FIG. 1.

The effect of CDV on KSHV RNA synthesis, cell viability, and virion production. (A) Real-time RT-PCR assay for KSHV ORF 17 RNA. RNA was harvested at 72 h after the induction of lytic replication from BCBL-1 cells pretreated with 0, 10, 25, 50, 75, and 100 μM CDV. The TPA-induced and CDV-treated ORF 17 cycle threshold (Ct) values were normalized against endogenous GAPDH Ct (dCt) values, and the Ct value for the latent background non-CDV-treated samples was subtracted to determine the net expression (ddCt). Data values were calculated as the amplification efficiency of the ORF 17 probe, one doubling per cycle, raised to the power of −ddCT (2^−ddCt) to determine the change (n-fold) in expression. Data values from three independent experiments are plotted as the average percentages for the maximal TPA-induced, non-CDV-treated (0 μM) conditions set at 100% expression. (B) BCBL-1 cells with 100 μM CDV pretreatment (indicated with a white X on a black circle and a stippled line) and non-CDV-treated cells (black diamond with solid line) were induced with 20 ng of TPA/ml and grown in parallel with untreated cells (open triangle with dashed lines). Samples were collected at serial time points, and viability was determined with trypan blue staining. Data shown are the averages of three separate experiments. The presence of CDV appeared to have no effect on BCBL-1 cell viability. (C) Real-time PCR assay for virion DNA. Supernatant was collected at the indicated time points from untreated (open bar), TPA-induced (black bar), and TPA-induced and 100 μM CDV-treated (gray-shaded bar) cell culture samples and assayed for KSHV DNA after an initial DNase treatment, followed by SDS-proteinase K treatment and DNA extraction. CDV suppresses late KSHV replication in a dose-dependent manner and inhibits free virion DNA to near-baseline levels.