Research ArticleInfectious Disease

Productive Replication of Ebola Virus Is Regulated by the c-Abl1 Tyrosine Kinase

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Science Translational Medicine  29 Feb 2012:
Vol. 4, Issue 123, pp. 123ra24
DOI: 10.1126/scitranslmed.3003500
  • Fig. 1

    Effect of c-Abl1 knockdown and kinase inhibition on Ebola VLP release in transfected 293T cells. (A) Knockdown of c-Abl1 (lanes 1 to 3) or c-Abl2 (lanes 4 to 6) using nontargeting siRNA control or siRNA targeting c-Abl1 or c-Abl2 confirmed by Western analysis in cell lysates with antibodies specific to either c-Abl1 (lanes 1 to 3) or c-Abl2 (lanes 4 to 6). β-Actin was used as a loading control. The results are representative of five independent experiments. 293T cells were transfected with plasmids encoding VP24, VP35, VP40, NP, and GP. In all cases, Ebola VLPs were analyzed by immunoprecipitation with GP followed by Western blotting for NP and VP40 (lanes 10 to 12). Cell lysates are shown in lanes 7 to 9. Data represent means ± SEM of individual measures with cells from four independent experiments. Significant differences by paired Student’s t test between test and control siRNAs are indicated. *P < 0.05. (B) Knockdown of c-Abl1 using a nontargeted siRNA control (lane 13) or three individual siRNAs (S9, S10, and S11) targeting c-Abl1 (lanes 14 to 16) was analyzed by Western analysis in cell lysates, with eIF4E as a loading control. NP and VP40 present in cell lysates were determined (lanes 17 to 20), and Ebola VLPs in supernatants were measured as in (A) after knockdown with c-Abl1 individual siRNAs (lanes 21 to 24). Data represent means ± SEM of individual measures with cells from three independent experiments. Significant differences by paired Student’s t test between test and control siRNAs are indicated. *P < 0.05. For (A) and (B), quantitation of NP and VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band (lower panels). (C) Western analysis of Ebola VLP release for NP and VP40 content with imatinib (lanes 25 to 27) or nilotinib (lanes 28 to 30). Water was used as control for imatinib and DMSO for nilotinib. Quantitation of NP and VP40 was performed relative to the intensity of the solution control band. Data are presented as means ± SEM of individual measures with cells from eight (for NP) and three (for VP40) independent experiments. Significant differences by paired Student’s t test between drug and vehicle control are indicated. *P < 0.05.

  • Fig. 2

    Toxicity analysis, morphology of Ebola intracellular nucleocapsids, and nucleocapsid formation. (A) 293T cell viability analysis. Toxicity of 10 or 20 μM imatinib or nilotinib or vehicle control was measured by exclusion of 7-aminoactinomycin D (7-AAD), a fluorescent chemical compound with a strong affinity for DNA, in untransfected cells 36 to 48 hours after the drug was added. Data are presented as means ± SEM of three independent experiments. (B) Morphology of Ebola virus nucleocapsids in 293T cells transfected with empty vector (left panel) or the five Ebola virus plasmids in combination with water or DMSO controls (middle panels) or 20 μM drug treatments (right panels) seen intracellularly (white arrows) by electron microscopy. Size standards are shown. (C) Nucleocapsid formation. Effect of nilotinib (lane 4) in nucleocapsid formation on cells transfected with VP24, VP35, and NP. Anti-NP antibody was used for immunoprecipitation (IP) of nucleocapsids. Quantitation of NP and VP35 was performed relative to the intensity of the DMSO control band (lane 3). Input amount of protein is shown in lanes 1 and 2. Data are presented as means ± SEM of individual measures with cells from three independent experiments.

