Calcineurin blockers inhibit Ca⁺⁺-dependent induction of KSHV production.

BCBL-1 cells were incubated with ionomycin (1 μg/mL) or TPA (15 nM) in the presence of calcineurin or PKC inhibitors. (A) On day 3, KSHV production was determined by enumerating virion-positive cells by TEM analyses. Results of 3 individual experiments are shown (x-axis: 1-3). Top panel: TPA (▪), TPA + SS (■), TPA + BIM (▧). Note that the result of TPA + BIM in experiment 1 and TPA + SS in experiment 3 is 0%. Bottom panel: ionomycin (▪), ionomycin + CsA (■), ionomycin + FK506 (▧). Without inducers, virion-positive cells were rarely observed (less than 0.5%). (B) On day 3 or 4, KSHV production was determined by measuring virus particle accumulation in cell culture supernatants by Western blot analyses. Cell culture supernatants were collected, and viral particles were pelleted by ultracentrifugation. Virus pellets were solubilized in equal volumes of sample buffer, subjected to gel electrophoresis, and blotted onto nitrocellulose membranes. The KSHV mCP was labeled with anti-mCP rabbit polyclonal antibody, followed by an antirabbit IgG antibody coupled to alkaline phosphatase and visualized with Western blue. A commercially available purified virus preparation was used as positive control. Data shown are representative of 3 separate experiments.