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

Journal name:
Nature
Volume:
477,
Pages:
344–348
Date published:
DOI:
doi:10.1038/nature10380
Received
Accepted
Published online

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.

At a glance

Figures

  1. Structure and function of Ebola virus entry inhibitors.
    Figure 1: Structure and function of Ebola virus entry inhibitors.

    a, Compounds 3.0 and 3.47. b, c, Vero cells were grown in media containing increasing concentrations of 3.0 (b) or 3.47 (c) for 90min before the addition of VSV particles encoding luciferase (b) or GFP (c) and pseudotyped with either EboV GP, VSV G or Lassa fever virus GP (LFV GP). Virus infection is reported as percent of luminescence units (RLU) or GFP-positive cells relative to cells exposed to DMSO vehicle alone. Data are mean±s.d. (n = 4) and is representative of three experiments. d, Vero cells were grown in media containing 3.0 (40μM), 3.47 (40μM), vehicle (1% DMSO) or the cysteine cathepsin protease inhibitor E-64d (150μM) 90min before the addition of replication competent Ebola virus Zaire-Mayinga encoding GFP (multiplicity of infection (m.o.i.) = 0.1). Results are mean relative fluorescence units (RFU)±s.e.m. (n = 3).

  2. NPC1 is essential for Ebola virus infection.
    Figure 2: NPC1 is essential for Ebola virus infection.

    a, HeLa cells were treated with 3.0 (20μM), 3.47 (1.25μM) or vehicle for 18h, then fixed and incubated with the cholesterol-avid fluorophore filipin. b, HeLa cells were transfected with siRNAs targeting Alix, ASM, NPC1, NPC2 and ORP5. After 72h, VSV EboV GP or LFV GP infection of these cells was measured as in Fig. 1c. Data are mean±s.d. (n = 3) and is representative of three experiments. c, CHOwt, CHOnull and CHOnull cells stably expressing mouse NPC1 (CHONPC1) or NPC1 mutants L657F, P692S, D787N were exposed to MLV particles encoding LacZ and pseudotyped with either EboV GP or VSV G. Results are the mean±s.d. (n = 4) and is representative of three experiments. FFU, focus forming units. d, CHOwt, CHOnull, and CHONPC1 cells were infected with replication competent Ebola virus Zaire-Mayinga encoding GFP (m.o.i. = 1). Results are mean relative fluorescence units±s.d. (n = 3). e, CHOwt and CHOnull cells were treated with the cathepsin B inhibitor CA074 (80μM) or vehicle. These cells were challenged with VSV G particles or VSV EboV GP particles treated with thermolysin (EboV GPTHL) or untreated control (EboV GP). Infection was measured as in Fig. 1b. Data are mean±s.d. (n = 9).

  3. Protease-cleaved EboV GP binds to NPC1.
    Figure 3: Protease-cleaved EboV GP binds to NPC1.

    a, Schematic diagram of EboV GP1 binding assay used in panel c. b, left, LE/LY membranes from CHONPC1, CHOnull and CHO NPC1 P692S cells were analysed by immunoblot using antibodies to NPC1 or V-ATPase B1/2. Right, VSV-EboV GP particles and EboV GPΔTM protein were incubated in the presence or absence of thermolysin (THL) and analysed by immunoblot for GP1. c, EboV GPΔTM or thermolysin-cleaved EboV GPΔTM (0.1, 0.5, or 1.0μg) was added to LE/LY membranes purified from CHOnull or CHONPC1 cells. Membrane bound and unbound GP1 were analysed by immunoblot. d, LE/LY membranes from CHOnull or CHOhNPC1 cells were incubated with EboV GPΔTM or thermolysin-cleaved EboV GPΔTM. Following binding, membranes were dissolved in CHAPSO, NPC1 was precipitated using an NPC1-specific antibody, and the immunoprecipitate and the input membrane lysate were analysed by immunoblot for NPC1 (top) or GP1 (bottom). * IgG heavy chain.

  4. NPC1 is a target of the small molecule inhibitors.
    Figure 4: NPC1 is a target of the small molecule inhibitors.

    a, LE/LY membranes from CHOnull or CHOhNPC1 cells were incubated at the indicated concentrations of 3.47, 3.18 or DMSO (5%) before the addition of the photo-activatable 3.98 (25μM). After incubation, 3.98 was activated by ultraviolet light and then conjugated to biotin. NPC1 was immunoprecipitated and analysed by immunoblot for conjugation of 3.98 to NPC1 using streptavidin–horseradish peroxidase (HRP) (top) and recovery of NPC1 (bottom). b, Thermolysin-cleaved EboV GPΔTM protein (1μg) was added to LE/LY membranes from CHOnull or CHONPC1 cells in the presence of DMSO (10%) or the indicated concentrations of 3.47, 3.0, or 3.18 (left panel), and 3.47 or U18666A (U18, right panel). Membrane-bound and unbound GP1 were analysed by immunoblot. c, Proposed model of EboV entry. Following EboV uptake and trafficking to late endosomes24, 25, EboV GP is cleaved by cathepsin protease to remove heavily glycosylated domains (CHO) and expose the putative receptor binding domain (RBD) of GP1 (refs 6, 17–19). Binding of cleaved GP1 to NPC1 is necessary for infection and is blocked by the EboV inhibitor 3.47.

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Author information

  1. These authors contributed equally to this work.

    • Marceline Côté,
    • John Misasi,
    • Tao Ren &
    • Anna Bruchez

Affiliations

  1. Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

    • Marceline Côté,
    • John Misasi,
    • Anna Bruchez,
    • Claire Marie Filone,
    • Qi Li,
    • Kartik Chandran &
    • James Cunningham
  2. Division of Infectious Disease, Department of Medicine, Children’s Hospital, Boston, Massachusetts 02115, USA

    • John Misasi
  3. New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Tao Ren &
    • Kyungae Lee
  4. United States Army Medical Research Institute of Infectious Diseases, Virology Division, Frederick, Maryland 21702, USA

    • Claire Marie Filone &
    • Lisa Hensley
  5. Diabetic Cardiovascular Disease Center, Washington University School of Medicine, Saint Louis, Missouri 63110, USA

    • Daniel Ory
  6. Department of Microbiology and Immunology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • James Cunningham
  7. Present address: Department of Microbiology and Immunobiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

    • Kartik Chandran

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.

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The authors declare no competing financial interests.

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    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.

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