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. 2011 Oct;85(19):9801-10.
doi: 10.1128/JVI.05100-11. Epub 2011 Jul 13.

The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocytes

Regina Feederle  1 , Janina Haar, Katharina Bernhardt, Sarah D Linnstaedt, Helmut Bannert, Helge Lips, Bryan R Cullen, Henri-Jacques Delecluse
Affiliations

The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocytes

Regina Feederle et al. J Virol. 2011 Oct.

Abstract

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP). Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.

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Figures

Fig. 1.
Fig. 1.
Schematic overview of the construction of the single and double BHRF1 miRNA viral mutants. The top panel shows a schematic representing the EBV wild-type genome. Shown are the latent EBV genes, the two promoters that initiate the latent EBNA transcripts (Cp and Wp), the lytic BHRF1 promoter, the BART regions, and the deletion in EBV strain B95.8. The EBV BHRF1 and BART microRNAs are presented as black bars. The middle panel shows an enlargement of the BHRF1 gene locus. We obtained the Δ1 mutant by mutating the miR-BHRF1-1 seed region (arrows) (bottom); this resulted in the introduction of an additional AclI restriction site that allowed the screening of the properly recombined mutants. MiR-BHRF1-2 and miR-BHRF1-3 were deleted by exchanging the mature miRNA sequences for a kanamycin resistance cassette flanked by flip recombinase target (Frt) sites. The excision of the kanamycin cassette by Flp recombinase led to the replacement of these miRNAs with an Frt site that includes a HindIII restriction site. The Δ13 mutant resulted from the sequential deletion of miR-BHRF1-1 and miR-BHRF1-3. ORF, open reading frame.
Fig. 2.
Fig. 2.
Restriction enzyme analysis of the BHRF1 miRNA single and double mutants. (A) Schematic overview of part of the EBV wt genome. Restriction enzyme recognition sites around BHRF1 and the size of the resulting restriction fragments are given. (B) For each recombinant virus, we analyzed the E. coli wt (lane 1), mutant (lane 2), and revertant (lane 3) BAC DNAs. Also included are the mutant (lane 4) and revertant (lane 5) EBV BACs isolated from the producer cell lines. The schematic inserted below each panel indicates the size of the predicted restriction fragments after successful mutagenesis, while the wild-type restriction profile is given in panel A. The resulting fragment changes observed after restriction analysis are indicated with arrows. H3, HindIII; M, molecular size marker (in kbp); frt, flip recombinase recognition site.
Fig. 3.
Fig. 3.
B cells transformed by the different single BHRF1 miRNA mutants display defective cell growth relative to that of their wild-type counterparts but evince normal BHRF1 expression levels. (A) Growth curve of EBV-infected B cells in the first weeks postinfection. Mean total cell numbers from three independent B-cell infection experiments are shown. Positive controls included LCLs generated with wild-type or revertant viruses. An LCL that carries Δ123 was also included in the panel as a reference. For clarity, the mean cell number measured at 29 days postinfection (dpi) is presented on the right at a higher resolution. (B) Cell transformation assays were carried out at a low B-cell concentration (102 cells/well) with an MOI of 0.1 infectious particles per B cell. The transformation rate is the ratio between the number of wells exhibiting B-cell outgrowth and the total number of seeded wells. Results are presented relative to the values obtained with the LCL generated with wt EBV. Averages of the results of three independent infection experiments are presented. (C) BHRF1 mRNA expression at day 30 postinfection in three unrelated primary B-cell samples infected with Δ1, Δ2, Δ3, their revertants, or wt EBV viruses. Results are presented relative to the values obtained with the LCL generated with wt EBV. The mean values from three independent analyses are given.
Fig. 4.
Fig. 4.
miR-BHRF1-2 is necessary for full miR-BHRF1-3 expression. We determined the BHRF1 miRNA expression profile from three LCL sets generated with Δ1, Δ2, Δ3, Δ13, Δ123, and their wild-type counterparts by stem-loop RT-qPCR using primers specific for miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3. Data were normalized to RNU48 expression levels and are expressed relative to the value observed for one wt LCL, which was set to 1. EBV-negative Elijah cells were used as a negative control.
Fig. 5.
Fig. 5.
B cells transformed by viruses expressing only miR-BHRF1-2 display defective cell growth relative to that of their wild-type counterparts. A B-cell transformation assay with Δ13 viruses was performed at high (A) or low (B) B-cell concentrations under the same conditions as those described in the legends of Fig. 3A and B. Controls included Δ123 and the wild-type counterparts.
Fig. 6.
Fig. 6.
Latent promoter usage and EBNA-LP synthesis in transformed B cells. EBNA-LP expression in B cells transformed with mutant and control viruses at day 11 (A) and day 36 (B) postinfection was determined by Western blot analysis (top panels). The intensity of the Western blot signals was measured by using ImageJ software. Results are given below each sample as a ratio of the intensity compared to that of the corresponding wt band, which was set as 1. RT-qPCR was performed to assess Cp- and Wp-initiated EBNA transcript levels at day 11 (A) or day 36 (B) postinfection (bottom panels). Analyses of the single mutant and corresponding revertant viruses are depicted in the left panel, and the double (Δ13) and triple (Δ123) mutants and the corresponding revertants are shown in the right panel. RT-qPCR results obtained at a given time point are given relative to those for wt EBV at the same time point and represent mean values obtained from three independent infection experiments.
Fig. 7.
Fig. 7.
miR-BHRF1-2 stimulates expression of miR-BHRF1-3 if both microRNAs are located on the same transcript. (A) The BHRF1 locus was cloned from a wt EBV BAC or from a Δ2 BAC onto an expression plasmid and was transfected into HEK293 cells to assess miR-BHRF1-2 and miR-BHRF1-3 expression by stem-loop RT-qPCR. Shown are means of data from three independent transfection experiments, and results are given relative to values obtained with one experiment with the wt. (B) Expression plasmids that carry the BHRF1 open reading frame (B) and miR-BHRF1-2 (B-2), miR-BHRF1-3 (B-3), or both miRNAs (B-2-3) were transfected into 293 cells alone or in combination (B2 + B3). The expression levels of both BHRF1 miRNAs are given as mean values from two independent transfection experiments relative to values obtained with one wt plasmid comprising the complete BHRF1 miRNA locus (1-B-2-3).
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