Fig 1.
NDV infection and membrane fusion induced syncytium formation and disturbed the spatial arrangement of cellular F-actin filaments.
(A) Representative images showing that oncolytic NDV infection altered the spatial arrangement of cellular F-actin filament in A549 cells. A549 cells were mock-infected and NDV-infected (Herts /33 strain) (MOI = 1), at the indicated timepoint. At 12, 18, 24, and 30 h.p.i., the coverslips were examined by IFA. After being fixed and permeabilized, the cells at a final concentration of 50 μg/ml fluorescent phalloidin conjugate the solution in PBS were inoculated for 40 min at room temperature. F-actin filament was labeled with phalloidin (green), nuclei with DAPI (blue), and NDV with the NP primary antibody (red). Arrows indicate the location of membrane fusion with nearby cells. Syncytia were marked with white dotted circles. Scale bars = 20 μm. (B) Representative images showing that F-HN co-expression altered the spatial arrangement of cellular F-actin filament in A549 cells. A549 cells were mock-transfected or co-transfected by both Flag-F and HA-HN plasmids at the indicated timepoints. At 6, 12, 18, 24, and 30 h.p.t., the coverslips were examined via IFA. Arrows indicate the location of membrane fusion with nearby cells. Syncytia were marked with dotted circles. Scale bars = 20 μm.
Fig 2.
NDV late replication and membrane fusion induced DNA double strand break (DSB) through ATM kinase activation.
(A) Virulent oncolytic NDV infection activated ATM-mediated DSB signaling in A549 cells. Western blot samples were prepared from A549 cells after virulent oncolytic NDV infection (Herts/33 strain) (MOI = 1) corresponding to the marked timepoints and analyzed in accordance with the procedures in the Materials and Methods section. The monomer ATM was marked with a black triangle. β-actin served as a loading control. (B) Growth curve of extracellular NDV in A549 cells. The extracellular NDV titers of culture supernatant were determined on DF-1 cells as TCID50 based on the Reed–Muench method. Error bars are indicated by red dashed lines. (C) NDV replication was required for ATM-mediated DSB signaling pathway activation. A549 cells were infected with virulent oncolytic NDV (Herts33/NDV strain) (MOI = 1) and UV-inactivated NDV. At 30 h.p.i., Western blot samples were collected and analyzed in accordance with the procedures in the Materials and Methods section. The monomer ATM was marked with a black triangle. (D) F and HN cooperated synergistically to activate ATM-mediated DSB in A549 cells. A549 cells were transfected with plasmids Flag-F, HA-HN, or both. Western blot samples corresponding to the marked timepoints were collected and analyzed in accordance with the procedures in the Materials and Methods section. (E) The cleavage site motif of F influenced the ATM-mediated DSB signaling pathway. In A549 cells, wild-type HN plasmids were co-transfected with wild-type F, F112G, F115G, F117L, F112G+115G, F112G+117L, F115G+117L, and F112G+115G+117L. At 36 h.p.t., the corresponding Western blot samples were collected and analyzed.
Fig 3.
Aggregation of γ-H2A.X punctuate foci in nuclei were induced by virulent NDV infection and membrane fusion.
(A) Representative images showing that virulent oncolytic NDV infection triggered the nuclear aggregation of γ-H2A.X in A549 cells. A549 cells were mock-infected, NDV-infected (MOI = 1), inoculated for 18, 24, and 30 h, UV exposed for 30 and 60 min, and treated with etoposide at a final concentration of 80 μm for 24 h. After treatment, the A549 cells were collected, fixed, and visualized by IFA. The UV and etoposide treatment groups were used as positive controls. γ-H2A.X (green); Nuclei (blue); NDV (red). Scale bars = 20 μm. (B) Statistical analysis of the nuclear aggregation punctate foci number of γH2AX in response to virulent oncolytic NDV infection in A549 cells. Numerical data were the average number of punctate foci in different microscope fields in at least three independent experiments. Significance was analyzed using two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. The UV and etoposide groups served as positive controls. (C) Representative images showing that F-HN co-expression triggered the nuclear aggregation of γ-H2A.X in A549 cells. A549 cells were mock-transfected or co-transfected with both Flag-F and HA-HN plasmids at the indicated timepoints. At 24 and 30 h.p.t., the coverslips were examined by IFA. γ-H2A.X (green); Nuclei (blue); NDV (red). Scale bars = 20 μm. (D) Statistical analysis of the nuclear aggregation punctate foci number of γ-H2A.X in response to F-HN co-expression.
