Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

While considerable lipid mixing also occurred in GFP-LAMP1 ϩ vesicles (75%), we found, surprisingly, that a significant fraction of dequenching VSV-EBOV GP⌬Muc particles (15%) colocalized with GFP-Rab5 puncta. Despite their slower rate of lipid mixing events (Fig. 2C) , 10% of particles bearing full-length EBOV GP also dequenched in Rab5 ϩ compartments (data not shown). Because Rab7 appears to be a nearly essential component of the EBOV fusion compartment, these Rab5 ϩ vesicles are likely intermediate endosomes, suggesting that GP triggering can occur earlier in the endocytic pathway than commonly believed (20, 50, 52, 53) . Our results also imply that some population of endosomes must display Rab5 and NPC1 concurrently, at least in the U2OS cell line used. We detected an average lag of 6 min (ranging from 40 s to more than 20 min) between colocalization of viral and NPC1 signals and dequenching, indicating that the occurrence of GP triggering is not immediate upon delivery to NPC1 ϩ compartments (Fig. 5K) .