Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3, 4, 5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.
At a glance
- Ebola haemorrhagic fever. Lancet 377, 849–862 (2010) &
- Ebolavirus glycoprotein structure and mechanism of entry. Future Virol. 4, 621–635 (2009) &
- Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol. 80, 4174–4178 (2006) et al.
- Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavirus bind a common receptor. J. Biol. Chem. 281, 15951–15958 (2006) et al.
- Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645 (2005) , , , &
- Vps-C complexes: gatekeepers of endolysosomal traffic. Curr. Opin. Cell Biol. 21, 543–551 (2009) , &
- Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228–231 (1997) et al.
- Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nature Biotechnol. 29, 542–546 (2011) et al.
- Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009) et al.
- A forward genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that alter its protease dependence during cell entry. J. Virol. 84, 163–175 (2010) , , , &
- Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007) et al.
- Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010) , , , &
- Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005) , , &
- Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex. J. Biol. Chem. 282, 23878–23891 (2007) et al.
- The building BLOC(k)s of lysosomes and related organelles. Curr. Opin. Cell Biol. 16, 458–464 (2004)
- Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase. Nature Med. 11, 1109–1112 (2005) et al.
- Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo. J. Biol. Chem. 285, 4983–4994 (2010) &
- Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nature Med. 14, 1247–1255 (2008) et al.
- Deficiency of Niemann-Pick type C-1 protein impairs release of human immunodeficiency virus type 1 and results in Gag accumulation in late endosomal/lysosomal compartments. J. Virol. 83, 7982–7995 (2009) , , , &
- Identification of HE1 as the second gene of Niemann-Pick C disease. Science 290, 2298–2301 (2000) et al.
- A system for functional analysis of Ebola virus glycoprotein. Proc. Natl Acad. Sci. USA 94, 14764–14769 (1997) et al.
- Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. J. Virol. 72, 3155–3160 (1998) &
- Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids 44, 477–487 (2009)
- Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease. Biochim Biophys Acta 1043, 123–128 (1990) et al.
- Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. Plos One 2, e851 (2007) et al.
- T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proc. Natl Acad. Sci. USA. 108, 8426–8431 (2011) et al.
- C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J. Virol. 76, 6841–6844 (2002) et al.
- Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog. 6, http://dx.doi.org/10.1371/journal.ppat.1001110 (2010) , , &
- Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 6, http://dx.doi.org/10.1371/journal.ppat.1001121 (2010) et al.
- Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol. J. Biol. Chem. 275, 4013–4021 (2000) , , &
- Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection. Am. J. Pathol. 163, 2347–2370 (2003) et al.
- Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells. Am. J. Pathol. 163, 2371–2382 (2003) et al.
- Identification of a minimal size requirement for termination of vesicular stomatitis virus mRNA: implications for the mechanism of transcription. J. Virol. 74, 8268–8276 (2000) , &
- Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl Acad. Sci. USA 92, 8388–8392 (1995) , , &
- Infectivity-enhancing antibodies to Ebola virus glycoprotein. J. Virol. 75, 2324–2330 (2001) , , , &
- Generation of iPSCs from cultured human malignant cells. Blood 115, 4039–4042 (2010) et al.
- Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990) &
- The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease. J. Biol. Chem. 261, 2772–2777 (1986) et al.
- Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J. Biol. Chem. 277, 24609–24617 (2002) , , &
- Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nature Chem. Biol. 1, 203–209 (2005) et al.
- Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5, e1000394 (2009) , , , &
- Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605 (2004) et al.
- The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. I. Analysis of neutralizing epitopes with monoclonal antibodies. Virology 121, 157–167 (1982) &
- Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664–1666 (2000) et al.
- Ebola virus can be effectively neutralized by antibody produced in natural human infection. J. Virol. 73, 6024–6030 (1999) et al.
- A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J. Infect. Dis. 178, 651–661 (1998) , , , &
- Development of a model for marburgvirus based on severe-combined immunodeficiency mice. Virol. J. 4, 108 (2007) et al.
- Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73, 2333–2342 (1999) , , , &
- Assembly of a functional Machupo virus polymerase complex. Proc. Natl Acad Sci USA 107, 20069–20074 (2010) et al.
- Supplementary Figures (14.4M)
This file contains Supplementary Figures 1-19 with legends.