Figure 5

EBV gene expression and apoptosis sensitivity in EBV-loss clones of BL reinfected with Akata virus or ΔCpWp-B95.8 rEBVs. (a and b) Western blot analysis of EBV latent protein expression in EBV-loss BL clones of Akata (An1-5), Kem (Kn1-3) and Mutu (Mn1-3) reinfected with Akata virus (Ak-V) (panel a, left) or ΔCpWp-B95.8 (B-V) virus (panel b, right). Latency III LCLs express EBNA1, EBNA2 and LMP1, but Latency I reinfectants express only EBNA1 protein. EBV-loss cells that had not been reinfected express only the cellular loading control, calregulin (CALR). (c and d) Expression of miR-BARTs in EBV-loss clones reinfected with Akata virus (Ak-V) (panel c, left) or ΔCpWp-B95.8 virus (B-V) (panel d, right) quantified using stem-loop real-time PCR. Representative data for three miRNAs from BART cluster 1 (miR-BARTs 3, 15 and 5) and two from BART cluster 2 (miR-BARTs 22 and 7) are shown relative to levels in the EBV-positive, Latency I cell line, Kem-BL. Note: the ΔCpWp-B95.8 virus genome harbours a deletion spanning miR-BARTs 22 and 7 (denoted as Δ) as well as the 3′ end of the miR-BART-5 pre-miRNA. (e) Survival of EBV-loss clones reinfected with Akata virus (Ak-V, purple) after challenge with ionomycin for 48 h relative to untreated controls and compared with EBV-positive parental BL cells (wt, blue) and EBV-loss BL cells from the same clonal background (−ve, red). (f) Survival of EBV-loss BL clones reinfected with ΔCpWp-B95.8 virus (B-V, purple) after challenge with ionomycin for 48 h relative to untreated controls and compared with EBV-positive parental BL cells (wt, blue) and EBV-loss BL cells from the same clonal background (−ve, red). Data are presented as the mean and S.D. of three independent experiments, each carried out in triplicate. Statistical significance was determined using an unpaired, two-tailed Student’s T-test, **P<0.01, *P<0.05