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:

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Abstract

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.

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: A haploid genetic screen identifies the HOPS complex and NPC1 as host factors for filovirus entry.
Figure 2: Viral infection mediated by filovirus glycoproteins requires NPC1 but not NPC2.
Figure 3: Virus entry is arrested at a late step in cells deficient for the HOPS complex and NPC1.
Figure 4: NPC1 function is required for infection by authentic Ebola and Marburg viruses.

Similar content being viewed by others

References

  1. Feldmann, H. & Geisbert, T. W. Ebola haemorrhagic fever. Lancet 377, 849–862 (2010)

    Article  Google Scholar 

  2. Lee, J. E. & Saphire, E. O. Ebolavirus glycoprotein structure and mechanism of entry. Future Virol. 4, 621–635 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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  PubMed  Google Scholar 

  5. 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  PubMed  PubMed Central  Google Scholar 

  6. Nickerson, D. P., Brett, C. L. & Merz, A. J. Vps-C complexes: gatekeepers of endolysosomal traffic. Curr. Opin. Cell Biol. 21, 543–551 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Carstea, E. D. et al. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228–231 (1997)

    Article  CAS  PubMed  Google Scholar 

  8. Carette, J. E. et al. Global gene disruption in human cells to assign genes to phenotypes by deep sequencing. Nature Biotechnol. 29, 542–546 (2011)

    Article  CAS  Google Scholar 

  9. Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Wong, A. C., Sandesara, R. G., Mulherkar, N., Whelan, S. P. & 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  PubMed  Google Scholar 

  11. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

    Article  CAS  PubMed  Google Scholar 

  12. Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010)

    Article  CAS  PubMed  Google Scholar 

  13. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005)

    Article  CAS  PubMed  Google Scholar 

  14. Sbrissa, D. et al. 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)

    Article  CAS  PubMed  Google Scholar 

  15. Dell’Angelica, E. C. The building BLOC(k)s of lysosomes and related organelles. Curr. Opin. Cell Biol. 16, 458–464 (2004)

    Article  PubMed  Google Scholar 

  16. Tiede, S. et al. Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase. Nature Med. 11, 1109–1112 (2005)

    Article  CAS  PubMed  Google Scholar 

  17. Goldman, S. D. & Krise, J. P. 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)

    Article  CAS  PubMed  Google Scholar 

  18. Lloyd-Evans, E. et al. Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nature Med. 14, 1247–1255 (2008)

    Article  CAS  PubMed  Google Scholar 

  19. Tang, Y. Y., Leao, I. C., Coleman, E. M., Broughton, R. S. & Hildreth, J. E. K. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Naureckiene, S. et al. Identification of HE1 as the second gene of Niemann-Pick C disease. Science 290, 2298–2301 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Takada, A. et al. A system for functional analysis of Ebola virus glycoprotein. Proc. Natl Acad. Sci. USA 94, 14764–14769 (1997)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wool-Lewis, R. J. & Bates, P. Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. J. Virol. 72, 3155–3160 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Cenedella, R. J. Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids 44, 477–487 (2009)

    Article  CAS  PubMed  Google Scholar 

  24. Rodriguez-Lafrasse, C. et al. Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease. Biochim Biophys Acta 1043, 123–128 (1990)

    Article  CAS  PubMed  Google Scholar 

  25. Sobo, K. et al. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. Plos One 2, e851 (2007)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  26. Kondratowicz, A. S. 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)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Alvarez, C. P. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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, http://dx.doi.org/10.1371/journal.ppat.1001110 (2010)

  29. Nanbo, A. et al. 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)

  30. Cruz, J. C., Sugii, S., Yu, C. & Chang, T. Y. Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol. J. Biol. Chem. 275, 4013–4021 (2000)

    Article  CAS  PubMed  Google Scholar 

  31. Geisbert, T. W. et al. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Geisbert, T. W. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Whelan, S. P., Barr, J. N. & Wertz, G. W. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Whelan, S. P., Ball, L. A., Barr, J. N. & Wertz, G. T. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl Acad. Sci. USA 92, 8388–8392 (1995)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Takada, A., Watanabe, S., Okazaki, K., Kida, H. & Kawaoka, Y. Infectivity-enhancing antibodies to Ebola virus glycoprotein. J. Virol. 75, 2324–2330 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Carette, J. E. et al. Generation of iPSCs from cultured human malignant cells. Blood 115, 4039–4042 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Morgenstern, J. P. & Land, H. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pentchev, P. G. et al. 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)

    CAS  PubMed  Google Scholar 

  39. Ebert, D. H., Deussing, J., Peters, C. & Dermody, T. S. Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J. Biol. Chem. 277, 24609–24617 (2002)

