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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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Construction of the BHRF1 miRNA viral mutant.Schematic representing part of the EBV wild type genome with a focus on the BHRF1 miRNA cluster (top panel; not to scale). The two promoters for latent EBNA transcripts (Cp, Wp) and the polyA site of the BHRF1 gene are indicated. Construction of the Δ123 mutant was performed in three sequential steps (lower panel). First, the seed region of miR-BHRF1-1 was mutated using chromosomal building. The seed region is indicated in red and introduced sequence changes are shown in blue. This also created a new AclI restriction enzyme site (underlined). Second, the mature miR-BHRF1-2 and miR-BHRF1-3 sequences were replaced by a kanamycin resistance cassette flanked by flip recombinase target (frt) sites. Third, transient expression of the flp recombinase led to the excision of the kanamycin cassette.
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ppat-1001294-g001: Construction of the BHRF1 miRNA viral mutant.Schematic representing part of the EBV wild type genome with a focus on the BHRF1 miRNA cluster (top panel; not to scale). The two promoters for latent EBNA transcripts (Cp, Wp) and the polyA site of the BHRF1 gene are indicated. Construction of the Δ123 mutant was performed in three sequential steps (lower panel). First, the seed region of miR-BHRF1-1 was mutated using chromosomal building. The seed region is indicated in red and introduced sequence changes are shown in blue. This also created a new AclI restriction enzyme site (underlined). Second, the mature miR-BHRF1-2 and miR-BHRF1-3 sequences were replaced by a kanamycin resistance cassette flanked by flip recombinase target (frt) sites. Third, transient expression of the flp recombinase led to the excision of the kanamycin cassette.

Mentions: The EBV genome encodes a large number of non-coding RNAs that include the Epstein-Barr encoded RNAs (EBERs), as well as 25 miRNAs and one small nucleolar RNA (snoRNA) [8]–[12]. MiRNAs bind to mRNAs that contain fully or partially complementary sequences and as a consequence usually impair their translation and reduce their stability [13]. The EBV miRNAs are distributed in two clusters, located in the BART region or within the BHRF1 gene locus [9], [10]. The latter cluster comprises three members, two of which are located in the BHRF1 3′ untranslated region and the third immediately 5′ to the BHRF1 lytic mRNA transcription start site [9], [10] (Fig. 1A). They are processed from introns located within ∼100-kb long RNA transcripts that initiate at the Cp or Wp promoters used by all EBNA genes [14]. Expression of the BHRF1 miRNAs is characteristic of EBV-transformed B cells that express all latent genes (latency III) [1], [10]. In contrast, Bcell tumours such as Burkitt's lymphomas or epithelial cell tumours that express a restricted number of latent proteins (latency I or II) do not express the BHRF1 miRNAs [10]. However, induction of virus replication in some Burkitt's lymphoma cells leads to re-expression of the miRNA BHRF1 cluster, suggesting that these miRNAs may serve functions not only during latency III but also upon induction of virus replication [15]. In an attempt to understand the function of the BHRF1 miRNA cluster in continuously growing lymphoblastoid B cell lines, we constructed viruses that lack these miRNAs and report here their phenotypic traits.


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)

Construction of the BHRF1 miRNA viral mutant.Schematic representing part of the EBV wild type genome with a focus on the BHRF1 miRNA cluster (top panel; not to scale). The two promoters for latent EBNA transcripts (Cp, Wp) and the polyA site of the BHRF1 gene are indicated. Construction of the Δ123 mutant was performed in three sequential steps (lower panel). First, the seed region of miR-BHRF1-1 was mutated using chromosomal building. The seed region is indicated in red and introduced sequence changes are shown in blue. This also created a new AclI restriction enzyme site (underlined). Second, the mature miR-BHRF1-2 and miR-BHRF1-3 sequences were replaced by a kanamycin resistance cassette flanked by flip recombinase target (frt) sites. Third, transient expression of the flp recombinase led to the excision of the kanamycin cassette.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001294-g001: Construction of the BHRF1 miRNA viral mutant.Schematic representing part of the EBV wild type genome with a focus on the BHRF1 miRNA cluster (top panel; not to scale). The two promoters for latent EBNA transcripts (Cp, Wp) and the polyA site of the BHRF1 gene are indicated. Construction of the Δ123 mutant was performed in three sequential steps (lower panel). First, the seed region of miR-BHRF1-1 was mutated using chromosomal building. The seed region is indicated in red and introduced sequence changes are shown in blue. This also created a new AclI restriction enzyme site (underlined). Second, the mature miR-BHRF1-2 and miR-BHRF1-3 sequences were replaced by a kanamycin resistance cassette flanked by flip recombinase target (frt) sites. Third, transient expression of the flp recombinase led to the excision of the kanamycin cassette.
Mentions: The EBV genome encodes a large number of non-coding RNAs that include the Epstein-Barr encoded RNAs (EBERs), as well as 25 miRNAs and one small nucleolar RNA (snoRNA) [8]–[12]. MiRNAs bind to mRNAs that contain fully or partially complementary sequences and as a consequence usually impair their translation and reduce their stability [13]. The EBV miRNAs are distributed in two clusters, located in the BART region or within the BHRF1 gene locus [9], [10]. The latter cluster comprises three members, two of which are located in the BHRF1 3′ untranslated region and the third immediately 5′ to the BHRF1 lytic mRNA transcription start site [9], [10] (Fig. 1A). They are processed from introns located within ∼100-kb long RNA transcripts that initiate at the Cp or Wp promoters used by all EBNA genes [14]. Expression of the BHRF1 miRNAs is characteristic of EBV-transformed B cells that express all latent genes (latency III) [1], [10]. In contrast, Bcell tumours such as Burkitt's lymphomas or epithelial cell tumours that express a restricted number of latent proteins (latency I or II) do not express the BHRF1 miRNAs [10]. However, induction of virus replication in some Burkitt's lymphoma cells leads to re-expression of the miRNA BHRF1 cluster, suggesting that these miRNAs may serve functions not only during latency III but also upon induction of virus replication [15]. In an attempt to understand the function of the BHRF1 miRNA cluster in continuously growing lymphoblastoid B cell lines, we constructed viruses that lack these miRNAs and report here their phenotypic traits.

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