Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

Live imaging revealed that the majority of virions did not engage in lipid mixing (Fig. 2D ). Of the total number of cellassociated VSV-EBOV GP⌬Muc particles, approximately 16% dequenched in Vero cells and 10% in U2OS cells within 2 h. Additionally, 9.1% of particles bearing full-length EBOV GP dequenched in U2OS cells in the same time span. VSV G particles showed similarly low probabilities for lipid mixing in the two cell types. Although we are unable to distinguish definitively between bound and internalized virions, nearly all cell-associated particles had trafficked to perinuclear regions by the end of experiments (data not shown), leading us to believe that uptake levels are high with both EBOV GP and VSV G. The low percentage of particles with actual relevance to infection validates our single-particle approach to elucidating aspects of EBOV entry. Viral lipid mixing is strictly GP mediated. In order to confirm that the observed dequenching events indeed represented GPmediated lipid mixing, we first assessed dequenching in the presence of NH 4 Cl. Failure to acidify endosomes precludes GP activation and infection (36, 37) , as low pH is required for cysteine cathepsin activity (38) and possibly for fusogenic conformational changes in GP (39, 40) . We found nearly a complete reduction of VSV-EBOV GP⌬Muc dequenching by NH 4 Cl treatment, which lends support to the observed lipid mixing being specifically GP mediated (Fig. 3A) .