    Article  CAS  PubMed  Google Scholar 

  40. Blum, G. et al. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nature Chem. Biol. 1, 203–209 (2005)

    Article  CAS  Google Scholar 

  41. Cureton, D. K., Massol, R. H., Saffarian, S., Kirchhausen, T. L. & Whelan, S. P. Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5, e1000394 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  42. Ehrlich, M. et al. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605 (2004)

    Article  CAS  PubMed  Google Scholar 

  43. Lefrancois, L. & Lyles, D. S. 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)

    Article  CAS  PubMed  Google Scholar 

  44. Wilson, J. A. et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664–1666 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  45. Maruyama, T. et al. Ebola virus can be effectively neutralized by antibody produced in natural human infection. J. Virol. 73, 6024–6030 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Bray, M., Davis, K., Geisbert, T., Schmaljohn, C. & Huggins, J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J. Infect. Dis. 178, 651–661 (1998)

    Article  CAS  PubMed  Google Scholar 

  47. Warfield, K. L. et al. Development of a model for marburgvirus based on severe-combined immunodeficiency mice. Virol. J. 4, 108 (2007)

    Article  PubMed  PubMed Central  Google Scholar 

  48. Muhlberger, E., Weik, M., Volchkov, V. E., Klenk, H. D. & Becker, S. Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73, 2333–2342 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Kranzusch, P. J. et al. Assembly of a functional Machupo virus polymerase complex. Proc. Natl Acad Sci USA 107, 20069–20074 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank M. Kielian, H. Ploegh, V. Prasad and D. Sabatini for critical reading of the manuscript and valuable advice; C. Guimaraes, V. Blomen and T. Peterson for suggestions; M. Bogyo for providing the CTSB/CATL activity probe (GB111); T.-Y. Chang for the gift of NPC1-null CHO cells; D. Lyles for the antibody to VSV M; M. Nibert for providing reovirus; J. de la Torre for providing rVSV-GP-BDV; J. Wojcechowskyj for providing RVF; E. Mühlberger for providing Ebola cDNA; and M. Ericsson for support with electron microscopy. This research was supported by NIH grants R01 AI088027 (K.C.), AI081842 and U54 AI057159 (NERCE-BEID) (S.P.W.), and R21 HG004938 (T.R.B.), and by the DTRA Project, CBM.VAXPLAT.05.10.RD.005 (J.M.D.). T.R.B. was additionally supported by the Whitehead Fellows Program. S.P.W. is a recipient of a Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award. A.C.W. was additionally supported by NIH-funded training programs T32 GM007288 and T32 AI070117 at the Albert Einstein College of Medicine. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army.

Author information

Authors and Affiliations

Authors

Contributions

K.C., S.P.W., T.R.B. and J.M.D. were the senior authors of this study and made equivalent contributions. The study was conceived by K.C., S.P.W. and T.R.B. J.E.C. and T.R.B. devised and implemented the haploid genetic screen, generated the HAP1 cells and identified hits by deep sequencing and cell cloning. P.D.C. carried out karyotype analysis on the HAP1 line. K.C. created and characterized the rVSV-GP-EboV virus used in the screen. A.M.G. created the rVSV-G-RABV. J.E.C., G.O. and K.C. performed entry and infection experiments with the HAP1 cells. A.C.W. and K.C. carried out entry and infection experiments with rVSVs in human fibroblasts, CHO and Vero cells. N.M. and K.C. carried out RNAi experiments with primary cells. M.R. was involved in experimental strategy and design and performed entry and infection experiments by high-resolution fluorescence and electron microscopy. N.M. carried out VLP entry experiments and P.J.K., the replicon assay. A.C.W. performed the cysteine cathepsin enzyme assays. A.S.H., A.I.K. and J.M.D. performed the infection and animal challenge experiments with the authentic viral agents. G.R. performed fluorescence microscopy and image analysis with filovirus-infected cell cultures. J.E.C., K.C., S.P.W. and T.R.B. wrote the paper.

Corresponding authors

Correspondence to John M. Dye, Sean P. Whelan, Kartik Chandran or Thijn R. Brummelkamp.

Ethics declarations

Competing interests

J.E.C., M.R., S.P.W., K.C. and T.R.B. have filed a patent on filovirus host factors identified in this study and T.R.B. is a co-founder of Haplogen, an early-stage company involved in haploid genetic approaches

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-19 with legends. (PDF 14814 kb)

Supplementary Table 1

This table shows the iindependent gene-trap insertions in genes in the unselected population (control). (XLS 5201 kb)

Supplementary Table 2

This table shows the enrichment of gene-trap insertions in the population treated with rVSV-GP-EboV. (XLS 82 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carette, J., Raaben, M., Wong, A. et al. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature 477, 340–343 (2011). https://doi.org/10.1038/nature10348

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10348

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

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing