Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik

Post-NPC1-binding cysteine cathepsin activity is needed for infection but not lipid mixing by EBOV GP. In order to separate possible requirements for cysteine cathepsins pre-and post-NPC1 binding, we evaluated lipid mixing by GP in various cleavage states in the presence and absence of E-64d. Thermolysin was used to generate particles either fully or partially lacking C-terminal GP1 sequences corresponding to the glycan cap and mucin domains (Fig. 8A) . While increased infectivity of particles bearing cleaved GP has been reported (19, 30, 35) , we did not observe this phenomenon or enhanced lipid mixing. When particles were bound to cells to the same extent, cleaved and uncleaved GP produced similar levels of lipid mixing and infection ( Fig. 8B and C) . Unexpectedly, we found that dequenching by particles with cleaved EBOV GP was much less susceptible to E-64d blockage and that the degree of lipid mixing correlated with the extent of GP cleavage (Fig. 8C) . Fully cleaved GP exhibited slight hyperfusogenicity that was unaffected in the absence of cathepsin activity. The in-ability of precleavage to rescue infectivity in E-64d-treated cells (Fig. 8B) , however, indicates that a specific requirement for cathepsins in fusion pore formation lies post-NPC1 binding.