Fig. 6.
GBF1 depletion impairs UUKV replication and particle production. A, AF488-labeled UUKV (UUKV-AF488) was bound to A549 cells silenced for GBF1 expression (si_GBF1_1 and _2) or transfected with a negative-control siRNA (si_Ctrl) for 2 h on ice before fixation and flow cytometry analysis. B, UUKV-AF488 (MOI ∼20) was bound to A549 cells treated with si_Ctrl or siRNAs against GBF1 on ice before warming to 37 °C for 30 min. Internalization was quantified by flow cytometry after cell fixation and trypan blue treatment to quench fluorescence of cell surface-bound viruses. Internalization was normalized to internalization in cells treated with si_Ctrl. Arithmetic means ± S.E. from three independent infection experiments shown. C, BHK-21 cells were transfected with Pol I-driven plasmid DNAs encoding the anti-genomic L and S.ΔNSsGFP UUKV RNA segments, and then, subjected to GCA. Cells were harvested 24 h post-transfection, and GFP expression was analyzed by flow cytometry. D, BHK-21 cells were transfected with UUK-GN/GC and UUK-N expression plasmids media was exchanged after 24 h to media containing GCA (10 μm) and cells and media were collected 16–18 h post media exchange. Supernatant were concentrated by ultracentrifugation before protein analysis with Western blotting. Left panel shows representative blots and right panel show quantification, mean values and standard deviation (n = 3) of the released GN/GC normalized to intracellular GN/GC levels. E, 293T cells were transfected with plasmids expressing wt UUK-GN/GC (lane 2), UUK-GN/GC mutant where position 46–50 (lane 3) in the cytoplasmic tail is exchanged to alanine and the cytoplasmic tail UUK-GN/GC mutant 23–24 (lane 4). 24 h post-transfection IP was performed using the mAb 6G9E5 against GN, proteins in IPs and cell lysates (CL) were separated and detected by Western blotting. Representative blots are shown from three independent experiments.