Article Figures & Data
Figures
- Fig. 1.
Complementation of gene expression in E4 deletion mutant infection by different mutant constructs of 34K. (A) Map of the 34K protein. 34K is depicted as a rectangle with the positions of the amino acids of the protein shown above. NES and NRS regions are shaded, and the regions are expanded below to show the amino acid sequence. Mutant and wild-type expression constructs were a kind gift of Matthias Dobbelstein and are described in reference 6. The mutations present in the NES and NRS are in bold and underlined. The NES mutation has a leucine and an isoleucine at positions 90 and 92 changed to alanines. The NRS mutation has an arginine at position 248 changed to glutamic acid. (B) HeLa cells were transfected and infected with an E4 deletion mutant as described in the text. Fixed cells were stained with M45 to detect 34K, and they were also stained with a polyclonal serum to detect the viral penton base and fiber proteins. Transfected cells were identified by the presence of 34K and were then scored for the presence or absence of late proteins. The columns represent the percent of transfected cells that also contained late proteins. Abbreviations used for the different constructs: WT34K, wild-type 34K; NES, double point mutation L90A/I92A in the nuclear export signal; NRS, point mutation R248E in the nuclear retention signal; NES/NRS, L90A/I92-R248E double mutation in the nuclear export and retention signals; β-gal, construct expressing β-galactosidase; UT, infected cells that were not transfected. (C) Cells transfected with constructs expressing wild-type 34K (panels a through c), NES-mutated 34K (panels d through f), or green fluorescent protein (panels g through i). Following infection with H5dl1011, cells were fixed and stained as described in Fig. 1B. Cells stained for the transfected protein are shown in panels a, d, and g; late viral proteins are shown in panels b, e, and h. The total number of cells in the microscope fields are visualized in panels c, f, and i. Bar, 10 μm.
- 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.
- Fig. 3.
34K shuttles during Ad infection and leptomycin B inhibits its shuttling. Cell fusions with PEG between infected and uninfected HeLa cells were made as described in the text. Following fusion and fixation, the number of 34K- or penton-positive nuclei was scored. The percent increase in 34K or penton-positive nuclei was calculated by dividing the average number of stained cells per field on coverslips that were treated with PEG by the average number of stained cells per field on coverslips that were not treated with PEG. This ratio was expressed as a percentage. LMB, leptomycin B.
