Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Journal name:
Nature
Volume:
477,
Pages:
340–343
Date published:
DOI:
doi:10.1038/nature10348
Received
Accepted
Published online

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.

At a glance

Figures

  1. A haploid genetic screen identifies the HOPS complex and NPC1 as host factors
                        for filovirus entry.
    Figure 1: A haploid genetic screen identifies the HOPS complex and NPC1 as host factors for filovirus entry.

    a, Genes enriched for gene-trap insertions in the rVSV-GP-EboV-selected cell population compared to unselected control cells. Circles represent genes and their size corresponds to the number of independent insertions identified in the rVSV-GP-EboV-selected population. Genes are ranked on the x-axis based on chromosomal position. b, RT–PCR analysis of the expression levels of NPC1, VPS33A and VPS11 in mutant clones. c, Infectivity of VSV pseudotyped with the indicated filovirus glycoproteins. IU, infectious units. Means±standard deviation (s.d.) (n = 3) are shown. EboV, Ebola virus (Zaire); MarV, Marburg virus. Asterisk indicates below detection limit. d, HAP1 clones were infected with viruses including recombinant VSV viruses carrying rabies or Borna disease virus glycoproteins (rVSV-G-RABV and rVSV-GP-BDV) and stained with crystal violet.

  2. Viral infection mediated by filovirus glycoproteins requires NPC1 but not
                        NPC2.
    Figure 2: Viral infection mediated by filovirus glycoproteins requires NPC1 but not NPC2.

    a, Primary skin fibroblasts from a healthy individual and patients carrying homozygous mutations in NPC1 or NPC2 were stained with filipin, or challenged with rVSV-G or rVSV-GP-EboV. Filipin-stained (black) and infected cells (green) were visualized by fluorescence microscopy. Filipin-stained images were inverted for clarity. Blue indicates Hoechst nuclear counterstain. b, Infectivity of VSV pseudotyped with the indicated viral glycoproteins in control and Niemann–Pick fibroblasts. Asterisk indicates below detection limit. SudV, Sudan virus. c, NPC1 patient fibroblasts expressing empty vector or human NPC1 were stained with filipin or challenged with rVSV-GP-EboV. d, Infectivity of rVSV-G and rVSV-GP-EboV in Vero cells pre-incubated for 30min with the indicated concentrations of U18666A. Scale bars, 200µm (a, c). Means±s.d. (n = 3–6) are shown (b, d).

  3. Virus entry is arrested at a late step in cells deficient for the HOPS
                        complex and NPC1.
    Figure 3: Virus entry is arrested at a late step in cells deficient for the HOPS complex and NPC1.

    a, Viral particles attach and internalize into HOPS- and NPC1-deficient cells. Indicated HAP1 clones were infected with Alexa-647-labelled rVSV-GP-EboV (blue) at 4°C. Non-internalized, bound viral particles (arrowheads, blue) were also stained with a GP-specific antibody (green) and the plasma membrane with Alexa-594-wheat germ agglutinin (red) (top panels). To assess viral internalization, cells were heated to 37°C (bottom panels). Internalized viral particles (blue puncta) are resistant to acid-stripping and inaccessible to a GP antibody. Original magnification, ×63. b, Cells were inoculated with rVSV-GP-EboV and examined by transmission electron microscopy. Representative images of early entry steps are shown. c, In vitro-cleaved rVSV-GP-EboV cannot bypass the infection block observed in VPS11GT, VPS33AGT and NPC1GT cells. GT, gene trap. Infectivity of thermolysin-cleaved rVSV-GP-EboV in the indicated HAP1 clones is shown. Asterisk indicates below the limit of detection. d, Viral escape into the cytoplasm is blocked in HOPS-complex- and NPC1-deficient cells. Wild-type HAP1 cells treated with U18666A (10μgml−1) and the indicated mutant clones were infected with rVSV-G or rVSV-GP-EboV virus for 3h and processed for VSV M staining (red). Punctate staining is indicated by arrows. Original magnification, ×20. e, Electron micrographs of rVSV-GP-EboV-infected VPS33A- and NPC1-deficient HAP1 cells and NPC1-deficient fibroblasts showing agglomerations of bullet-shaped VSV particles in vesicular compartments. All images were taken at 3h after inoculation. Asterisks highlight rVSV-GP-EboV particles in cross-section.

  4. NPC1 function is required for infection by authentic Ebola and Marburg
                        viruses.
    Figure 4: NPC1 function is required for infection by authentic Ebola and Marburg viruses.

    a, NPC1 patient fibroblasts were exposed to Ebola virus (EboV) or Marburg virus (MarV) at a multiplicity of infection (MOI) of 0.1. Supernatants were harvested and yields of infectious virus were measured. Asterisk indicates below detection limit. p.f.u., plaque-forming units. b, Vero cells treated with DMSO or U18666A (20μM) were infected with Ebola virus or Marburg virus at a MOI of 0.1 and yields of infectious virus were measured. c, Human peripheral blood monocyte-derived dendritic cells (DC) and umbilical-vein endothelial cells (HUVEC) were infected in the presence or absence of U18666A at a MOI of 3 and the percentage of infected cells was determined by immunostaining. d, HUVECs were transduced with lentiviral vectors expressing a non-targeting short hairpin (sh)RNA (Ctrl) or an shRNA targeting NPC1, infected with Ebola virus or Marburg virus at a MOI of 3 and the percentage of infected cells was determined. Representative images of cells 48h after infection are also shown: green, viral antigen; blue, nuclear counterstain. For panels ad, Means±s.d. are shown (n = 2–3). In panels a, b, error bars are not visible because they are within the symbols. For panels c, d, **P<0.01; ***P<0.001. e, Survival of Npc1+/+ and Npc1+/− mice (n = 10 for each group) inoculated intraperitoneally with ~1,000p.f.u. of mouse-adapted Ebola virus or Marburg virus. f, A proposed hypothetical model for the roles of CTSB, the HOPS complex and NPC1 in Ebola virus entry.

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Author information

  1. These authors contributed equally to this work.

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

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
  6. 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,
    • Gregor Obernosterer &
    • Thijn R. Brummelkamp

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 financial 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

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Supplementary information

PDF files

  1. Supplementary Figures (14.4M)

    This file contains Supplementary Figures 1-19 with legends.

Excel files

  1. Supplementary Table 1 (5M)

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

  2. Supplementary Table 2 (82K)

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

Additional data