Multiple Phosphatidylinositol 3-Kinases Regulate Vaccinia Virus Morphogenesis
Figure 8
PI3K absence or inhibition reduces IHD-J virion production and release.
A-B. Multistep growth curves of cell-associated (A) or released virus (B) conducted at an MOI of 0.01. AS1 and AS2 were used at 50 µM, Rifampicin 0.1 mg/mL, and the carrier DMSO at 0.5%. A. Multistep growth curves of cell-associated infectious virions, MOI of 0.01. AS1 and rifampicin reduce the amount of virus produced over time relative to DMSO control cells, (20.8-fold and 15.4-fold reduction at 36 hours). AS2 caused a modest reduction in the amount of virus produced over time relative to DMSO controls (5-fold reduction at 36 hours). B. Multistep growth curves of released infectious virions, MOI of 0.01. Note that AS1, AS2, and rifampicin reduce the amount of virus released into the supernatant (Fold reduction at 36 hours: AS1- 127.8-fold, AS2- 28.8-fold, Rifampicin- 55.6-fold). C-D. Multistep growth curves of cell-associated or released virus conducted at an MOI of 0.01 in p85-deficient cells. C. Multistep growth curves of cell-associated infectious virions, MOI of 0.01. From 8 to 36 hours, a reduction in the amount of virus produced was apparent in p85α−/−β−/− cells compared to the p85WT and p85α−/− cells (19.3-fold reduction at 36 hours). D. Multistep growth curves of released infectious virions, MOI of 0.01. A reduction in the amount of virus released from the p85α−/−β−/− cells compared to the p85WT and p85α−/− cells was apparent (20-fold reduction at 36 hours). Results are from one trial conducted in triplicate, and match growth curves conducted using WR. E. PI3K inhibitors reduce comet tails formed by VV, strain IHD-J. Monolayers were infected with 100PFU of virus, and plaques were visualized 48 hours later with crystal violet stain. Experiments were conducted in three independent trials. F,G. Toxicity assays of PI3K inhibitors on uninfected cells. F. MTS assay on BSC-1 cells treated with PI3K inhibitors for different periods of time. MTS production is a measure of mitochondrial metabolism. Values represent the concentration of drug (in µM) required to reduce formazan production by half. Experiments were conducted in two independent trials. G. Trypan blue exclusion assays of BHK cells treated with PI3K inhibitors (AS1 and AS2, 50 µM) for different time periods.