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.

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References

  1. 1.

    & Ebola haemorrhagic fever. Lancet 377, 849–862 (2010)

  2. 2.

    & Ebolavirus glycoprotein structure and mechanism of entry. Future Virol. 4, 621–635 (2009)

  3. 3.

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

  4. 4.

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

  5. 5.

    , , , & Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645 (2005)

  6. 6.

    , & Vps-C complexes: gatekeepers of endolysosomal traffic. Curr. Opin. Cell Biol. 21, 543–551 (2009)

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    , , , & 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)

  11. 11.

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

  12. 12.

    , , , & Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010)

  13. 13.

    , , & Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005)

  14. 14.

    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)

  15. 15.

    The building BLOC(k)s of lysosomes and related organelles. Curr. Opin. Cell Biol. 16, 458–464 (2004)

  16. 16.

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

  17. 17.

    & 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)

  18. 18.

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

  19. 19.

    , , , & 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)

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

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

  25. 25.

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

  26. 26.

    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)

  27. 27.

    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)

  28. 28.

    , , & Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog. 6, (2010)

  29. 29.

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

  30. 30.

    , , & Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol. J. Biol. Chem. 275, 4013–4021 (2000)

  31. 31.

    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)

  32. 32.

    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)

  33. 33.

    , & 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)

  34. 34.

    , , & Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl Acad. Sci. USA 92, 8388–8392 (1995)

  35. 35.

    , , , & Infectivity-enhancing antibodies to Ebola virus glycoprotein. J. Virol. 75, 2324–2330 (2001)

  36. 36.

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

  37. 37.

    & 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)

  38. 38.

    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)

  39. 39.

    , , & Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J. Biol. Chem. 277, 24609–24617 (2002)

  40. 40.

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

  41. 41.

    , , , & Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 5, e1000394 (2009)

  42. 42.

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

  43. 43.

    & 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)

  44. 44.

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

  45. 45.

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

  46. 46.

    , , , & A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J. Infect. Dis. 178, 651–661 (1998)

  47. 47.

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

  48. 48.

    , , , & Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73, 2333–2342 (1999)

  49. 49.

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

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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

Author notes

    • Jan E. Carette
    • , Gregor Obernosterer
    •  & Thijn R. Brummelkamp

    Present addresses: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94304, USA (J.E.C.); Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands (G.O., T.R.B.).

    • Jan E. Carette
    • , Matthijs Raaben
    •  & Anthony C. Wong

    These authors contributed equally to this work.

Affiliations

  1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA

    • Jan E. Carette
    • , Gregor Obernosterer
    •  & Thijn R. Brummelkamp
  2. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Matthijs Raaben
    • , Philip J. Kranzusch
    • , April M. Griffin
    •  & Sean P. Whelan
  3. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Anthony C. Wong
    • , Nirupama Mulherkar
    •  & Kartik Chandran
  4. US Army Medical Research Institute of Infectious Diseases, 1425 Porter St, Fort Detrick, Maryland 21702-5011, USA

    • Andrew S. Herbert
    • , Ana I. Kuehne
    • , Gordon Ruthel
    •  & John M. Dye
  5. Center for Advanced Molecular Diagnostics, Shapiro 5-058, 70 Francis Street, Boston, Massachusetts 02115, USA

    • Paola Dal Cin

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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.

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

Corresponding authors

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

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1-19 with legends.

Excel files

  1. 1.

    Supplementary Table 1

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

  2. 2.

    Supplementary Table 2

    This table shows the enrichment of gene-trap insertions in the population treated with rVSV-GP-EboV.

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DOI

https://doi.org/10.1038/nature10348

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