Figure 5.
The pH Threshold for UUKV Membrane Fusion Is 5.4
(A) Confluent monolayer of cells was exposed to UUKV (moi ∼1000) on ice and then treated for 5 min at 37°C with buffers at indicated pH. Nuclei and cytoplasm were stained 3 hr postwarming with bisbenzimide and CellMask Deep Red, respectively.
(B) Shows the quantification of data obtained in (A). Fusion index is given as f = (1 − [c/n]), where c is the number of cells in a field after fusion and n the number of nuclei. Error bars indicate SD.
(C) Confluent monolayers of cells were exposed to UUKV (moi ∼5–30) on ice, treated with varying pH for 1.5 min at 37°C, and then incubated for up to 18 hr at 37°C in the presence of NH4Cl (50 mM) to block further penetration from endocytic vesicles. The infection was determined by FACS. Error bars indicate SD.
(D) Shows the fusion of UUKV-RED (moi ∼2) with the plasma membrane in one confocal plane using the protocol described in (C). Higher magnifications of the plasma membrane are shown in the corners (white squares).