Figure 4.

GBF1 complementation assay in cells treated with brefeldin A (BFA). Huh‐7.5 cells were transfected with a plasmid expressing the YFP protein or with plasmids expressing YFP‐fused wildtype GBF1 (GBF1wt), M832L BFA‐resistant GBF1 mutant (GBF1ML), or E794K inactive GBF1 mutant (GBF1EK). Two days post‐transfection, expression levels of YFP‐fused GBF1 proteins, and YFP protein were controlled by western blotting with an anti‐GFP antibody (a) and microscopy (b). Expression of constructs in cells treated with BFA (75 ng/ml) is shown in (b). (c) Two days post‐transfection, cells were electroporated with the full‐length infectious p6 RNA and cultured for 16 hr in the presence of dimethyl sulfoxide (DMSO) or 75 ng/ml BFA. At 96 hr p.e., cells were fixed and analysed by immunofluorescence with an anti‐ORF2 capsid protein antibody. For each construct, the percentage of ORF2‐positive cells in BFA‐treated cells is compared to cells cultured in the absence of BFA. Results are presented as mean ± standard deviation of three independent experiments. ** and *** mean p‐values below .01 and .001, respectively