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Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment

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Science  27 Feb 2015:
Vol. 347, Issue 6225, pp. 995-998
DOI: 10.1126/science.1258758
  • Fig. 1 Inhibitors of NAADP signaling block EBOV infection.

    Dose-response curves for verapamil (A), tetrandrine (B), gabapentin (C), and Ned19 (D) were determined by pretreating HeLa cells with the indicated doses of each compound and then infecting the cells with a recombinant EBOV encoding green fluorescent protein (GFP) as a marker of infection (EBOV-GFP). Infection efficiencies were calculated by dividing the numbers of GFP-positive cells by those of total cells and normalizing the infectivity to untreated cells (mean ± SD, n = 3). Each data set is representative of three independent experiments. (E) The effect of the indicated compounds on NAADP-stimulated calcium release was measured by stimulating cells with 1 μM NAADP-AM (30, 31) or control dimethyl sulfoxide and imaging cell fluorescence after addition of the calcium-sensitive dye Fluo-4. Cells showing Fmax/F0 > 2 (Fmax: maximum fluorescence intensity; F0: mean fluorescence intensity before stimulation) were counted as responsive cells. At least 800 cells were analyzed for each treatment, and data averaged over three experiments ± SD. (F) Pseudotyped viruses bearing the glycoproteins of EBOV (rVSV-EBOV-GP) or VSV (rVSV-VSV-G) and encoding firefly luciferase as an infection marker were used to show entry dependence of EBOV on NAADP signaling. Cells were treated with tetrandrine (2 μM) or Ned19 (100 μM) and then infected with either pseudotyped virus. Luciferase activities were normalized to those of untreated controls.

  • Fig. 2 The endosomal calcium channels TPC1 and TPC2 are necessary for EBOV infection.

    (A) MEFs from wild-type (WT), Tpcn1−/−, or Tpcn2−/− mice (25, 32) were infected with EBOV-GFP. The frequency of GFP-positive cells in the total cell population was normalized to that of total cells. (B) HeLa cells were transfected with either two independent nontargeting, TPC1-specific, or TPC2-specific siRNAs and infected with EBOV-GFP. The frequency of GFP-positive cells in the total cell population was normalized to that of mock-transfected cells. (C) HeLa cells overexpressing a dominant-negative form of TPC2 (L265P) tagged with GFP were infected with WT EBOV. Cells expressing GFP alone were used as a control. Infected cells were detected with antibody against EBOV GP. The proportion of cells showing GFP fluorescence that were infected was calculated. All data for (A), (B), and (C) are the mean ± SD (n = 3) and representative of three independent experiments. (D) Colocalization of Ebola VLPs with TPC1 or TPC2 was determined by incubating VLPs (red) for 2 hours with cells transfected with TPC1 or TPC2 tagged with GFP (green). Colocalized particles were indicated by arrowheads. Scale bars, 10 μm. (E) Whole endolysosomal currents were recorded from TPC2-expressing human embryonic kidney 293T (HEK293T) cells by using modified conventional patch-clamp with PI(3,5)P2 (3335). Current-voltage relations were recorded in the presence or absence of tetrandrine (500 nM). (F) Bar diagram summarizing data of current amplitude of TPC2 or TPC1 in the presence of gabapentin (100 μM), Ned19 (200 μM), or tetrandrine (500 nM), normalized to those before drug application. *P < 0.001 using analysis of variance, compared to current in the presence of gabapentin for TPC2 or without inhibitors for TPC1. Data are the mean ± SEM.

  • Fig. 3 Blocking TPC function affects EBOV entry through endosomal compartments.

    (A) VLPs loaded with β-lactamase were used to measure membrane fusion and virus capsid release into the cytoplasm after each treatment (as for fig. S10). The number of cells showing signal was divided by the number of total cells. (B) Evaluation of EGF trafficking in TPC knockout cells. Representative confocal images of WT, Tpcn2−/−, and Tpcn1−/− MEFs incubated with AlexaFluor555-EGF. (C) Evaluation of EGF trafficking in tetrandrine or U18666A-treated cells. Representative confocal images of HeLa cells incubated with AlexaFluor555-EGF (red) in the presence or absence of tetrandrine or U18666A. (D) Colocalization of Ebola VLPs and EGF. HeLa cells were incubated with AlexaFluor555-EGF (red) for 30 min followed by Ebola VLPs (green) for 3.5 hours in the presence of tetrandrine. VLPs were stained with a GP-specific antibody. Examples of colocalized particles are indicated by arrowheads. Scale bars (B to D): 10 μm. (E) Effect of tetrandrine on colocalization of Ebola VLPs with TPC2- and/or NPC1-positive endosomes was measured. HeLa cells overexpressing GFP-tagged TPC2 (green) and Myc-tagged NPC1 (red) were pretreated with inhibitors and incubated with VLPs (blue) for 4 hours. Insets show magnified areas of the image, and arrowheads indicate examples of VLPs that are associated with the TPC2(+)/NPC1(–) compartment (left panel) or the TPC2(+)/NPC1(+) compartment (right panel). Scale bars, 5 μm. (F) In the presence of the indicated inhibitors, the ratio of VLPs colocalizing with the TPC2(+)/NPC1(+) compartment (left) or the TPC2 (+)/NPC1(–) compartment (right) was calculated. *P < 0.05 and **P < 0.005, using unpaired Student’s t test to compare treated to untreated cells. Data are the mean ± SEM (n = 3 or 4).

  • Fig. 4 Tetrandrine inhibits EBOV infection both in vitro and in vivo.

    (A) Macrophages were treated with tetrandrine (8 μM) and then infected with EBOV-GFP. After 48 hours, the frequency of GFP-positive cells was calculated and normalized to that of untreated controls. The data are the mean ± SD (n = 3) and representative of two independent experiments. (B) Female Balb/c mice injected intraperitoneally with mouse-adapted EBOV were treated with 30 mg of tetrandrine per kilogram of body weight or control saline on days 0, 1, 3, 5, and 7 (n = 8 for each group). Survival curves are shown. *P = 0.0008 by log-rank (Mantel-Cox) test. (C and D) Clinical scores of EBOV-infected mice. Disease signs included weight loss, rough hair coat, squinty eyes, hunched back, moderate unresponsiveness, labored breathing, and persistent prostration. Based on these criteria, a clinical score for each day was calculated and plotted (individually indicated by symbols) for the untreated animals (C) or tetrandrine-treated animals (D). (E) Virus titer in sera of infected mice was measured by plaque assays. *P = 0.006 by unpaired Student’s t test. (F) Delayed treatment of EBOV-challenged mice. Female Balb/c mice injected intraperitoneally with mouse-adapted EBOV were treated with tetrandrine (30 or 90 mg/kg) or control saline on days 1, 3, 5, and 7 (n = 7 for each group). Survival curves are shown. *P = 0.04 by log-rank (Mantel-Cox) test comparing treated to untreated animals.

Supplementary Materials

  • Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment

    Yasuteru Sakurai, Andrey A. Kolokoltsov, Cheng-Chang Chen, Michael W. Tidwell, William E. Bauta, Norbert Klugbauer, Christian Grimm, Christian Wahl-Schott, Martin Biel, Robert A. Davey

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S13

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