Article Figures & Data
Figures
- FIG 1
Establishing a high-throughput screening (HTS) assay to identify inhibitors of VP35. (A) Schematic for high-throughput screening assay of VP35 function. Stable VP35 cells were dispensed in 384-well plates using an automated dispenser. Two hours later, cells were treated with SeV (negative control) or SeV plus doxorubicin (positive control). Compound addition was done via pin tool transfer. Twenty hours posttreatment, a luciferase assay was performed. (B) Results of HTS. A total of 2,080 bioactive compounds were screened (8 screening plates). Each screening plate was run in duplicate (indicated by A or B). Data points indicate relative luciferase units (RLU) for each sample. Controls were as described for panel A. The overall Z factor for the screen was greater than 0.5, and the signal-to-background ratio (S/B) was greater than 100. (C) Z values for each 384-well plate in the pilot screen are plotted. (D) The 5 hits identified by the pilot screen that had a Z score greater than 5 in both replicates are listed along with the average Z score for the two replicates. See also Fig. S1 in the supplemental material.
- FIG 2
IFN induction and cytotoxicity of doxorubicin and daunorubicin in reporter cell lines. (A to D) (A and B) Dose response in VP35 cells for activation of the IFN-β reporter luciferase reporter gene (bars) and for cytotoxicity (triangles) of doxorubicin (A) or daunorubicin (B). (C and D) Dose response in control-FF cells for activation of the IFN-β reporter luciferase reporter gene by doxorubicin (C) and daunorubicin (D). (E and F) The effect of doxorubicin (E) and daunorubicin (F) on expression of a constitutively expressed firefly luciferase gene. Percent luciferase activity is relative to that with no drug treatment. Data represent means ± standard deviations and are representative of three independent experiments. RLU, relative luciferase units.
- FIG 3
Induction of an IFN response by doxorubicin and daunorubicin is not cell type specific. Dose response for activation of the IFN-β reporter gene (bars) and for cytotoxicity (triangles) of doxorubicin (A) or daunorubicin (B) in A549 cells transfected with empty vector (vector) or VP35. Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was performed for endogenous IFN-β (C) or ISG54 (D) mRNA levels in A549 cells transfected with empty vector (vector) or VP35 and treated with doxorubicin. The RNA was isolated 12 h after treatment with the indicated concentrations of drug, and levels were normalized to levels of β-actin mRNA. Data represent means ± standard deviations and are representative of three independent experiments.
- FIG 4
An ATM-dependent IFN response that is not blocked by VP35 is stimulated by doxorubicin and daunorubicin. (A) An IFN-β promoter assay was performed as described above except that cells (control or VP35) were treated with DMSO, ATM kinase inhibitor Ku55933 (10 μM), or mirin (10 μM) for 2 h before doxorubicin (1 μM) or daunorubicin (1 μM) treatment or SeV infection. ****, P value < 0.0001 (one-way analysis of variance followed by Tukey’s test). (B) IFN-β promoter reporter gene assays were performed as described above except that cells were transfected with scrambled shRNA (sh Scrm.) or ATM-specific shRNA (sh ATM) plasmids. ****, P value < 0.0001 (one-way analysis of variance followed by Tukey’s test). A Western blot for ATM and VP35 is shown in the inset. M, mock treated (medium + DMSO); S, SeV infected; D, doxorubicin (1 μM) treated. (C) Phospho-ATM (S1981), phospho-p53 (S15), total ATM, total p53, and VP35 levels were assessed by Western blotting in HEK293T cells transfected with empty vector or VP35 and mock treated (mock), treated with doxorubicin (Doxo), or infected with SeV (SeV) at 4 h posttreatment. β-Tubulin served as a loading control. (D) Phospho-IRF-3 (p-IRF-3) and total IRF-3 (IRF-3) levels were assessed in HEK293T cells transfected with FLAG-IRF-3 plasmid and either empty vector (vector) or VP35 plasmid. The cells were either mock treated or treated with doxorubicin or infected with SeV for 8 h. β-Tubulin served as a loading control. Total IRF-3 levels were assessed by using anti-FLAG, p-IRF-3 levels were assessed by using anti-p-IRF-3 (Ser396), and VP35 levels were assessed by using anti-VP35 antibodies. (E) NF-κB firefly luciferase reporter gene activity in mock- or VP35-transfected cells that were mock treated (medium + DMSO), treated with doxorubicin, or infected with SeV in the presence or absence of ATM kinase inhibitor Ku55933. Cells treated with 50 ng/ml of TNF-α for 2 h served as a known NF-κB activation control. Fold induction is relative to the mock-treated, vector control. Data represent means ± standard deviations and are representative of three independent experiments. **, P value < 0.01. (F) IFN-β reporter gene assays were performed as described above in control cells or VP35 cells but in the presence of scrambled siRNA (scrm.) or Top2A-specific siRNA (Top2a). ***, P value < 0.001 (one-way analysis of variance followed by Tukey’s test). The inset shows Western blotting assays to detect Top2A and β-tubulin. M, mock treated; D, doxorubicin treated; S, SeV infected.
