Mechanism of receptor recognition by Ebola virus, and its implications

Abstract

Filoviruses are the causative agents of an increasing number of disease outbreaks in human populations, including the current unprecedented Ebola virus (EBOV) outbreak in Western Africa. One obstacle to controlling these epidemics is our poor understanding of the biological factors that influence the host range and spillover of filoviruses. For my thesis work, we investigated the role of the intracellular filovirus receptor, Niemann-Pick Cl (NPC1) as a molecular determinant of filovirus host range at the cellular level. We had previously shown that unlike human cells that can be infected by EBOV, a cell line derived from a Russell's viper (Daboia russellii) (VH-2) is resistant to infection in an NPC1-dependent manner. We found that VH-2 cells are resistant to EBOV infection because the Russell's viper NPC1 ortholog bound poorly to the EBOV spike glycoprotein (GP). Analysis of panels of viper-human NPC1 chimeras and point mutants led us to identify a single amino acid residue in NPC1, at position 503, that bidirectionally influenced both its binding to EBOV GP as well as its viral receptor activity in cells. Significantly, this single residue change perturbed neither NPC1's endosomal localization nor its housekeeping role in cellular cholesterol trafficking. This subtle change in the viral receptor suggested that there might be other similar NPC1 sequence polymorphisms occurring in nature that control susceptibility to filovirus infection.;A number of bat species have been shown to be possible reservoirs of a number of viruses, including filoviruses. We tested cell lines derived from four species of African fruit bats and found that cells from African straw-colored fruit bats (Eidolon helvum) are selectively refractory to EBOV infection. Intriguingly, this could be explained by a single amino acid change, at residue 502, in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. This residue is right next door to the one that was responsible for the difference in affinity in viper NPC1 (503). We further analyzed a number of bat derived NPC1 sequences and found evidence of signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. This identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. Together with the viper NPC1 work, the residues 502 and 503 identify a hotspot in NPC1 that is important for viral receptor activity by virtue of their direct interaction with EBOV GP, and suggest that they may influence filovirus host range in nature.;The sole protein studded on the filovirus membrane is the spike glycoprotein (GP), which mediates all steps of entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) and only then can it interact with NPC1. To definitively map the RBS, we solved the crystal structure of proteolytically cleaved Ebola virus GP and performed functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins. This study revealed that the RBS is shaped like a wave, with two interacting surfaces that are crucial for NPC1 binding: electrostatic interactions on the crest and hydrophobic interactions in the trough. Finally, we demonstrated that monoclonal antibodies targeting the well conserved filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of pan-filovirus therapeutics.;The very recent structure of the cleaved EBOV GP bound to NPC1 corroborates our work, showing how NPC 1 interacts with GP by two loops, one that contacts the crest and the other the trough. In addition, one of these loops, "loop 2" contains residue 502 and 503 which explains why changes in these residues would affect this interaction. Our work thus defines the interaction between the virus GP and the receptor, identifying residues in both that are crucial for the interaction and can therefore be targeted for therapeutics or used as a guide to understand filovirus tropism.