Fig 1.
H84T inhibits Ebola virus infection in cell cultures.
Huh 7 or Vero E6 cells were pretreated for 1 h with the indicated concentrations of H84T. Cells were infected with EBOV/Mak at an MOI of 2.5. After 48 h, cells were fixed and stained with an antibody to the EBOV VP40 protein followed by secondary staining with an Alexa 594-labeled antibody. The experiment was run on duplicate plates with triplicate wells per dose (mean ± SD; n = 3). One representative graph (from one of the two plates) is shown. Percent inhibition of infection was calculated as described in the Methods section. Abbreviations: EBOV/Mak, Ebola virus (Makona); MOI, multiplicity of infection; SD, standard deviation; VP40, viral protein 40.
Fig 2.
H84T inhibits human- and mouse-adapted Ebola virus replication in tissue culture.
Huh 7 or Vero E6 cells were pretreated for 1 h with the indicated concentrations of H84T. Cells were subsequently infected with (A) EBOV/Mak at an MOI of 0.5 or (B) ma-EBOV at an MOI of 0.25 for 48 h. Cells were fixed and stained with an antibody to the EBOV VP40 protein followed by secondary staining with an HRP-labeled antibody. Antiviral activity of H84T, shown in blue, and cytotoxicity in uninfected cells, shown in red, were calculated as described in the Methods section. The experiment depicted in (A) was run twice for each cell line, and each experiment comprised duplicate plates with triplicate wells per dose (mean ± SD; n = 3). One representative graph from the two independent experiments (total of 4 plates) is shown. In (B), one experiment was run on a single plate with triplicate wells per dose (mean ± SD; n = 3). Average cytotoxicities (with 20 μM H84T) were 4.2% and 3.0% for Huh 7 and Vero cells, respectively (from n = 5 plates each). Abbreviations: EBOV/Mak, Ebola virus (Makona); HRP, horseradish peroxidase; ma-EBOV, mouse-adapted Ebola virus; MOI, multiplicity of infection; SD, standard deviation; VP40, viral protein 40.
Fig 3.
H84T inhibits Ebola virus infection in mice.
(A) Survival of mice (n = 13 per group) exposed IP to ma-EBOV (756 PFU) on day 0. H84T treatments were administered IP at 50 mg/kg in PBS at the indicated time points before or after exposure to virus. The control group was treated once daily with vehicle (PBS, Group 2). Treatment for Groups 1 and 5 was discontinued on day 5 due to apparent toxicity. (B, C) Survival of mice (n = 13 per group) exposed IP to ma-EBOV (770 PFU) on day 0. (B) Mice were treated IP with 50, 25, 10, or 5 mg/kg of H84T in PBS at 6 h before exposure to virus and on days 3, 6, and 9 post-exposure. (C) Mice were treated IP with 50 mg/kg H84T in PBS at the indicated time points before or after exposure to virus. Control groups in (B) and (C) received vehicle (PBS, Group 5) or no treatment (None, Group 6). Abbreviations: IP, intraperitoneal; ma-EBOV, mouse-adapted Ebola virus; PFU, plaque forming unit.
Fig 4.
H84T blocks Ebola VLP entry, but less potently than it blocks infection by trVLPs.
(A) VLPs and trVLPs or (B,C) 293T/17 cells were pretreated with the indicated concentration of H84T for 1 h at 37°C. Cells were then processed and analyzed for VLP entry and trVLP infection, with H84T present throughout, as described in the Methods section. VLP entry (blue bars) and trVLP infection (red bars) were scored at 3 and 48 h post addition of VLPs or trVLPs, respectively. (D) VLPs and trVLPs were pretreated with the indicated concentration of the anti-EBOV single chain antibody KZ52-scFv for 1 h at 37°C. Cells were then processed and analyzed as in panels A-C. Experiments depicted in panels C and D were performed on the same day with the same set of target cells. Data are averages of triplicate samples. Error bars represent the SD of the triplicates. Asterisks represent: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 based on two-tailed student t-tests.
Fig 5.
H84T blocks Ebola virus genome transcription/replication, but less potently than it blocks infection by trVLPs.
(A) Parallel sets of 293T/17 cells (one set of trVLP target cells and one set of untransfected cells, at 50–75% confluency) were pretreated for 1 h at 37°C with ribavirin (Riba, 50 μg/mL), Toremifene (Tomf, 0.625, μM), or H84T (25, 100, or 250 μM) and maintained in the same concentration of inhibitor for the remainder of the experiment. After the preincubation period, trVLPs were either added to the trVLP target cells for the p4cis “trVLP infection” assay (red bars) or cells were transfected with the p1cis monocistronic mini-genome and associated RNP plasmids, for the p1cis “genome replication” assay (green bars). Luciferase activity was measured 48 h post infection or transfection. Both assays are described in the Methods section. (B) A repeat experiment was conducted as in (A), but with the addition of two higher concentrations of toremifene (1.25 and 2.5 μM). Data are the averages of triplicate samples, and error bars represent SD of the triplicate means. Asterisks indicate (**) p<0.01 and (****) p< 0.0001, based on two-tailed student t-tests, relative to respective mock treated controls.
Fig 6.
H84T does not block Ebola VLP budding or release.
293T/17 cells were pretreated for 1 h at 37°C with the indicated concentration of H84T and then transfected with plasmids encoding VP40 and Ebola GP. 24 h post transfection (in the continued presence of H84T), particles that had been released into the medium were collected, cleared of cell debris, and concentrated by centrifugation. The cells were then lysed (as described in the Methods section). Samples of cell lysates and released particles were then subjected to SDS-PAGE and analyzed by western blotting for the presence of VP40. Tubulin was used as a loading control for cell lysate samples. The extent of VLP release was calculated as the ratio of the VP40 band intensity in the supernatant divided by the VP40 band intensity in the supernatant + lysate. Data in (A) are the averages (+/- SEM) from 4 independent experiments (one set of samples per experiment). The blot from a representative experiment is shown in (B). ns: not statistically significant (p = 0.27, 25 μM; p = 0.9, 100 μM) based on two-tailed student t-tests.
Fig 7.
Comparative effects of WT, H84T and glycan binding-deficient D133G BanLec on EBOV trVLP infection and cytotoxicity.
293T/17 cells were pretreated with the indicated concentrations of WT (triangles; solid lines), H84T (circles; dashed lines), or D133G (squares; dotted lines) BanLec for 1 h and then processed for trVLP infection (blue) as in the legend to Fig 4 and for cytotoxicity (red) as described in the Methods section. Data (y-axis) are the averages of triplicate samples +/- SD: % cytotoxicity (red), % inhibition of infection (blue).