  • Fig. 3

    c-Abl1–specific siRNA and nilotinib effects on VP40 VLP egress. 293T cells were transfected with plasmid encoding VP40. (A and B) VP40 VLPs were analyzed by immunoprecipitation with mAb anti-FLAG followed by Western analysis for VP40 after transfection with a pool (A) (lanes 4 to 6) or individual (B) (lanes 11 to 14) siRNAs specific to c-Abl1. Cell lysates are shown in lanes 1 to 3 and 7 to 10. VP40 levels in VLPs were normalized to inputs. Quantitation of VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band. Data represent means ± SEM of individual measures with cells from three independent experiments for (A). Significant differences by paired Student’s t test between test and control siRNAs are indicated. *P < 0.05. (C) Western analysis of VP40 VLP release after immunoprecipitation with mAb anti-FLAG (lanes 17 and 18). Cells were incubated with 20 μM nilotinib (lanes 16 and 18). DMSO was used as the solution control (lanes 15 and 17). Cell lysates are shown in lanes 15 and 16. Quantitation of VP40 was performed relative to the intensity of the solution control band. Data are presented as means ± SEM of individual measures with cells from three independent experiments. Significant differences by paired Student’s t test between drug and vehicle control are indicated. *P < 0.05.

  • Fig. 4

    Effect of c-Abl1 expression on VP40 phosphorylation and sensitivity to TK antagonist inhibition and siRNA. (A) Western analysis of transfected cell lysates (lanes 1 to 4) or VP40 immunoprecipitates (IP) (lanes 5 to 8) of cells transfected with empty vector control (lanes 1, 3, 5, and 7) or wild-type (WT) c-Abl1 (lanes 2, 4, 6, and 8) in the presence of VP40 expression vector. DMSO control (lanes 1 and 2 and lanes 5 and 6) or 20 μM nilotinib (lanes 3 and 4 and lanes 7 and 8) was added 12 to 18 hours after transfection. Kinase activity on VP40 was measured by Western analysis of inputs using an anti-phosphotyrosine (pTyr) antibody (upper panel). Western analysis was also performed for c-Abl1 and FLAG-tagged VP40 (middle panels). eIF4E was used as a loading control (lower panel). Analyses are representative of three independent experiments. (B) Western analysis of input cellular lysates (lanes 9 to 11) or VP40 immunoprecipitates (lanes 12 to 14) in cells transfected with nontargeting siRNA control (lanes 9, 10, 12, and 13) or a smart pool of siRNA targeting c-Abl1 (lanes 11 and 14) in the absence (lanes 9, 11, 12, and 14) or presence (lanes 10 and 13) of 20 μM imatinib. Kinase activity on VP40 was measured by Western analysis of inputs using an anti-phosphotyrosine antibody (upper panel). Western analysis was also performed for c-Abl1 and FLAG-tagged VP40 (middle panels). β-Actin was used as a loading control (lower panel). Analyses are representative of three independent experiments. (C) Western analysis of phosphotyrosine and c-Abl1 immunoprecipitates. Cells were transfected with Ebola virus VP40 and then treated with DMSO vehicle (lanes 15, 16, 18, 19, 21, and 22) or 20 μM nilotinib (lanes 17, 20, and 23). Cell lysates (lanes 15 to 17) were immunoprecipitated with an anti-phosphotyrosine antibody (PY20) (lanes 18 to 20) or anti–c-Abl1 antibody (8E9) (lanes 21 to 23), and Western analysis was performed for FLAG-tagged VP40 and c-Abl1. β-Actin was used as a loading control. The results are representative of two independent experiments. (D) Western analysis of phosphotyrosine immunoprecipitates. Cells were transfected with nontargeting siRNA control (lanes 24 and 27) or siRNA targeting c-Abl1 (lanes 25 and 28) or c-Abl2 (lanes 26 and 29) for a day before Ebola virus VP40 transfection. Cell lysates (lanes 24 to 26) were immunoprecipitated with an anti-phosphotyrosine antibody (PY20) (lanes 27 to 29), and Western analysis was performed for FLAG-tagged VP40. The results are representative of three independent experiments. (E) Western blotting analysis of cell lysates and extracellular VLPs with an anti-phosphotyrosine antibody (PY20) and anti–FLAG-tagged VP40 upon expression of empty vector (lanes 31 and 34) or WT VP40 (lanes 32 and 35) in the five-plasmid mixture in 293T cells. Lanes 30 and 33 show no transfection. Inputs are shown in lanes 30 to 32 with β-actin as a loading control. VLPs were purified by sucrose density sedimentation gradients. The results are representative of two independent experiments. (F) Coimmunoprecipitation of NP and VP40. Cells were cotransfected with NP and VP40 and then treated with DMSO (lanes 39 and 44) or 20 μM nilotinib (lanes 40 and 45). NP and FLAG-tagged VP40 in cell lysates (lanes 36 to 40) and VP40 immunoprecipitates (lanes 41 to 45) were detected by Western analysis using rabbit anti-NP antiserum and anti-FLAG mAb. β-Actin was used as a loading control. NP levels in VLPs were normalized to NP levels in lysates and then expressed as a ratio of the DMSO control sample (lower panel). Data are presented as means ± SEM from four independent experiments, and significance was analyzed by paired Student’s t test.

