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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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.


  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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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

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    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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