FIG. 2.

(A) EMCV infection did not alter PML mRNA levels. CHO cells transfected with the empty vector (CHO-pSG5) or overexpressing PMLIII were not infected or were infected with EMCV at an MOI of 5. Total RNA extracted as described in Materials and Methods at 4 and 8 h postinfection for cells overexpressing PMLIII and at 8 h postinfection for control samples (20 μg of RNA by lane) was analyzed for PML and GAPDH. (B) The proteasome inhibitor did not alter viral protein expression and abrogated EMCV-induced PMLIII degradation. Total cell extracts were prepared from CHO-PMLIII cells noninfected or infected for 4 h with EMCV at an MOI of 5 in the absence or presence of epoxomicin. Twenty micrograms of protein extract of each sample was analyzed by Western blotting using antiviral protein antibodies (left panel; virus antigens are indicated), anti-PML, or anti-Actin antibodies (right panel); the unmodified form of PML is indicated by an arrowhead. (C) Confocal microscopy analysis of PML and 20S in EMCV-infected cells. U373MG-PMLIII cells were not infected or were infected with EMCV at an MOI of 5 for 2 h. Double-immunofluorescence staining was performed using monoclonal anti-PML antibody visualized by Alexa Fluor 594 and rabbit anti-20S antibody followed by Alexa Fluor 488 labeling.