Elucidating and Inhibiting Endosomal Events in Ebola Virus Entry

Abstract

Ebola virus (EBOV) causes a rapidly fatal hemorrhagic fever for which there are no treatments. The EBOV glycoprotein, GP, is necessary and sufficient to mediate entry of the virus into host cells. The EBOV entry begins when virions attach to the cell surface and are endocytosed. Once within the endocytic pathway, GP is cleaved by host cysteine proteases called cathepsins, exposing a putative receptor binding site (RBS) in GP that is hypothesized to bind to an unknown endosomal receptor. Viral-host membrane fusion is thought to occur in late endosomes, however the exact location as well as the fusion trigger is not known.;This thesis describes work showing that Niemann-Pick C1 (NPC1) is the long sought endosomal receptor for EBOV. NPC1, a large transmembrane protein present in late endosomes, contains three domains that protrude into the endosomal lumen. Only one of these domains, domain C, is required for EBOV entry. Further only protease cleaved GP binds to domain C, which is necessary and sufficient for binding. We propose that the interaction between GP and NPC1 occurs within the endocytic pathway, however we can engineer a synthetic cell surface receptor by redirecting domain C to the plasma membrane. Only virus containing cleaved GP is able to bind to cell surface domain C and utilize it for entry. This work demonstrates that NPC1 is a new type of viral receptor that does not engage its target at the cell surface, but rather within an intracellular compartment after GP has been extensively processed.;Membrane fusion, the final step in EBOV entry, occurs within the cell, however much remains unknown about this process. To begin to examine fusion, we developed a peptide-based fusion inhibitor analogous to the GP C-terminal heptad repeat (CHR) sequence. We conjugated the EBOV CHR peptide to a cell penetrating peptide (cpp), and show that this peptide localizes to late endosomes. The cpp-EBOV peptide is capable of inhibiting infection by EBOV suggesting that we are capturing a previously unidentified fusion intermediate. This peptide can be used in the future as a probe for the location of the fusion reaction and for possible triggers of this conformational change.