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

Abstract

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 infection 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 EboV. 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 domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

Figure 1. Structure and function of ebolavirus entry inhibitors

a, Compounds 3.0 and 3.47. b,c, Vero cells were cultured in media containing increasing concentrations of 3.0 (b) or 3.47 (c) for 90 minutes prior to 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 is mean ± s.d. (n=4) and is representative of 3 experiments. d, Vero cells were cultured in media containing 3.0 [40 μM], 3.47 [40 μM], vehicle (1% DMSO) or the cysteine cathepsin protease inhibitor E-64d [150μM] 90 minutes prior to the addition of replication competent ebolavirus Zaire-Mayinga encoding GFP (moi = 0.1). Results are mean relative fluorescence units ± s.e.m. (n=3).

Figure 2. NPC1 is essential for ebolavirus infection

a, HeLa cells were treated with 3.0 (20 μM), 3.47 (1.25 μM) or vehicle for 18 hours, then fixed and incubated with the cholesterol-avid fluorophore filipin. b, HeLa cells were transfected with siRNAs targeting ASM, Alix, NPC1, NPC2, and ORP5. After 72 hours, VSV EboV GP or LFV GP infection of these cells was measured as in Fig 1c. Data is mean ± s.d. (n=3) and is representative of 3 experiments. c, CHO_wt, CHO_null and CHO_null cells stably expressing mouse NPC1 (CHO_NPC1) 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 3 experiments. d, CHO_wt, CHO_null, and CHO_NPC1 cells were infected with replication competent ebolavirus Zaire-Mayinga encoding GFP (moi = 1). Results are mean relative fluorescence units ± s.d. (n=3). e, CHO_wt and CHO_null cells were treated with the cathepsin B inhibitor CA074 (80 μM) or vehicle. These cells were challenged with VSV G particles or VSV EboV GP particles treated with thermolysin (EboV GP_THL) or untreated control (EboV GP). Infection was measured as in Fig 1b. Data is mean ± s.d. (n=9).

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 CHO_NPC1, CHO_null and CHO NPC1 P692S cells were analyzed by immunoblot using antibodies to NPC1 or V-ATPase B1/2. (right) VSV-EboV GP particles and EboV GP_Δ™ protein were incubated in the presence or absence of thermolysin (THL) and analyzed by immunoblot for GP1. c, EboV GP_Δ™ or thermolysin-cleaved EboV GP_Δ™ (0.1, 0.5, or 1.0 μg) was added to LE/LY membranes purified from CHO_null or CHO_NPC1 cells. Membrane bound and unbound GP1 were analyzed by immunoblot. d,. LE/LY membranes from CHO_null or CHO_hNPC1 cells were incubated with EboV GP_Δ™ or thermolysin-cleaved EboV GP_Δ™. Following binding, membranes were dissolved in CHAPSO, NPC1 was precipitated using an NPC1-specific antibody, and the immunoprecipitate and the input membrane lysate were analyzed by immunoblot for NPC1 (top) or GP1 (bottom). * IgG heavy chain.

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

a, LE/LY membranes from CHO_null or CHO_hNPC1 cells were incubated at the indicated concentrations of 3.47, 3.18 or DMSO (5%) prior to the addition of the photoactivatable 3.98 (25 μM). After incubation, 3.98 was activated by UV light and then conjugated to biotin. NPC1 was immunoprecipitated and analyzed by immunoblot for conjugation of 3.98 to NPC1 using streptavidin-HRP (top) and recovery of NPC1 (bottom). b, Thermolysin-cleaved EboV GP_Δ™ protein (1 μg) was added to LE/LY membranes from CHO_null or CHO_NPC1 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 analyzed by immunoblot. c, Proposed model of EboV entry. Following EboV uptake and trafficking to late endosomes^,, EboV GP is cleaved by cathepsin protease to remove heavily glycosylated domains (CHO) and expose the putative receptor binding domain (RBD) of GP1^,–. Binding of cleaved GP1 to NPC1 is necessary for infection and is blocked by the EboV inhibitor 3.47.