Figure 4

Nitazoxanide alters SeV-F protein intracellular distribution and prevents transport to the host cell surface. (A) Confocal images of SeV-F (red) and ER-marker calnexin (CNX) (green) in mock- and SeV-infected AGMK cells treated with NTZ (10 μg/ml) or vehicle for 24 h. Nuclei are stained with DAPI (blue). Merge images are shown. Scale bar, 20 μm. Orange arrows indicate F-protein plasma membrane localization in untreated cells (SeV); yellow arrows indicate perinuclear F-protein aggregates in NTZ-treated cells (SeV + NTZ). (B) Confocal images of large SeV-F-protein (red) perinuclear aggregates in SeV-infected AGMK cells treated with NTZ (10 μg/ml) for 24 h. ER-marker calnexin (CNX) (green) is shown. Nuclei are stained with DAPI (blue). Merge images are shown. Scale bar, 7 μm. The enlarged area in the inset highlights F-protein/calnexin colocalization. (C) SeV-F/calnexin (CNX) interactions (visualized as red spots) detected at 24 h p.i. by in situ proximity ligation assay (PLA) in SeV-infected AGMK cells treated with NTZ (5 μg/ml) or vehicle. Nuclei are stained with DAPI (blue). Scale bar, 20 μm (zoom, 7 μm). (D) Confocal images of SeV-F (red) distribution on plasma membrane in SeV-infected AGMK cells treated as in (C) and processed for plasma membrane staining, as described in Methods. F-protein could not be detected on the plasma membrane of NTZ-treated cells, despite the presence of high intracellular levels (see Fig. 3C). Scale bar, 20 μm (zoom, 7 μm).