FIG. 7.

Effect of proteasome inhibitors on VACV genome and plasmid replication. (A, B) Effects of MG132 and epoxomicin on genome replication. HeLa cells were infected with VACV and incubated in the presence of DMSO, 10 μM MG132, or 2 μM epoxomicin. The level of viral DNA was determined at the indicated hours postinfection by slot blot analysis with a radioactive viral DNA probe. The intensities of the radioactive bands were quantified using ImageQuant software and plotted. (C) Effect on genome replication of MG132 addition following early gene expression. HeLa cells infected with vpF17R-CAT were treated with HU for 3 h to allow early gene expression. At the end of this time, the HU was removed and the cells were resuspended in medium containing MG132 or DMSO. At 2, 4, 6, and 8 h after the removal of HU, DNA was extracted from the cells and subjected to slot blot analysis. (D) Effect of MG132 on plasmid replication. HeLa cells were transfected with pUC19. After 24 h, the cells were infected with VACV in the absence (−) or presence (+) of 10 μM MG132. Mock-infected cells (Mock) and infected cells (vWR) were harvested at the indicated times, and the total DNA was digested with BamHI and DpnI and resolved by agarose gel electrophoresis. DNA fragments were transferred to a nylon membrane and probed for plasmid-specific sequences.