Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure and function of Ebola virus entry inhibitors.
Figure 2: NPC1 is essential for Ebola virus infection.
Figure 3: Protease-cleaved EboV GP binds to NPC1.
Figure 4: NPC1 is a target of the small molecule inhibitors.

Similar content being viewed by others

References

  1. Zampieri, C. A., Sullivan, N. J. & Nabel, G. J. Immunopathology of highly virulent pathogens: insights from Ebola virus. Nature Immunol. 8, 1159–1164 (2007)

    Article  CAS  Google Scholar 

  2. Geisbert, T. W. & Jahrling, P. B. Exotic emerging viral diseases: progress and challenges. Nature Med. 10, S110–S121 (2004)

    Article  CAS  Google Scholar 

  3. Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P. & Cunningham, J. M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645 (2005)

    Article  ADS  CAS  Google Scholar 

  4. Schornberg, K. et al. Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol. 80, 4174–4178 (2006)

    Article  CAS  Google Scholar 

  5. Lee, J. E. et al. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177–182 (2008)

    Article  ADS  CAS  Google Scholar 

  6. Dube, D. et al. The primed ebolavirus glycoprotein (19-kilodalton GP1,2): Sequence and residues critical for host cell binding. J. Virol. 83, 2883–2891 (2009)

    Article  CAS  Google Scholar 

  7. Hood, C. L. 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)

    Article  CAS  Google Scholar 

  8. Harrison, S. C. Viral membrane fusion. Nature Struct. Mol. Biol. 15, 690–698 (2008)

    Article  CAS  Google Scholar 

  9. Wong, A., Sandesara, R., Mulherkar, N., Whelan, S. & Chandran, K. 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)

    Article  CAS  Google Scholar 

  10. Kolter, T. & Sandhoff, K. Lysosomal degradation of membrane lipids. FEBS Lett. 584, 1700–1712 (2010)

    Article  CAS  Google Scholar 

  11. Du, X. et al. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J. Cell Biol. 192, 121–135 (2011)

    Article  CAS  Google Scholar 

  12. Chevallier, J. et al. Lysobisphosphatidic acid controls endosomal cholesterol levels. J. Biol. Chem. 283, 27871–27880 (2008)

    Article  CAS  Google Scholar 

  13. Ko, D. C., Gordon, M. D., Jin, J. Y. & Scott, M. P. Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events. Mol. Biol. Cell 12, 601–614 (2001)

    Article  CAS  Google Scholar 

  14. Millard, E. E. et al. 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)

    Article  CAS  Google Scholar 

  15. Ohgami, N. 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)

    Article  ADS  CAS  Google Scholar 

  16. Towner, J. S. et al. Newly discovered Ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 4, e1000212 (2008)

    Article  Google Scholar 

  17. Kuhn, J. H. et al. Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavirus bind a common receptor. J. Biol. Chem. 281, 15951–15958 (2006)

    Article  CAS  Google Scholar 

  18. Kaletsky, R. L., Simmons, G. & Bates, P. Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity. J. Virol. 81, 13378–13384 (2007)

    Article  CAS  Google Scholar 

  19. Brindley, M. A. et al. Ebola virus glycoprotein 1: Identification of residues important for binding and postbinding events. J. Virol. 81, 7702–7709 (2007)

    Article  CAS  Google Scholar 

  20. Dube, D. 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)

    Article  ADS  CAS  Google Scholar 

  21. Ban, H. S. et al. Identification of HSP60 as a primary target of o-carboranylphenylphenoxyacetanilide, an HIF-1α inhibitor. J. Am. Chem. Soc. 132, 11870–11871 (2010)

    Article  CAS  Google Scholar 

  22. Sobo, K. et al. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. PLoS ONE 2, e851 (2007)

    Article  ADS  Google Scholar 

  23. Huynh, K. K., Gershenzon, E. & Grinstein, S. Cholesterol accumulation by macrophages impairs phagosome maturation. J. Biol. Chem. 283, 35745–35755 (2008)

    Article  CAS  Google Scholar 

  24. Saeed, M. F., Kolokoltsov, A. A., Albrecht, T. & Davey, R. A. 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)

    Article  Google Scholar 

  25. Nanbo, A. et al. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 6, e1001121 (2010)

    Article  Google Scholar 

  26. Gelsthorpe, M. E. 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)

    Article  CAS  Google Scholar 

  27. Millard, E. E., Srivastava, K., Traub, L. M., Schaffer, J. E. & Ory, D. S. Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis. J. Biol. Chem. 275, 38445–38451 (2000)

    Article  CAS  Google Scholar 

  28. Soneoka, Y. et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633 (1995)

    Article  CAS  Google Scholar 

  29. Towner, J. S. 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)

    Article  CAS  Google Scholar 

  30. Weidmann, M., Mühlberger, E. & Hufert, F. T. Rapid detection protocol for filoviruses. J. Clin. Virol. 30, 94–99 (2004)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Considine, A. Nilsson and S. Wilkes for assistance, S. Chiang for critical reading of the manuscript, G. Beltz, N. Gray, S. Grinstein, Y. Iannou, R. Infante, J. Kornhuber, F. Sharom and S. Whelan for discussion. This work was supported by grants from U54 AI057159, R01 CA104266 to J.C., PIDS-Sanofi-Pasteur Fellowship, K12-HD052896 and 5K08AI079381 to J.M., 5-T32- HL007623 to A.B., and fellowship from Fonds de la Recherche en Santé du Québec to M.C.; C.M.F. was supported by the Postgraduate Research Participation Program at the US Army Medical Research and Material Command administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC.

Author information

Authors and Affiliations

Authors

Contributions

M.C., J.M., T.R. and A.B. equally contributed to this work. K.C. and T.R. performed the inhibitor screen. K.L. synthesized and purified 3.0 analogues and T.R. tested them. T.R., A.B., J.M., Q.L. and M.C. carried out infection assays with pseudotyped viruses. A.B. performed microscopy. J.M. purified recombinant glycoprotein. M.C. and J.M. designed and performed binding assays. M.C. performed immunoprecipitation. D.O. provided NPC1 constructs, antibodies and CHO cell lines. Ebola virus infections were performed in the lab of L.H. by C.M.F.; J.C. supervised the project and wrote the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to James Cunningham.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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. (PDF 4560 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Côté, M., Misasi, J., Ren, T. et al. Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection. Nature 477, 344–348 (2011). https://doi.org/10.1038/nature10380

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10380

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research