FIG. 3.
Role of GBF1 in Ebolavirus VP40-mediated VLP formation. (A) Functionality of a dominant-negative GBF1 mutant. 293 cells were transfected with a plasmid encoding FLAG-GBF1 or its dominant-negative mutant. After 24 h, cells were fixed and stained with an anti-GM130 antibody (green; GE Health Care, England) and an anti-FLAG antibody (red). Hoechst 33342 (Invitrogen, CA) was used to stain the nuclei (blue). Arrows indicate the GM130 redistribution in cells expressing dominant-negative GBF1_E794K, confirming the functionality of this mutant. (B) Reduction of Ebolavirus VP40-mediated VLP production by a dominant-negative GBF1 mutant. VP40 was expressed in the absence (lane 1) or presence of wild-type (lane 2) or dominant-negative (lane 3) GBF1 in 293T cells. VLP production efficiency was calculated as described in the legend to Fig. 2B. The results shown are representative of two independent experiments. (C) A dominant-negative GBF1 mutant reduces the frequency of VP40-induced formation of cell protrusions. Venus-VP40 was coexpressed with FLAG-GBF1 or its mutant in 293 cells. The cells were imaged 24 h posttransfection by confocal microscopy.