Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection1 and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV2. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann–Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain3, 4, 5, 6, 7, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit8. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.
At a glance
- Immunopathology of highly virulent pathogens: insights from Ebola virus. Nature Immunol. 8, 1159–1164 (2007) , &
- Exotic emerging viral diseases: progress and challenges. Nature Med. 10, S110–S121 (2004) &
- Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645 (2005) , , , &
- Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol. 80, 4174–4178 (2006) et al.
- Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177–182 (2008) et al.
- The primed ebolavirus glycoprotein (19-kilodalton GP1,2): Sequence and residues critical for host cell binding. J. Virol. 83, 2883–2891 (2009) et al.
- Biochemical and structural characterization of cathepsin L-processed Ebola virus glycoprotein: Implications for viral entry and immunogenicity. J. Virol. 84, 2972–2982 (2010) et al.
- Viral membrane fusion. Nature Struct. Mol. Biol. 15, 690–698 (2008)
- 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) , , , &
- Lysosomal degradation of membrane lipids. FEBS Lett. 584, 1700–1712 (2010) &
- A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J. Cell Biol. 192, 121–135 (2011) et al.
- Lysobisphosphatidic acid controls endosomal cholesterol levels. J. Biol. Chem. 283, 27871–27880 (2008) et al.
- Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events. Mol. Biol. Cell 12, 601–614 (2001) , , &
- The sterol-sensing domain of the Niemann-Pick C1 (NPC1) protein regulates trafficking of low density lipoprotein cholesterol. J. Biol. Chem. 280, 28581–28590 (2005) et al.
- Binding between the Niemann–Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain. Proc. Natl Acad. Sci. USA 101, 12473–12478 (2004) et al.
- Newly discovered Ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 4, e1000212 (2008) 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.
- Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity. J. Virol. 81, 13378–13384 (2007) , &
- Ebola virus glycoprotein 1: Identification of residues important for binding and postbinding events. J. Virol. 81, 7702–7709 (2007) et al.
- Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry. Proc. Natl Acad. Sci. USA 107, 16637–16642 (2010) et al.
- Identification of HSP60 as a primary target of o-carboranylphenylphenoxyacetanilide, an HIF-1α inhibitor. J. Am. Chem. Soc. 132, 11870–11871 (2010) et al.
- Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. PLoS ONE 2, e851 (2007) et al.
- Cholesterol accumulation by macrophages impairs phagosome maturation. J. Biol. Chem. 283, 35745–35755 (2008) , &
- Cellular entry of Ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog. 6, e1001110 (2010) , , &
- Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 6, e1001121 (2010) et al.
- Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding. J. Biol. Chem. 283, 8229–8236 (2008) et al.
- Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis. J. Biol. Chem. 275, 38445–38451 (2000) , , , &
- A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633 (1995) et al.
- Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening. Virology 332, 20–27 (2005) et al.
- Rapid detection protocol for filoviruses. J. Clin. Virol. 30, 94–99 (2004) , &
- Supplementary Information (4.4M)
This file contains Supplementary Figures 1-8 with legends and Supplementary Text and Data detailing the synthesis and characterization of the novel chemicals used in the manuscript.