Fig. 7.
Molecular requirements for SV40 passage through the late endosomal pathway and to the ER. (A) Percentage of SV40 particles internalized in the presence of 5 μM nocodazole (Noc) compared to mock-treated controls. (B and C) EM images of SV40 particles added to CV-1 cells in the presence of 5 μM nocodazole for 4 h. Viruses are indicated by black arrows. (D) SV40-AF647 was incubated with CV-1 cells transfected with Rab7-mRFP and Rab5-EGFP in the continuous presence of 5 μM nocodazole. A confocal slice of a representative cell imaged after 230 min is shown. (E) Percentage of virus particles colocalizing with Rab7-mRFP and Rab5-EGFP at different time points postwarming in the presence of 5 μM nocodazole, as quantified from images such as those shown in D. Error bars are SEM of data from each time point for 5 to 15 cells from three different experiments, with an average of 50 to 100 particles per cell. (F) CV-1 cells incubated with 5 μM nocodazole were infected with SV40 at an MOI of 5 for 24 h. The drug was washed out at 8 h postwarming, or different drugs were added after the washout. The infection level was determined by the immunostaining of the SV40 T antigen and flow cytometry normalized to drug-free controls. Shown are means ± SEM of data from three independent experiments, each with triplicate samples. Abbreviations: Baf, bafilomycin A1; Mon, monensin; BFA, brefeldin A; OV, orthovanadate; Gen, genistein; OA, okadaic acid; CalC, calphostin C; Dyn, dynasore. (G) Percentage of SV40 particles internalized in the presence of 80 μM dynasore compared to mock-treated controls.