- FIG 5
cGAS and STING enhance IFN induction by doxorubicin. (A) IFN-β reporter gene assays were performed as described above in control cells or cell lines with stable expression of STING. These were transfected with empty vector, cGAS-wt, NTase mutant cGAS (cGAS-NTM), or DNA binding cGAS mutant (cGAS-DBM). Some cells were also transfected with VP35 plasmid, as indicated. The next day, cells were mock treated, treated with doxorubicin (Doxo), or infected with SeV. Twenty hours later, reporter gene activity was measured. The Western blot indicates expression of STING, cGAS, VP35, and β-tubulin as a loading control. (B and C) IFN-β reporter control cells or cells stably expressing STING and wt-cGAS were transduced with empty vector or VP35-expressing lentiviruses. Three days later, cells were pretreated with ATM kinase inhibitor Ku55933 (10 μM) for 2 h (B) or transfected with scrambled short hairpin RNA (sh scrnm.) or ATM-specific short hairpin RNA plasmid (sh ATM) to knock down ATM expression (C) and mock treated (medium + DMSO), treated with doxorubicin (Doxo, 3 μM), induced with c-di-GMP (20 μg), or infected with SeV. Twenty hours later, IFN-β reporter activation was measured by luciferase assay. The Western blots show expression of STING, cGAS, ATM, VP35, and β-tubulin. ****, P value < 0.0001 (one-way analysis of variance followed by Tukey’s test). Error bars represent means ± standard deviations, and values are representative of three independent experiments. See also Fig. S2 and S3 in the supplemental material.
- FIG 6
Effect of doxorubicin in vitro. (A) The toxicity of doxorubicin was evaluated in A549 cells using the CellTiter-Glo assay at 48 h after treatment with the drug. (B) Titers of an Ebola virus that expresses GFP (EBOV) after infection at a multiplicity of 2 of A549 cells in the presence of DMSO or doxorubicin (10 μM) (Doxo). The cells were pretreated with doxorubicin prior to infection for 1 h, and doxorubicin was added back to the medium after the infection. The error bars indicate the standard deviations from three independent replicates. **, P value < 0.01 (Student’s two-tailed t test). Data represent means ± standard deviations from two independent experiments (each performed in triplicate). (C and D) qRT-PCR for endogenous IFN-β (C) and ISG54 (D) mRNA levels normalized to β-actin mRNA at indicated postinfection time points. The error bars indicate the standard deviations from three independent replicates. ***, P value < 0.001; ****, P value < 0.0001 (Student’s two-tailed t test). hpi, hours postinfection.
- FIG 7
Doxorubicin bypasses multiple RNA virus IFN antagonists. IFN-β promoter (A) or ISG54 promoter (B) firefly luciferase reporter gene assays were performed. The empty vector (vector) or expression plasmids for the indicated viral IFN antagonists were transfected. The next day, cells were mock treated, treated with doxorubicin, or infected with SeV. Eighteen hours later, luciferase activity was determined. Fold induction was determined by setting the mock-treated (medium + DMSO) empty-vector controls to 1. Error bars indicate standard deviations from three independent replicates. Experiments similar to those described for panels A and B were performed to detect IFN-β promoter (C) or ISG54 promoter (D) reporter gene activity but with ATM kinase inhibitor pretreatment. An experiment similar to that described for panel C was performed using either the control-FF cells (E) or the cGAS-wt-STING-FF stable IFN-β reporter cells described in the legend to Fig. 5B (F). Data represent means ± standard deviations and are representative of three independent experiments. Error bars indicate standard deviations from three independent replicates. **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001 (one-way analysis of variance followed by Tukey’s test). See also Fig. S4 in the supplemental material.
- FIG 8
Proposed model for activation of IFN by doxorubicin bypassing IFN antagonism by Ebola virus VP35 protein. Ebola virus VP35 antagonizes IFN responses triggered by RIG-I-like receptors (RLR), which include RIG-I and melanoma differentiation-associated protein 5 (MDA5). RLR detect cytoplasmic double-stranded RNAs (dsRNAs) or RNAs with 5′ triphosphate (5′ pppdsRNA), products of RNA virus replication. The activation of RLR is further facilitated by protein kinase R activator (PACT). Upon activation, RLR signal through the mitochondrial antiviral signaling protein (MAVS) to activate kinases IκB kinase ε (IKKε) and TBK1. These kinases phosphorylate IFN regulatory factor 3 (IRF-3) or IRF-7, which then accumulates in the nucleus and promotes expression of type I IFNs. Doxorubicin treatment results in IFN induction by two independent pathways: the DNA damage repair response pathway involving ATM and DNA sensor machinery cGAS-STING. The DNA damage leads to activation of ATM that triggers activation of an IRF-3 and/or NF-κB response, thus leading to IFN activation. Furthermore, damaged DNA can also be detected by a cytoplasmic DNA sensor, cGAS, which through the STING–TBK1–IRF-3 axis leads to activation of IFN responses. Interestingly, these DNA-mediated IFN activation pathways are not subverted by the presence of Ebola virus VP35 protein. Thus, these data suggest novel avenues for developing antiviral therapeutics.
