FIG. 4.

Examination of input VP16-GFP in newly infected cells shows that the nuclear accumulation of VP16-GFP is enhanced by the presence of UL14 protein. (A to H) U2OS cells were inoculated with 14R- and 14D-VP16G viruses at an MOI of 40 PFU/cell in the presence of 100 μM cycloheximide. Adsorption was performed at 4°C with continuous rocking of the culture dishes. After 2 h of virus binding, the cells were washed three times in fresh medium containing cycloheximide and either fixed in 4% paraformaldehyde in PBS or cultured in the presence of cycloheximide at 37°C. Cells were fixed at 1, 2 and 4 h after shifting up to 37°C and analyzed by confocal immunofluorescence microscopy. Samples were analyzed by z-axis sectioning, and a single representative plane is shown. The nuclear accumulation of VP16-GFP was apparent in 14R-VP16G-infected cells at 1 h postinfection. In contrast, 14D-VP16G-infected cells accumulated VP16-GFP with slower kinetics and a lower peak nuclear fluorescence. Identical results were obtained using HSV-1 MP44 in place of 14R-VP16G (not shown). Bars, 10 μm. (I) Quantification of the nuclear fluorescence intensity of VP16-GFP. The nuclear fluorescence intensity of the cells shown in the photographs in panels A to H was analyzed using LSM version 3.2 software. The average absolute value and standard deviation is shown. (J) Cycloheximide sufficiently inhibits de novo protein synthesis in cells synchronously infected with 14R-VP16G at an MOI of 40 PFU/cell. Prior to infection, cells were chilled at 4°C for 15 min. Vero cells were infected with the virus in the presence (lanes 1 to 4) or absence (lanes 5 to 7) of 100 μM cycloheximide. Viruses were bound to the cell surface for 2 h at 4°C. Cells were either washed in cold PBS and harvested (lanes 2 and 5) or washed extensively (three times) in cold fresh medium in the presence (lanes 1 to 4) or absence (lanes 5 to 7) of cycloheximide, followed by incubation at 37°C in fresh medium in the presence (lanes 1 and 2) or absence (lanes 3 to 7) of cycloheximide. Cells were harvested after 2 h (lanes 3 and 6) or 4 h (lanes 1, 4, and 7) of incubation. M, mock-infected cells. Whole-cell lysates were separated by SDS-PAGE and Western blotting and detected with anti-UL42, anti-GFP, and anti-tubulin antibodies. The early protein UL42 was undetectable in cells infected in the presence of cycloheximide throughout the experiment (lane 1). Input VP16-GFP was readily detectable (lanes 2 and 5), and there was no marked change (due to de novo synthesis) in the levels of VP16-GFP (a late protein) in lanes 1 to 6.