Fig 2.

Entry of RVFV_ns depends on vacuolar acidification. (A) BHK-21 or A549 cells were pretreated for 1 h with different concentrations of bafilomycin A1 or ammonium chloride (NH₄Cl) and subsequently infected with RVFV_ns (MOI of ∼0.1) or VSV_ns (MOI of ∼0.8). Infection (GFP-positive cells) was quantified by FACS. Results shown are representative of three individual experiments performed in duplicate. NI, not infected; ND, no drugs. (B) RVFV_ns was bound to BHK-21 cells for 1 h in the cold and subsequently warmed to 37°C. NH₄Cl (10 mM) was added at indicated times to instantly raise the endosomal pH, thereby inhibiting further infection. Eighteen hours after warming, infection (GFP-positive cells) was analyzed by fluorescence microscopy (right panel; size bars represent 400 µM) and quantified by FACS (left panel). Untreated cells yielded ∼70% infection. Graphical data shown are representative of two independent experiments performed in duplicate. C, NH₄Cl control (not infected).