FIG. 2.

Late viral gene expression is not sensitive to leptomycin B treatment. (A) HeLa cells were grown, infected, fixed, and stained for viral late proteins as described in the text. Infected cells (Ad5, ∼20 FFU/cell) were either not treated (−LMB), treated for 6 h with 10 nM leptomycin B (+LMB), or treated with 5 μg of actinomycin D (+ActD) per ml between 12 and 18 hpi. More than 150 cells/coverslip were counted and scored for the presence of late viral proteins. The columns represent the mean values of four different experiments and show the percent increase in cells expressing late proteins. This was calculated by subtracting the percent late protein-positive cells at the time the drug was added to the culture from the percent late protein-positive cells in the culture following incubation with or without the drug. (B) HeLa cells were infected with Ad5 (∼20 FFU/cell) and were either untreated (−LMB) or treated with 10 nM leptomycin B between 12 and 18, 12 and 24, or 1.5 and 24 hpi (+LMB). Infected cells were harvested at 12, 18, and 24 hpi, and an equal amount of cell extract was electrophoresed on a sodium dodecyl sulfate–10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and incubated with antiserum against viral late proteins for immunoblotting. The viral proteins penton (63.3 kDa) and fiber (62 kDa) were visualized by the ECL detection kit according to the manufacturer's instructions. LMB, leptomycin B; UI, uninfected.