Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

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

As neutralizing antibodies directed against the EBOV GP base, such as KZ52 (7, 42) or c4G7 and c2G4 in the ZMapp antibody cocktail (43) (44) (45) , have been hypothesized to inhibit fusogenic structural rearrangement, we evaluated their effects on lipid mixing (Fig. 3C ). Our results confirm that KZ52 and ZMapp treatments specifically render EBOV GP less able to engage in lipid mixing while leaving viral internalization and trafficking unaltered (see Fig. S1A in the supplemental material). Preincubation of VSV-EBOV GP⌬Muc with KZ52 resulted in inhibition of dequenching by 60% at 50 g/ml and 85% at 100 g/ml. ZMapp also had a concentration-dependent, if slightly more potent, effect on dequenching, with lipid mixing inhibited by 83% at 50 g/ml and 91% at 100 g/ml. Neither antibody treatment was able to abolish dequenching, even though the concentrations used were approximately 30-fold to 40-fold higher than the reported IC 90 values for viral neutralization by KZ52, c2G4, and c4G7 individually (42, 45) . This supports the idea of the existence of a significant barrier to fusion, with the number of unencumbered GP molecules needed for simple lipid mixing being much lower than the number needed to bring about membrane fusion and genome release. Collectively, our results signify that lipid mixing is the express product of GP triggering, which entails some degree of conformational change by proteolytically primed EBOV GP and which may lead to or encompass full fusion. NPC1 is required for fusogenic triggering of EBOV GP. The endosomal/lysosomal membrane protein NPC1, involved in cholesterol homeostasis, serves as an essential intracellular receptor for filovirus infection, with a cysteine cathepsin-cleaved form of GP directly recognizing the lumenal domain C of NPC1 (20) (21) (22) 46) . Because the precise role of NPC1 in filovirus entry has not been fully defined, we evaluated lipid mixing in the absence of NPC1 (see Fig. S2 in the supplemental material). Dequenching of VSV-EBOV GP⌬Muc particles was inhibited almost entirely in NPC1-deficient U2OS cells, whereas VSV G dequenching was unaffected, indicating that NPC1 expression is vital for EBOV fusion triggering (Fig. 4A) .