Fig 10.

Effects of silencing ORF50 on KSHV replication. (A) ORF50 silencing in BCBL-1 cells treated with TPA and DCPE to induce KSHV replication. BCBL-1 cells were transfected with an ORF50 siRNA and control GAPDH siRNA and then treated with TPA or DCPE to induce KSHV replication. RNAs were assayed for ORF50 RNA at 24 h via a TaqMan real-time RT-PCR assay (results normalized to actin control and a nontargeting siRNA pool). Transfection of ORF50 siRNA resulted in a decrease in ORF50 RNA expression in both cells treated with TPA (86% decrease) and DCPE (93% decrease). (B) ORF50 protein levels in normal BCBL-1 cells after induction by TPA and DCPE and ORF50 silencing. ORF50 siRNA knocks down expression of ORF50 when cells are treated with both TPA and DCPE. (Note that the DCPE lanes were exposed longer, because TPA is a potent inducer of ORF50 expression, while DCPE is not). (C) Effects on KSHV replication of silencing ORF50 in BCBL-1 cells induced with TPA or DCPE. Cells were transfected with siRNA and induced with either TPA or DCPE and assayed for KSHV virions using a real-time PCR assay for protected KSHV DNA after 48 h. Silencing ORF50 led to a statistically significant decrease in virus production when cells were induced with TPA (P = 0.004 [indicated by an asterisk in the figure]), but no statistically significant decrease when ORF50 was silenced in DCPE-treated cells.