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. 2011 Feb;7(2):e1001294.
doi: 10.1371/journal.ppat.1001294. Epub 2011 Feb 17.

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

Regina Feederle  1 , Sarah D Linnstaedt, Helmut Bannert, Helge Lips, Maja Bencun, Bryan R Cullen, Henri-Jacques Delecluse
Affiliations

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

Regina Feederle et al. PLoS Pathog. 2011 Feb.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. 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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