Fig 4.
Aggregation of p53 binding protein 1 (53BP1) punctate foci in nuclei was induced by NDV infection and membrane fusion.
(A) Representative images showing that virulent oncolytic NDV infection triggered nuclear aggregation of endogenous 53BP1 in A549 cells. A549 cells were mock-infected, NDV-infected (MOI = 1), inoculated for 12, 18, 24, and 30 h, and treated with etoposide at a final concentration of 80 μm for 24 h. The etoposide treatment served as the positive control. 53BP1 (green); nuclei (blue); NDV (red). Arrows indicate 53BP1 localization in the nuclei. Scale bars = 20 μm. (B) Statistical analysis of the nuclear aggregation punctate foci number of endogenous 53BP1 in response to virulent oncolytic NDV infection in A549 cells. Numerical data were the average number of punctate foci in different microscope fields in at least three independent experiments. Significance was analyzed using two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Representative images showing that F-HN co-expression triggered nuclear aggregation of endogenous 53BP1 in A549 cells. A549 cells were mock transfected or co-transfected with both Flag-F and HA-HN plasmids at the indicated time. At 24 and 30 h.p.t., the coverslips were examined by IFA. 53BP1 (green); nuclei (blue); NDV (red). Arrows indicate 53BP1 localization in the nuclei. Scale bars = 20 μm. (D) Statistical analysis of the nuclear aggregation punctate foci number of 53BP1 in response to F-HN co-expression.
Fig 5.
NDV late infection and membrane fusion activated the ATM-Chk2 axis in A549 cells.
(A) Representative images showing that virulent oncolytic NDV infection triggered nuclear aggregation of phosphorylated Chk2 on Thr48 in A549 cells. A549 cells were mock-infected and NDV-infected (MOI = 1) for 12, 18, 24, and 30 h, and UV exposed for 30 and 60 min. After treatment, the A549 cells were collected, fixed, and visualized by IFA. The UV-exposure group served as the positive control. Phosphorylated Chk2 (green); nuclei (blue); NDV (red). Scale bars = 20 μm. (B) Statistical analysis of the nuclear aggregation punctate foci numbers of phosphorylated Chk2 on Thr 48 in response to virulent oncolytic NDV infection in A549 cells. Numerical data were the average number of punctate foci in different microscope fields in at least three independent experiments. Significance was analyzed using two-tailed Student’s t-test. The UV-exposure group served as the positive control. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Representative images showing that F-HN co-expression triggered nuclear aggregation of phosphorylated Chk2 on Thr 48 in A549 cells. A549 cells were mock transfected or co-transfected with both Flag-F and HA-HN plasmids at the indicated time. At 24 and 30 h.p.t., coverslips were examined by IFA. Phosphorylated Chk2 (green); nuclei (blue); NDV (red). Scale bars = 20 μm. (D) Statistical analysis of the nuclear aggregation punctate foci number of phosphorylated Chk2 on Thr 48 in response to F-HN co-expression.
Fig 6.
Inhibition of ATM kinase activity suppressed NDV replication and syncytium formation.
