Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

GP fusion triggering occurs in NPC1-positive (NPC1 ؉ ), Rab7 ؉ compartments. Colocalization of dequenching virions with fluorescently labeled endosomal markers was performed in order to elucidate the compartment in which EBOV GPdependent membrane fusion is triggered ( Fig. 5 ; see also Movies S2 to S5 in the supplemental material). Although a recent report suggested that EBOV fuses exclusively in compartments devoid of NPC1 (50), we found that all dequenching particles in NPC1-mNG-expressing cells colocalized with this marker at the time of dequenching ( Fig. 5J ; see also Movie S5). Additionally, the high percentage of dequenching particles colocalized with green fluorescent protein (GFP)-Rab7 (98%) indicates the ubiquity of this protein in endosomes supporting GP triggering. Further evidence that EBOV must traffic to a suitably mature compartment for fusion was provided by inhibition of dequenching by the twopore calcium channel (TPC)-blocking small molecule tetrandrine (Fig. 6) . Tetrandrine, which may impair the activity of TPC in endocytic trafficking and heterotypic fusion of vesicles (51) , was recently shown to inhibit EBOV infection in vitro and in vivo (50) and decreased GP-mediated lipid mixing in our assay by 95% at 10 M.