  • Fig. 5

    Localization of VP40 c-Abl1–mediated tyrosine phosphorylation and effect of tyrosine mutation on VLP release. (A) Expanded region of the MALDI-TOF MS spectrum of the VP40 (amino acids 3 to 21) acquired for the nonphosphorylated (left) versus the phosphorylated (right) peptides from 293T cells cotransfected with VP40 and c-Abl1. Analysis was performed on a gel slice of a FLAG-tagged VP40 immunoprecipitate. Arrow indicates site of Y13 phosphorylation of VP40. (B) Western analysis of VP40 phosphorylation using WT or VP40 mutants created by site-directed mutagenesis on Y13. Cells were transfected with empty vector control (lanes 1, 3, 5, and 7) or WT c-Abl1 vector (lanes 2, 4, 6, and 8) in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7, and 8) of Y13A VP40 labeled with a FLAG tag. WT VP40 was used as a reference in both cases. Phosphorylation on tyrosine was measured by Western analysis (upper panel) in cell lysates (lanes 1 to 4) or in VP40 immunoprecipitates (lanes 5 to 8). Western analysis was also carried out for VP40 by FLAG labeling (middle panel). β-Actin was used as a loading control (lower panel). The results are representative of four independent experiments. (C) Western analysis of cell lysates and extracellular VLPs upon expression of empty vector (lanes 9 and 12), WT VP40 (lanes 10 and 13), or Y13A VP40 (lanes 11 and 14) Ebola virus VP40 in the five-plasmid mixture in 293T cells. Inputs are shown in lanes 9 to 11 with eIF4E as a loading control. VLPs were purified by sucrose density sedimentation gradients. The results are representative of three independent experiments. (D) Quantitation of the ratio of NP in VLPs relative to cell lysate NP based on (C) (lanes 9 to 11 and 12 to 14). Data are presented as means ± SEM of individual measures with cells from three independent experiments, and significance was analyzed using paired Student’s t test.

  • Fig. 6

    Effect of siRNA knockdown and c-Abl1 TK inhibition on Ebola virus replication. (A) Effect of a nontargeted siRNA control or individual siRNAs targeting c-Abl1 (S9, S10, and S11) on Zaire strain Ebola virus (EBOV) release from Vero cells on day 7 after infection. Cells were infected at a multiplicity of infection (MOI) of 1. Background viral load for day 1 was subtracted. Data are presented as means ± SEM of individual measures with cells from two independent experiments. (B) Viral load was measured in supernatant fluids of Vero cells infected with Zaire strain Ebola virus and treated with nilotinib (20 μM). Viral load was measured by TCID50 on days 0, 1, 2, 7, and 8 after infection compared to DMSO vehicle control. Background viral load for day 0 was subtracted. Data are presented as means ± SEM of four individual measures, and significance was analyzed by paired Student’s t test.

Additional Files

  • Supplementary Material for:

    Productive Replication of Ebola Virus Is Regulated by the c-Abl1 Tyrosine Kinase

    Mayra García, Arik Cooper, Wei Shi, William Bornmann, Ricardo Carrion, Daniel Kalman, Gary J. Nabel*

    *To whom correspondence should be addressed. E-mail: gnabel{at}nih.gov

    Published 29 February 2012, Sci. Transl. Med. 4, 123ra24 (2012)
    DOI: 10.1126/scitranslmed.3003500

    This PDF file includes:

    • Fig. S1. Transient transfection of NP, VP40, VP35, VP24, and GP gives rise to Ebola VLPs in 293T cells.
    • Fig. S2. GP levels in 293T cell lysates after drug treatment.
    • Fig. S3. Tyrosine phosphorylation of c-Abl1 and VP40 after expression of c-Abl1 in 293T cells.
    • Fig. S4. Analysis of VP40 modification in transfected 293T cells.
    • Fig. S5. Nedd4 and c-Abl1 interaction with VP40.
    • Fig. S6. Infection assays.

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