Fig. 7.

Nitazoxanide inhibits the fusogenic activity of the SARS-CoV-2 D614G spike variant. A, B HEK293T cells transfected with plasmids for production of SARS-CoV-2 D614 or G614 spike-pseudotyped-LUC LV particles, as described in Fig. 4A, were treated with 2.5 µg/ml of NTZ (+) or vehicle (−). At 48 h post-transfection, pseudovirus-containing supernatants were collected for infection of 293T hACE2 or Caco-2 hACE2 cells. SARS-CoV-2 S expression was detected in WCE of HEK293T pseudovirus-producing cells by IB using anti-spike antibodies (A). 293T hACE2 or Caco-2 hACE2 cells were infected with SARS-CoV-2 spike-pseudotyped LV particles obtained as described above and pseudovirus entry was analyzed by measuring LUC activities 72 h post-infection (B). Data are expressed as percent of relative untreated controls. Error bars indicate means ± SD. **P < 0.01; Student’s t test. C A549 hACE2 cells were transiently transfected with the C-Flag tagged SARS-CoV-2 D614 or G614 spike constructs for 4 h and treated with NTZ (5 µg/ml) or vehicle (Control) for 36 h. Immunofluorescence analysis was performed using an anti-Flag antibody (red). Nuclei are stained with Hoechst (blue). Merge and zoom images are shown. Scale bar, 200 μm (zoom, 50 μm). The number of nuclei involved in syncytia formation is expressed as percent of total nuclei in transfected cells in the same sample (right panel). D HEK293T cells co-transfected with plasmids encoding the C-Flag tagged SARS-CoV-2 D614 or G614 spike and GFP for 4 h, and treated with NTZ (5 µg/ml) or vehicle (Control) for 36 h were overlaid on A549 hACE2 cell monolayers. After 4 h, cell–cell fusion was assessed by fluorescence microscopy (left panel). Bright field (BF) and merge images are shown. Scale bar, 200 μm. Cell–cell fusion was measured and expressed as percentage relative to the D614 control (right panel). C, D Data represent means ± SD of 5 fields from three replicates. *P < 0.05, **P < 0.01; ANOVA