Fig. 3.

Nitazoxanide does not affect SARS-CoV-2 spike stability and oligomerization, but impairs its maturation at an Endo-H-sensitive stage. A A549 cells were transfected with the SARS-CoV-2 spike (SARS-2 S) construct for 4 h and treated with NTZ (2.5 µg/ml) or vehicle. After 16 h, cycloheximide (CHX, 100 µg/ml) was added (time 0), and WCE were analyzed for S protein levels at different times after CHX addition by IB using anti-spike antibodies (top) and quantitated by scanning densitometry. Relative amounts of S protein were determined after normalizing to β-actin (bottom). The plot of percentage of initial S protein levels in control (blue) and NTZ-treated (red) cells versus time is shown. B Confocal images of Flag-tagged S protein (red) in A549 cells transfected with the C-terminal Flag-tagged SARS-CoV-2 spike (SARS-2 S-CF) construct for 4 h and treated with NTZ (5 µg/ml) or vehicle for 16 h. ER-marker calnexin (CNX) (green) is shown. Nuclei are stained with Hoechst (blue). Merge and zoom images are shown. Scale bar, 20 μm (zoom, 7 μm). Yellow arrows in the enlarged areas (zoom) highlight S protein/calnexin colocalization in NTZ-treated cells. Data from a representative experiment of two with similar results are shown. C Gel electrophoresis (4% polyacrylamide) of WCE from A549 cells transfected with SARS-2 S construct or empty vector and treated with different concentrations of NTZ for 16 h. The different forms (trimers, dimers and monomers) of the S protein were visualized by IB with anti-spike antibodies. The slower- and faster-migrating forms of S protein are identified by asterisks and red arrowheads respectively. D Diagram of substrate specificities of endoglycosidase H (Endo-H) and peptide-N-glycosidase F (PNGase-F). Red and blue scissors represent the cleavage sites of Endo-H (red) and PNGase-F (blue), respectively. Mannose (blue circles), N-acetylglucosamine (GlcNAc, green squares) and asparagine (Asn, yellow triangle) residues are shown. E A549 cells transfected with C-Flag tagged SARS-CoV-2 spike (SARS-2 S-CF) construct or empty vector (Mock) for 4 h were treated with NTZ (2.5 μg/ml) or vehicle. After 16 h, CHX (100 μg/ml) was added (time 0); at different times after CHX addition proteins were digested with Endo-H or PNGase-F (+) or left untreated (−) and processed for IB analysis using anti-Flag antibodies and quantitated by scanning densitometry. The Endo-H- or PNGase-F-cleaved faster-migrating S and S2 forms are indicated by white arrowheads. The percentage of Endo-H-resistant (Endo-H R) S protein in the different samples is indicated. F IB of S protein (α-spike), calreticulin (CRT), calnexin (CNX), GRP78 (anti-KDEL), ERp72 and ERp57 in WCE from A549 cells transfected with the SARS-2 S construct or empty vector for 4 h and treated with 2.5 µg/ml NTZ (+) or vehicle (−) for different times. Black arrows indicate bands corresponding to uncleaved S protein (S0), whereas gray arrows indicate bands corresponding to the S1 or S2 subunits