The deletion of BART miRNAs enhances BZLF1 expression in infected B cells.

(A) We performed immunoblot analyses on LCLs transformed with M81, M81/ΔAll or its revertant with antibodies specific for BZLF1 and actin. The upper picture shows the results of this assay for three different donors. The relative intensity of the signals were quantified using the ImageJ software and are given as a graph of bars. (B) A LCL transformed by M81/ΔAll was stably transfected with a plasmid that encodes a truncated form of NGFR and both BART-miRNA clusters (C1C2) or with a plasmid that encodes NGFR only (empty). After 30 days, the NGFR-positive cells were purified with a specific antibody. We determined the BART-miRNA expression in these cells relative to M81 LCL (left panel) and their BZLF1 protein expression (right panel). (C) This figure depicts the BZLF1 expression pattern in LCLs transformed by M81 or M81/ΔAll as determined by immunofluorescence staining. One staining example is shown in the top pictures, whilst the percentage of BZLF1-positive cells in LCLs from multiple B-cell donors at different days post infection (dpi) is given in the scatter plot underneath. The p values obtained from paired t tests performed with LCLs infected by the two types of virus are given. Both pictures were taken at the same magnification and replicating cells appear larger, presumably as the result of cytopathic effects induced by the lytic replication. (D) This western blot analysis was performed with protein lysates from LCLs generated with M81 or M81/ΔAll that contain the same number of BZLF1-positive cells and stained with a BZLF1-specific and an actin-specific antibody. (E) We used qPCR with a BZLF1-specfic Taqman probe to determine the BZLF1 mRNA levels in LCLs transformed with M81 and M81/ΔAll. The graph of bars shows the ratio between the expression levels in the 2 types of LCLs at different time points post-infection. (F) This immunoblot shows the variation of BZLF1 protein expression in B cells infected with wild type virus or with the BART miRNA knockout mutant over a period of 76 days. Actin staining was used as a loading control. An LCL generated with the same B cells and a virus depleted with lytic transactivators BZLF1 and BRLF1 (M81ΔZR) served as a negative control. Please also see S1, S2 and S3 Figs.