Figure 6.

Effect of dynamitin overexpression on virus infection and protein export. (A) Vero cells transfected with dynamitin (top panels) or GFP (bottom panels) were infected with ts-O45 VSV for 1 h at 17–20°C and maintained at 39.5°C to retain VSV-G protein in the ER. After 3 h, cells were fixed and stained for dynamitin (HA; top left) or GFP (bottom left) and VSV-G protein (right top and bottom, mAb P5D4 and polyclonal serum). Cells overexpressing HA-dynamitin are marked with asterisks. Bar, 20 μm. (B) Quantitation of infection. Vero cells were microinjected (gray bars) with HA-dynamitin or Sar1p (a yeast rab) or transfected (black bars) with HA-dynamitin, GFP, or β-gal and infected for 1 h at room temperature (RT; 17–20°C). Control cells that received no DNA (white bar) were infected in parallel. Cells were stained, and the percent expressing both the exogenous protein and ts-O45-G protein was determined. At least 220 dynamitin-overexpressing cells, in four independent experiments, were counted for each condition. Sar1p overexpression was documented on two separate coverslips in a single experiment. The GFP bar shows data from GFP and β-gal transfections. In the remaining five black bars, cells were infected for 1 h at RT, 1.5 h at 20°C, 2 h at 20°C, 1 h at 37°C, or 2 h at 37°C.