Silent mutations in BGLF4 override the effect of siG4 on late gene expression.

(A) Western blot analysis of 2089 cell lysates transfected with the indicated expression vectors. Two concentrations of siRNA specific to BGLF4 (siG4), 10 and 20 nM, were co-transfected with expression vectors encoding ZEBRA and wild-type BGLF4 or kinase dead BGLF4(K102I) (lanes 4, 5, 10 and 11). To demonstrate the specificity of siG4, silent mutations were introduced into the kinase active (rG4) and kinase dead (rG4(K102I)) bglf4 genes that rendered the BGLF4 transcript resistant to siG4. Lysates of cells harvested 48 h after transfection were analyzed by immunoblotting using antibodies specific to BGLF4, ZEBRA and the late BFRF3 protein (FR3). (B) Quantitative PCR to measure the extent of EBV genome amplification in each condition.