The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells
Fig 3
The miR-BART control expression of DICER and recruit BZLF1 mRNAs to the RISC.
(A) LCLs generated with B cells from independent donors exposed to M81 and M81/ΔAll were subjected to western blot analysis with an antibody specific for DICER. Actin was used as a loading control. The intensity of the observed signals was quantified with the ImageJ software. The results of this assay are shown in a graph of bars and are given relative to the expression in LCLs transformed by wild type M81. (B) We used stem loop qPCR to gauge the expression levels of cellular miRNAs in LCLs transformed by M81 or M81/ΔAll. (C and D) RISC immunoprecipitation in LCLs generated with M81 and M81/ΔAll. We measured the expression levels of the GADPH, HPRT, BZLF1, LMP1 and IPO7 mRNAs in untreated cells or after immunoprecipitation with an anti-Ago2 antibody or with a BrdU-specific antibody. The expression of HPRT, BZLF1, LMP1 and IPO7 mRNAs were first normalized to GADPH levels. Fig 3C shows the ratio of the values obtained after immunoprecipitation with the anti-Ago2 antibody with those obtained with a BrdU-specific antibody in three experiments and Fig 3D show the values of the same experiment normalized to the mRNA expression levels in untreated total mRNAs. (E) Luciferase activity assays were performed in triplicate for 2 sets of independent LCLs generated with M81 or M81/ΔAll. We cotransfected a plasmid that encodes the rat-CD2 protein together with luciferase expression plasmids. The latter included the unmodified luciferase vector (Luc2) or a luciferase expression plasmid fused with the 3’UTR of BZLF1 (Luc2-BZLF1 3UTR) or BALF5 (Luc2-BALF5 3’UTR). We performed luciferase assays 48 hours post-electroporation and the recorded values were normalized to the percentage of CD2-positive cells that had been determined by immunofluorescence staining. The graph shows the ratios of luciferase activity between M81 and M81/ΔAll LCLs for each transfected plasmid. Error bars indicate standard deviation for the values obtained in the three technical replicates. We show the results of statistical analyses performed by two-tailed student t-test (* indicates p<0.05 and ** indicates p<0.01).