Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

The interaction between cysteine cathepsin-primed GP1 and NPC1 has been previously shown to be vital for filovirus infection (22) ; however, the precise role of NPC1 in this process was unknown. Here, we used an NPC1-deficient cell line and a GP mutant unable to bind NPC1 to prove that the GP-NPC1 interaction, and not just virus-NPC1 colocalization, is indispensable for EBOV fusion triggering and lipid mixing ( Fig. 3 and 4) . We found that the appearance of NPC1 in virus-bearing endosomes always preceded GP triggering, though the time span between the two events varied (Fig. 5K) , as also reported by Simmons and colleagues through a live-cell imaging assay with GP-pseudotyped retroviruses while the current article was in preparation (53). Mingo et al. proposed that delivery to NPC1 Ï© vesicles represented a key rate-limiting step for EBOV fusion (52), but we found a lag ranging from 40 s to more than 20 min between virus-NPC1 colocalization and dequenching. It is unknown at present whether NPC1 binding directly promotes fusogenic conformational changes in GP or primes it for interaction with additional host factors. Therefore, while the lag may represent the span of time needed for the virus to bind NPC1 as its lumenal domains appear in the compartment, it may also signify a need for one or more host factors to act on GP prior to or following GP1-NPC1 interaction.