NS1 is essential for the formation of vesicle packets.

(A) Schematic representation of the expression constructs containing a partial deletion in NS1 (pΔNS1) or lacking NS1 completely (cΔNS1). Huh7-Lunet_T7 cells were transfected with pΔNS1, cΔNS1 or the wildtype (WT) construct, fixed 18 h post transfection and processed for electron microscopy, western blot or immunofluorescence. (B) Western blot of cell lysates prepared 18 h post transfection and analyzed by using antibodies indicated on the right. A representative experiment of three repetitions is shown. (C) Transfection efficiency of Huh7-Lunet_T7 cells as determined by immunofluorescence. Data are the mean from three independent experiments using two independent plasmid preparations and counting each time at least 200 cells per sample. The error bars indicate the SEM. (D) Representative transmission electron microscopy images of Huh7-Lunet_T7 cells transfected with expression constructs containing a partial or complete NS1 deletion. Scale bars (upper left of each panel) correspond to 500 nm in the overview and 100 nm in the cropped sections that are indicated with black rectangles in the overviews. (E) Quantification of the EM analysis. The percentage of cells with VPs is shown. Note the absence of regular VPs in cells expressing either NS1 deletion mutant. Data are based on 4 independent experiments, using two independent plasmid preparations and counting at least 20 cells per construct and per repetition.