A genetic analysis of NPC1 receptor activity in Ebola virus infection

Abstract

Filoviruses are enveloped, negative-sense, single-stranded RNA viruses that are the causative agents of numerous sporadic outbreaks of viral hemorrhagic fever diseases with case fatality rates up to 90%. Recently, the unprecedented 2013-2016 Ebola virus outbreak in West Africa re-emphasized the grave, epidemic potential that filovirus outbreaks can have on a global scale and resulted in renewed efforts to develop useful and effective therapeutics to combat these deadly diseases.;Filovirus infections require the presence of the intracellular, host protein Niemann-Pick Cl (NPC1). NPC1 is a large, multi-pass 13 transmembrane protein with three luminal domains (A, C, and I) that resides in late endosomes/lysosomes (LE/LY) and is involved in the cellular distribution of free cholesterol to post-LE/LY compartments. Mutations in NPC1 can cause Niemann-Pick Disease Type C (NPC), a rare and fatal inherited disease marked by the endosomal accumulation of free cholesterol and sphingolipids in the brain and other tissues.;Since the breakthrough identification of NPC1 as a critical entry receptor for filoviruses in 2011, it has been the focus of many studies aiming to clarify the filovirus entry mechanism through an investigation of the virus-host interaction. Additionally, because NPC1 is required for the entry of all filoviruses, it is an especially attractive target for antiviral therapeutics because disruption of NPC1 receptor activity could confer pan-filovirus protection.;NPC1 luminal domain C is necessary and sufficient to mediate binding to the EBOV surface glycoprotein (GP), which is responsible for mediating viral entry into host cells. NPC1 domain C has been shown to directly bind GP through the use of two membrane-distal loops that interact directly with the hydrophobic binding pocket of GP. Although it is well established that luminal domain C plays a major role in the virus-host interaction, multiple lines of evidence have indirectly implicated the other luminal domains in the filovirus entry pathway.;In this thesis, we employ a genetic screen using a library of NPC-causing NPC1 mutant proteins in order to investigate the full-length NPC1 protein for residues that may play a role in filovirus entry. Our work identified three mutations, P401L (domain C); Y890C (domain I); and Y899D (domain I), that constitute a new class of NPC1 proteins -- ones that reduce EBOV infection without abrogating NPC1-GP binding. Recently published structures of NPC1 show that the domain C mutant, P401L, exists at an interface between the luminal domains C and I. Furthermore, our work highlighted specific residues, Y890C and Y899D, that are located outside of domain C that influence NPC 1 receptor activity. Together, these findings build on the notion that other luminal domains are, in fact, important in NPC1 receptor activity.