FIG 8.

Binding and entry activity of several mutant viruses. BHK-21 cells were incubated with WT or mutant viruses (DE mutant, N154A, H144A, H319A, T410A, and Q258A) at 10,000 GCPs per cell for 1 h at 4°C. (A) The bound viral genome RNA of each mutant relative to that of WT was analyzed by qPCR using GAPDH as an internal control (bottom). After the unbound virus was removed, the cells were kept at 37°C for 1 h and then trypsinized and replated into a 24-well plate. To detect productive virus entry, the synthetic viral genome RNA of each mutation relative to that of WT was determined by qPCR at 16 h postinfection (top). (B) The entry activity of each mutation was measured by the ratio of synthetic viral RNA to bound viral RNA. Asterisks denote a statistically significant reduction in entry activity compared to that of the WT (**, P < 0.01; ***, P < 0.001). (C) qPCR was performed as described for panel A (top) in the presence of bafilomycin A1 or chloroquine. Means and standard errors are from three independent experiments.