Fig 2. NPC1-C-loop and low pH and are not sufficient to trigger detectable forced fusion at the plasma membrane (FFPM) by infectious pseudoviruses bearing EBOV-GP_cl.

(A): VSV pseudoviruses bearing the indicated GP were bound at 4°C to target (COS7) cells with (+) or without (-) NPC1-C-loop at their surface that had been pre-cooled (to prevent pseudovirus internalization). The cells were then pulsed at pH 5.0, 5.7 or 7.2 for 5 min at 37°C after which time the buffer was replaced with pH 7.4 medium containing 40 mM NH₄Cl to raise endosomal pH. (FFPM was not monitored at pH 4.5 due to excessive cell loss.) After 24 h, cells were lysed and assessed for the ratio of Renilla luciferase (virus entry and replication) over firefly luciferase (total cells), as described in the Methods section. (B): VSV pseudoviruses bearing the indicated GP (equal input as for the FFPM experiment in A) were added to COS7 cells (+/- C-loop, as indicated) as described above and then either mock-treated or treated with 40 mM NH₄Cl, as indicated. The cells were then incubated at 37°C for 24 h and analyzed for Renilla divided by firefly luciferase as in A. Data represent averages +/- SEM from four independent experiments each performed with triplicate samples.