FIG. 6.

Bax translocation, cytochrome c release, and cell death following PV infection depend on JNK activation. (A) Inhibition of JNK activity during PV infection in IMR5 cells treated with SP600125 (25 μM). Cells were incubated with the JNK inhibitor for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Levels of phospho (Thr183/Tyr185)-JNK and phospho (Ser63)-c-Jun in whole-cell lysates were determined at 30 min p.i. by Western blotting. Blots were subsequently stripped and reprobed with antibodies recognizing all JNK and c-Jun forms to confirm equal protein loading. (B) IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Whole-cell lysates (8 h p.i.) were subjected to Western blot analysis with anti-Bax and anti-cytochrome c (Cyt c) antibodies. Actin was used as a control for protein loading. (C) Inhibition of Bax translocation from the cytosol to mitochondria by the JNK inhibitor SP600125. IMR5 cells were uninfected or were infected with PV in the presence or the absence of SP600125 (25 μM). Eight hours p.i., the cytosolic and heavy membrane fractions were assayed for Bax by Western blotting. Actin and Cox IV were used as controls for protein loading of cytosolic and heavy membrane fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (D) Cytochrome c release is reduced by JNK inhibitor. IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Cytosolic extract proteins were analyzed at 14 h p.i. by immunoblotting with anti-cytochrome c (Cyt c) antibody. Actin was used as a control for protein loading. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin. (E) Inhibition of PV-induced cytopathic effect by the JNK inhibitor SP600125. Cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM) and visualized by light microscopy at the indicated times p.i. Magnification, ×350. (F) The JNK inhibitor SP600125 does not affect PV growth but affects PV release. IMR5 cells were infected with PV in the presence or absence of SP600125 (25 μM). Total virus yield (extracellular and intracellular) was determined by TCID₅₀ assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. The titers of extracellular virus were determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titer for two independent experiments. Standard errors of the means are indicated. *, P < 0.05 by Student's t test comparing untreated IMR5 cells to treated IMR5 cells.