FIG 6.
Cooperative resistance to entry inhibition. (A) Surface biotinylation of BSR T7/5 cells transiently transfected with expression plasmids encoding IAV-WSN HA with the specified mutations, carried out as described in the legend to Fig. 4A. Bars show average results ± SD for densitometric quantitation of signal intensities of three independent repeats. One-way ANOVA with Sidak's multiple-comparison posttest was used for statistical analysis (ns, not significant [P ≥ 0.05]; standard HA, unchanged IAV-WSN HA). (B) Immunoblotting of samples prepared as for panel A after deglycosylation with PNGase F. (C) Activity testing of recombinant IAV-WSN strains harboring reintroduced specific HA mutations against all three hit compound classes, as described in the legend to Fig. 5B to E. Values represent averages for three independent experiments ± SD. (D) Single-step growth curves for IAV-WSN-nanoLuc harboring the specified mutations in the HA protein on BEAS-2B cells. Luciferase activities were determined at the specified time points postinfection and are expressed relative to the maximal signal observed for the standard IAV-WSN-nanoLuc strain (standardmax). Values represent averages for three independent experiments ± SD. The dotted line at 0.01% of standardmax marks the signal cutoff (baseline observed at 0 h postinfection).