A Study of Endosomal Factors Involved in Membrane Fusion Mediated by the Ebola Virus Glycoprotein

Ebola virus (EBOV) causes hemorrhagic fever associated with fatality rates of up to 90%. Its entry process is complex and incompletely understood yet could provide clues for novel therapeutics. EBOV entry into cells is mediated by its glycoprotein (GP) comprised of two subunits, GP1, which is responsible for cell attachment, and GP2, which drives membrane fusion. Following attachment and internalization into host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP1 subunit) is processed to a 19-kDa form by endosomal cathepsins B and L (CatB and L), allowing GP to bind to its intracellular receptor Niemann Pick C1 (NPC1). Evidence suggests that an additional factor following EBOV GP-NPC1 binding is required to trigger conformational changes in GP that drive fusion of the viral and endosomal membranes. The cellular factor(s) that act on GP to induce these conformational changes remain unknown, as low pH and NPC1 binding appear insufficient to trigger fusion. A cathepsin protease inhibitor, E-64d, as well as pH raising agents have been shown to block infection by pseudoparticles bearing 19-kDa GP1, suggesting further cathepsin action is needed to trigger fusion. The identification of the final fusion trigger, however, has been impeded, in part due to a lack of tools to assess the biochemical requirements for EBOV GP-mediated fusion within endosomes. To address this limitation, I developed a new in vitro system to study fusion in the endosomal milieu. In this system, late endosomes are isolated from cells and used to prepare supported planar endosomal membranes (SPEMs) and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy (TIRFm) (Chapter 2). The system was validated by recapitulating pH-dependent fusion of influenza and Lassa viruses, the latter with endosomes both positive and negative for the Lassa virus intracellular receptor, Lamp1. Additionally, I explored the effect of the late endosomal lipid, bis(monoacylglycero)phosphate (BMP), on docking and lipid mixing of HIV particles pseudotyped with Lassa virus GP (Appendix A). Using SPEMs, I show in Chapter 2 that fusion mediated by 19-kDa EBOV GP depends on low pH and is enhanced by Ca2+, as reported in other studies. I further demonstrate that SPEMs retain cathepsin activity and that E-64d inhibits 19-kDa EBOV GP-mediated fusion at the hemi- and full fusion stages. Moreover, addition of cathepsins augments both hemi- and full fusion. Collectively the findings support the proposal that additional cathepsin activity is needed beyond generating 19 kDa GP1.
In Chapter 3, I contributed to exploring the role of cholesterol on EBOV fusion and entry. Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties, including intrinsic curvature. We found that EBOV GP interacts directly with cholesterol via several glycines in the membrane-proximal external region and transmembrane (MPER/TM) domains. We also demonstrated that cholesterol in the viral membrane promotes membrane fusion and cell entry. Further, compared to the wild-type counterpart, a mutation in the cholesterol binding site of the TM domain (G660L) resulted in a higher probability of stalling GP2 proteoliposome fusion at the hemifusion stage and lower cell entry of virus-like particles bearing this mutation.
In Chapter 4, the crystal structure of the luminal domain C of NPC1 (NPC1-C) in the space group P21 is described. The crystallization conditions were different from those of other published NPC1-C crystal structures and new purification protocols for glycosylated and non-glycosylated NPC1-C are described. The effect of glycosylation on the thermal stability of NPC1-C has also been explored (Appendix B).