Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

Mutations at the putative GP1-NPC1 interface have been previously demonstrated to reduce GP1-NPC1 interaction and greatly impair infectivity (22, 47, 48) . Recent work has shown that proteolytic cleavage of GP1 exposes the NPC1-binding site, com-prising a basic/polar crest overhanging a deep hydrophobic trough (65) . We investigated entry by VSV-EBOV GP⌬Muc particles bearing three mutations in the NPC1-binding site (T83M in the trough, K114E/K115E in the crest) that abolish GP1-NPC1 interaction and viral infectivity (65) . Lipid mixing was reduced by 97% with this triple mutant (Fig. 4B ). The inhibition of lipid mixing by VSV-EBOV GP in NPC1-deficient U2OS cells and by VSV-EBOV GP (T83M/K114E/K115E) in wild-type (wt) U2OS cells was not explained by altered delivery to endosomal/lysosomal compartments, since postinfection colocalization of viral particles and endocytic markers was not significantly reduced (see Fig. S1B in the supplemental material). NPC1's functionality as a cholesterol transporter may also have some bearing on EBOV entry, as GP-mediated lipid mixing is selectively inhibited by the cationic amphiphile U18666A (Fig. 4C) , which induces an NPC1 disease phenotype marked by cholesterol accumulation within late endosomes and lysosomes (20, 49) . GP1-NPC1 binding is not affected by U18666A (20, 24), but short-term U18666A treatments that do not cause substantial cholesterol accumulation (see Fig. S3 in the supplemental material) nevertheless prevent EBOV entry (20) , suggesting that this block is due to the drug's specific effect on NPC1 rather than to the resulting increase in endosomal/lysosomal cholesterol levels. Taken together, our results provide direct evidence that NPC1 facilitates one or more early steps in EBOV membrane fusion.