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Fig. 6. Characteristics of Golgi dispersion during poliovirus infection. (A) Golgi-specific glycosylation of VSVG-ts045-GFP was monitored under various conditions. COS-7 cells were transfected with plasmid that expressed VSVG-ts045-GFP, incubated overnight at 40°C, pulse-labeled with [35S]-methionine, and incubated with unlabeled methionine under the conditions described below. Cells were either mock-infected and kept at 40°C (lanes 1 and 2), shifted to 32°C for 30 minutes (lanes 3 and 4), treated with 2 µg/ml BFA for 1 hour at 40°C (lanes 5 and 6) or infected with poliovirus at 40°C for 4 hours (lanes 7 and 8). Labeled VSVG-ts045 was immunoprecipitated from cell extracts and digested with Endo H or not as indicated. Protein samples were analyzed by SDS-PAGE and visualized by autoradiography. Arrow indicates Endo-H-sensitive VSVG-ts045-GFP. (B) Membrane association of Golgi marker galactosyl transferase fused to GFP (GT-GFP) in uninfected and poliovirus-infected cells. COS-7 cells were transfected with plasmid that expressed GT-GFP and then either mock-infected or infected with poliovirus at an MOI of 10 PFU/ml for 3.5 hours. Whole-cell extracts were brought to 59% sucrose, overlaid with one 2-ml layer of 52% sucrose and a second 2-ml layer of 29% sucrose and centrifuged for 90 minutes at 196,000 g in an SW41 rotor. Fractions were collected and subjected to TCA precipitation. Proteins were displayed by SDS-PAGE, and the GT-GFP protein visualized by immunoblot. Fractions that contained 29% sucrose (lanes 1, 5), the 29%/52% interface (2, 6), 52% sucrose (lanes 3, 7) and 59% sucrose (lanes 4, 8) are shown.
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