Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

We initially investigated dequenching kinetics in Vero cells, due to their high permissivity and established use in filovirus infection studies. The time span needed for 50% of particles bearing EBOV GP⌬Muc to dequench (t 1/2 ) was 42 min, considerably greater than the t 1/2 of 17 min for similar particles bearing VSV G, which is believed to undergo fusion triggering in early endosomes ( Fig. 2A ) (33, 34) . We found that the dequenching kinetics for VSV-EBOV GP⌬Muc particles were very similar in U2OS cells (t 1/2 ϭ 44 min, Fig. 2B) , and, because autofluorescence is much lower than in Vero cells, we used U2OS cells in the bulk of our experiments. Regardless of cell type, virtually all dequenching events occurred within 2 h of the synchronized onset of infection. We also examined the dequenching kinetics of VSV particles bearing full-length EBOV GP. Although the onset and conclusion of dequenching events showed similar timings, the overall rate of dequenching by VSV-EBOV GP (t 1/2 ϭ 64 min) was slower than that of VSV-EBOV GP⌬Muc, which may reflect differences in internalization or the need for additional proteolytic processing (Fig. 2C) . Precleavage of EBOV GP⌬Muc by thermolysin, which structurally and functionally mimics the required cleavage of EBOV GP by endosomal/lysosomal cysteine protease cathepsin B (19, 35) , did not accelerate dequenching kinetics compared to uncleaved EBOV GP⌬Muc.