FIG. 5.

bUL47 nucleocytoplasmic shuttling is resistant to leptomycin B. (A) COS-1 cells were cotransfected with plasmids expressing GFP-bUL47 and β-galactosidase fused to both the SV40 NLS and the Rev NES. Heterokaryon assays were then carried out between the transfected cells and mouse NIH 3T3 cells in the presence of cycloheximide, with or without LMB. Cells were fixed and stained with DAPI and an anti-βgal antibody. Arrows indicate mouse nuclei within a heterokaryon. (B) Line drawing of constructs used to examine the shuttling capability of the bUL47 NES when transferred to β-galactosidase. (C) COS-1 cells were transfected with plasmids expressing β-galactosidase fusion proteins as shown in panel B, and heterokaryon assays were carried out in the presence of cycloheximide. Cells were fixed and stained with DAPI and an anti-βgal antibody. Each fusion protein was scored for its ability to shuttle in a heterokaryon, as summarized in panel B. Arrows indicate mouse nuclei. (D) COS-1 cells were transfected with NLS β-galactosidase containing either the Rev NES or the bUL47 NES, and heterokaryon assays were carried out in the presence of cycloheximide and leptomycin B. Cells were fixed and stained with DAPI and an anti-βgal antibody. Arrows indicate mouse nuclei.