FIG 2.

Depletion of retrograde transport factors reduces virus spread but not entry or formation of infectious virus. (A) HeLa cells were transfected with the indicated siRNA for 48 h, followed by infection with 0.01 PFU/cell of VACV strain IHDJ expressing the A4 core protein with GFP fused to its N terminus for 18 h. GFP-positive cells were scored by flow cytometry, and results are presented as percentage of control siRNA values. (B) HeLa cells were transfected with the indicated siRNA for 48 h. Cells were infected with 5 PFU/cell of VACV IHDJ expressing firefly luciferase for 1 h at 4°C. After attachment, cells were incubated at 37°C for 90 min and luciferase activity determined. The latter was plotted as percentage of that obtained with control siRNA. (C) HeLa cells were transfected for 48 h with the indicated siRNA, followed by infection with 3 PFU/cell of VACV. After 24 h, the cells were lysed and virus titers determined by plaque assay on BS-C-1 cells. (D) The indicated siRNAs were transfected into HeLa cells for 72 h, and virus spread was determined as for panel A. (E) The indicated siRNAs were transfected into HeLa cells for 72 h, and entry and early gene expression were determined as for panel B. (F) HeLa cells were transfected for 72 h with the indicated siRNA, and virus titers were determined as for panel C. Infections were carried out in triplicate, and error bars indicate standard deviations.