incubated Raji B cells with these supernatants at various dilutions. Three days later the number of gfp-positive Raji cells was determined to assess functional infectious titers (Fig. 2B). The ratio between structural titers (geq/ml) and functional titers (gru/ml) was found to be 7.8 and 10.3 for wt and Δ123 viruses, respectively. We therefore conclude, that the BHRF1 miRNAs are not essential for EBV infection but we cannot rule out a minor contribution to this process.


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)

Characterization of viral recombinants.(A) EBV-wt, Δ123, and Δ123 Rev restriction enzyme analysis. A schematic map of the EBV-wt genome segment encompassing the BHRF1 region with cleavage sites for AclI and HindIII and the expected fragment sizes are shown on the top panel. The recombinant viral genomes isolated either from E.coli cells or rescued from stably transfected HEK293 cells were cleaved with AclI (left) or HindIII (right). While the exchange of miR-BHRF1-2 and miR-BHRF1-3 against a frt-site introduced a HindIII restriction site, the mutation of miR-BHRF1-1 introduced an AclI restriction site. The fragment size predictions for each enzyme are given and the resulting fragment changes observed after restriction analysis are indicated with arrows (lower panel). H3: HindIII; M: molecular weight marker; frt: flp recombinase target site. (B) Viral genome DNA equivalents (geq) per ml of supernatant were quantified by qPCR amplification of the viral BALF5 gene. Concentration of infectious particles per ml of supernatant were calculated by counting gfp-positive Raji units (gru). Mean values of 3 different supernatants are presented.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001294-g002: Characterization of viral recombinants.(A) EBV-wt, Δ123, and Δ123 Rev restriction enzyme analysis. A schematic map of the EBV-wt genome segment encompassing the BHRF1 region with cleavage sites for AclI and HindIII and the expected fragment sizes are shown on the top panel. The recombinant viral genomes isolated either from E.coli cells or rescued from stably transfected HEK293 cells were cleaved with AclI (left) or HindIII (right). While the exchange of miR-BHRF1-2 and miR-BHRF1-3 against a frt-site introduced a HindIII restriction site, the mutation of miR-BHRF1-1 introduced an AclI restriction site. The fragment size predictions for each enzyme are given and the resulting fragment changes observed after restriction analysis are indicated with arrows (lower panel). H3: HindIII; M: molecular weight marker; frt: flp recombinase target site. (B) Viral genome DNA equivalents (geq) per ml of supernatant were quantified by qPCR amplification of the viral BALF5 gene. Concentration of infectious particles per ml of supernatant were calculated by counting gfp-positive Raji units (gru). Mean values of 3 different supernatants are presented.
Mentions: We deleted the BHRF1 miRNA cluster from the B95.8 genome cloned in E.coli in a sequential manner (Fig. 1). We first exchanged the miR-BHRF1-1 seed region for an unrelated sequence by chromosomal building using a shuttle plasmid that carries an ampicillin resistance cassette [16]. The modified miR-BHRF1-1 seed region introduces an AclI restriction site that allows unequivocal identification of the properly recombined mutant. In a second step, we replaced the DNA region that spans the miR-BHRF1-2 and miR-BHRF1-3 mature miRNAs by a kanamycin resistance cassette flanked by Flp recombinase target sites using recA-mediated homologous recombination. The last step consisted in excising the kanamycin resistance cassette by transient expression of the Flp recombinase. As a result, the miR-BHRF1-2 and miR-BHRF1-3 miRNAs were exchanged against one Flp recombinase target site (Fig. 1 and Fig. S1). The modified viral DNA, which carries a hygromycin resistance cassette, hereinafter referred to as Δ123, was then transfected into 293 cells. Clones from these stably transfected 293 cells (293/Δ123) were obtained by hygromycin selection and the viral mutant genomes present in these producing cell lines were transferred back into E.coli and their global integrity was confirmed by restriction enzyme analysis (Fig. 2A). Furthermore, sequencing the DNA fragments that were modified during virus construction confirmed the exactitude of the introduced alterations (Fig. S2) and the complete identity of sequences outside the miRNAs with wild type genome. Next, the producer cell clones were tested for their ability to sustain viral lytic replication. The viral structural titers were detected by quantitative PCR and found to be similar to those observed with wild type producer cell lines. The mean values ranged between 2.2×107 and 2.9×107 genome equivalents per ml of supernatant for Δ123 and wt, respectively, showing that the BHRF1 miRNAs are not required for virus production (Fig. 2B). We then incubated Raji B cells with these supernatants at various dilutions. Three days later the number of gfp-positive Raji cells was determined to assess functional infectious titers (Fig. 2B). The ratio between structural titers (geq/ml) and functional titers (gru/ml) was found to be 7.8 and 10.3 for wt and Δ123 viruses, respectively. We therefore conclude, that the BHRF1 miRNAs are not essential for EBV infection but we cannot rule out a minor contribution to this process.

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