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 provide direct evidence that EBOV GP fusion triggering occurs in NPC1 Ï© /Rab7 Ï© vesicles, which may, in fact, encompass a range of endosomal compartments. Previously thought to undergo triggering only quite late in the endocytic pathway (50, 53) , EBOV GP could mediate lipid mixing in intermediate endosomes, as indicated by their Rab5 signal, in our live assays. Because the presence of NPC1 is an absolute requirement for dequenching in our assay, we infer that some population of Rab5 Ï© vesicles must contain NPC1 in U2OS cells. Such colocalization is not impossible, as immunofluorescence and fractionation studies have shown that NPC1 localizes primarily, but not entirely, to late endosomes, lysosomes, and the trans-Golgi network (49) ; therefore, NPC1 levels in some intermediate endosomes may be sufficient to support GP triggering. However, it is unclear whether viral genome release from relatively immature endosomes can occur following GP fusion triggering. Indeed, Simmons et al. found no evidence for GP-mediated retroviral content release from Rab5 Ï© compartments (53) , raising the possibility that virions must traffic further in the endocytic pathway to encounter host factors at levels necessary for complete fusion and genome release.