Supplemental Material
FIG S1
Related to Fig. 1. Establishment of a high-throughput screening assay to identify inhibitors of VP35. (A) Generation of stable VP35 cells. HEK293T cells stably transfected with a firefly luciferase gene under the control of the IFN-β promoter (293T-FF) were transduced with lentiviruses that express either GFP alone (control-FF) or GFP and VP35 (VP35-FF) to generate stable cell lines for HTS assays. The Western blot shows the expression of GFP and VP35 in the stable cell lines. (B) Characterization of stable cell lines. Control-FF or VP35-FF cells were plated on 384-well plates and, the next day, mock treated or treated with SeV or doxorubicin (1 µM). Eighteen hours posttreatment, luciferase activity was determined. Control-FF or VP35-FF cells were treated with doxorubicin (1 µM) or infected with Sendai virus. Twelve hours posttreatment, total RNA was extracted using Trizol. qRT-PCR was performed for endogenous IFN-β (C) or ISG54 (D) mRNA levels, which were normalized to β-actin mRNA. (E) The HTS assay in a 384-well format. The VP35 cells were plated in 384-well plates. Two hours later, cells were infected with SeV in the presence of either DMSO (SeV + DMSO) or 3 µM doxorubicin (SeV + Doxo). Twenty hours later, luciferase activity was measured. (F) Knockdown of VP35 restores responsiveness of cells to SeV infection. The VP35 cells were mock transfected (untreated) or transfected with scrambled or VP35-specific (si349 and si219) small interfering RNAs. Seventy-two hours posttransfection, cells were mock treated, treated with doxorubicin (doxo), or infected with SeV. Twenty hours later, luciferase activity was measured. The Western blot demonstrates knockdown of VP35 expression. Download FIG S1, EPS file, 3.3 MB.
Copyright © 2017 Luthra et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
FIG S2
Related to Fig. 5. STING enhances IFN induction by doxorubicin. (A) Western blot for endogenous STING and cGAS in control-FF, VP35-FF, A549, and dendritic (DC) cells. (B) Steady-state levels of IFN-β in human wild-type fibroblasts (healthy control) or ATM-deficient fibroblasts (AT cells). Human wild-type fibroblasts (healthy control) (C) or ATM-deficient fibroblasts (AT cells) (D) were transduced with vector control or VP35-expressing lentiviruses. The cells were treated with doxorubicin, c-di-GMP, or SeV. The RNA was harvested at the indicated times, and endogenous levels of IFN-β were determined. Primary human monocyte-derived DCs were transduced with lentiviruses expressing vector control or VP35 Ebola virus protein (control or VP35). Seventy-two hours posttransduction, the cells were treated with doxorubicin (1 μM) or c-di-GMP or infected with SeV. After the indicated times, qRT-PCR was performed for endogenous IFN-β (E) or ISG54 (F) mRNA levels and values were normalized to β-actin mRNA levels. Download FIG S2, EPS file, 2.5 MB.
Copyright © 2017 Luthra et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
FIG S3
Related to Fig. 5. Activation of IFN response by DNA generated by doxorubicin. (A) Colocalization of cGAS with ssDNA. HeLa cells were transfected with vector or cGAS. The next day, cells were mock treated or treated with doxorubicin (doxo, 1 μM) for 4, 8, or 12 h. Cells were fixed, processed for immunofluorescence, and analyzed by confocal microscopy. cGAS was detected using anti-FLAG antibody and ssDNA using anti-ssDNA with Alexa Fluor 488- and 647-labeled secondary antibodies, respectively (as shown in the green and red panels). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bars, 10 µm. The insets show the zoomed-in images of cGAS and ssDNA colocalization regions. (B) The fluorescence intensity profiles for confocal images. The line scans of confocal images showing the overlap of peaks among cGAS, ssDNA, and DAPI as indicated with black arrows (red, green, and blue, respectively). The insets in panel A were used for quantification. (C) Effect of Trex1 expression on doxorubicin-mediated IFN activation. The control or STING-FF cells were transfected with vector or cGAS and also with Trex1 or Trex1 D18N mutant. The next day, cells were treated with doxorubicin (doxo, 1 μM) or SeV or transfected with interferon stimulatory DNA (ISD; 30 μg/ml) for 24 h. The following day, cells were lysed and luciferase activities were measured. Download FIG S3, PDF file, 0.4 MB.
Copyright © 2017 Luthra et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
FIG S4
Related to Fig. 7. Expression of interferon antagonists. (A and B) Expression of Ebola virus VP35 (eVP35), Marburg virus VP35 (mVP35), influenza A virus NS1 protein, Nipah virus V and W proteins (NiV V and NiV W), and respiratory syncytial virus NS1 and NS2 proteins (RSV NS1 and RSV NS2) for the luciferase experiments described for Fig. 7A and B. Expression is shown for the mock-treatment samples. The proteins were detected using anti-FLAG antibody, and anti-β-tubulin was used as the loading control. Download FIG S4, EPS file, 2.8 MB.
Copyright © 2017 Luthra et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
TEXT S1
Supplemental experimental procedures and supplemental references. Download TEXT S1, DOCX file, 0.1 MB.
Copyright © 2017 Luthra et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
