1% crystal violet, and the viral plaques were counted. For the 96-h assays and learn more for experiments using recombinant VACV-WR expressing mutated F13L, 1% 2-methylcellulose was added to the medium at 0 h. The percentage of inhibition of plaque formation was calculated as follows: 100 − [(mean number of plaques in test × 100)/(mean number of plaques in control)]. The EC50 values (effective concentration of drug required to inhibit 50% of virus replication) were derived from the plots. In some experiments, cytopathic effect reduction assays were conducted to measure the effective concentration

of compound that inhibited 50% of the virus induced CPE. BSC-40 monolayers were seeded in 96-well plates at 1 × 104 cells per well in 180 μl of growth media. ST-246 was added directly to the assay plates at 24 concentrations (0.001–5 μM) using the HP D300 digital titration instrument (Hewlett Packard, Corvallis, OR). Cell monolayers were infected with wild-type vaccinia virus or the vaccinia virus recombinants containing the D217N amino acid substitution using an amount of virus that would cause 95% CPE at 3 days post-infection. The assay was terminated at 3 days post-infection by fixing the cells in 5% glutaraldehyde solution and the amount of CPE was visualized by staining the monolayers with 0.1% crystal

violet. Virus-induced CPE were quantified by measuring absorbance at 570 nm. I BET 762 The EC50 values were calculated by fitting the data to a four-parameter logistic model Amobarbital to generate dose–response curve using XLfit 4.1 (IBDS, Emeryville, CA). Monolayers of BSC-40 cells (1 × 106 cells/well) were infected with 200 PFUs of the recombinant viruses CTGV-βGal or VACV-WR-βGal

and cells were either treated with 0.01, 0.02 or 0.05 μM ST-246 or with 0.05% DMSO (control). At 48 h post-infection, the monolayers were fixed with 4% paraformaldehyde, washed twice with PBS 1× and incubated 18 h at room temperature with a solution containing 0.4 mg/ml X-Gal, 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, and 2 mM MgCl2 (Sanes et al., 1986). The sites of enzyme activity were detected through the visualization of blue viral plaques. For measurement of β-galactosidase activity, the monolayers were infected and treated with ST-246 as described above, and after 48 h the cells were processed as described (Chakrabarti et al., 1985). Cellular extracts were mixed vigorously with chloroform/SDS, and incubated with 4 mg/ml ONPG [O-nitrophenyl-B-d-galactopyranoside] until a light yellow color was developed. The samples were quantified at A420nm. BSC-40 cells grown in 6-well plates (1 × 106 cells/well) were infected with 50 PFU of CTGV or VACV-WR and either treated with 0.05% DMSO (control) or with different concentrations of ST-246. The plates were incubated tilted at a 5° angle for 3–4 days at 34.6 °C and then stained with 0.1% crystal violet. Comet tail formation in vehicle-treated group and ST-246 treated cells was compared by visual inspection.