(A) The ATM-dependent signaling pathway was required for NDV intracellular replication in a dose-dependent manner. A549 cells were pretreated at a working concentration of KU55933 at 10, 20, and 40 μm for 1 h prior to NDV infection. A549 cells were mock-infected, NDV-infected (MOI = 5) for 24 h. At 24 h.p.i., Western blot samples were prepared and analyzed in accordance with the procedures in the Materials and Methods section. The monomer ATM was marked with a black triangle. β-actin served as a loading control. (B) The intensity band ratio of intracellular NDV NP to β-actin. Data were presented as means from three independent statistical experiments. The intensities of protein bands were quantified using Image J. Significance was analyzed using a one-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p <0.001. (C) The ATM-dependent signaling pathway was required for NDV extracellular replication in a dose-dependent manner. A549 cells were mock-infected, NDV-infected (MOI = 5) for 24 h. At 24 h.p.i., the cell-culture supernatants were collected and the extracellular NDV titers were determined on DF-1 cells as TCID50 based on the Reed-Muench method. Significance was analyzed using a two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D) The ATM-dependent signaling pathway was required for NDV intracellular replication in a time-dependent manner. A549 cells were pretreated at a working concentration of KU55933 at 30 μm for 1 h prior to NDV infection. A549 cells were mock-infected and NDV-infected (MOI = 5) for 18 and 24 h. At 18 and 24 h.p.i., Western blot samples were prepared and analyzed. (E) The intensity band ratio of intracellular NDV NP to β-actin. The intensities of protein bands were quantified using Image J. (F) The ATM-dependent signaling pathway was required for NDV extracellular replication in a time-dependent manner. A549 cells were mock-infected and NDV-infected (MOI = 5) for 18 h and 24 h. At 18 and 24 h.p.i., the cell-culture supernatants were collected and the extracellular NDV titers were determined. (G) Statistical analysis of the effect of ATM inhibitor KU55933 on syncytium formation in A549 cells. A549 cells were pretreated at a working concentration of KU55933 at 30 μm for 1 h prior to transfection and co-transfected with both Flag-F and HA-HN plasmids in the absence or presence of KU55933. Syncytium formation was observed on the basis of the number of nuclei. Numerical data were the average numbers of syncytia in different microscope fields in at least three independent experiments. Significance was analyzed using two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (H) Western blot analysis of the effect of ATM inhibitor KU55933 on syncytium formation in A549 cells. A549 cells were pretreated at a working concentration of KU55933 at 30 μm for 1 h prior to transfection and co-transfected with both Flag-F and HA-HN plasmids. Western-blot samples corresponding to the marked timepoints were collected and analyzed.
Fig 7.
Virulent NDV infection and F-HN co-expression activated the Mre11-Rad50-NBS1 (MRN) complex sensor in A549 cells.
(A) Virulent oncolytic NDV infection activated the MRN sensor of ATM-dependent DSB signaling in A549 cells. Western blot samples were prepared from A549 cells after virulent oncolytic NDV infection (Herts 33/NDV strain) (MOI = 1) corresponding to the marked timepoints, and analyzed in accordance with the procedures in the Materials and Methods section. β-actin served as a loading control. (B) NDV replication was essential for the activation of MRN sensor. A549 cells were inoculated with virulent oncolytic NDV (Herts33/ NDV) (MOI = 1) and UV-inactivated NDV. At 30 h.p.i., Western blot samples were collected and analyzed in accordance with the procedures in the Materials and Methods section. (C) F with HN cooperated synergistically to activate the MRN sensor of ATM-mediated DSB signaling in A549 cells. A549 cells were transfected with the plasmids Flag-F, HA-HN, or both. Western blot samples corresponding to the marked timepoints were collected and analyzed in accordance with the procedures in the Materials and Methods section. (D) Representative images showing that virulent oncolytic NDV infection and F-HN co-expression triggered nuclear aggregation of phosphorylated NBS1 on Ser343 in A549 cells. A549 cells were mock-infected, or NDV-infected (MOI = 1) for 18 and 24 h, UV-exposed for 30 min, and treated with etoposide at a final concentration of 80 μm for 24 h. The UV and etoposide treatment groups served as the positive controls. A549 cells were co-transfected with both Flag-F and HA-HN plasmids at the indicated timepoints. At 18 and 24 h.p.t., coverslips were examined by IFA. p-NBS1 (green); nuclei (blue); NDV (red). Scale bars = 20 μm. (E) MRN complex activity was required for intracellular NDV replication in a time-dependent manner. A549 cells were pretreated with a working concentration of Mirin at 100 μm for 1 h prior to NDV infection. A549 cell were mock-infected and NDV-infected (MOI = 5) for 18 and 24 h. At 18 and 24 h.p.i., Western blot samples were prepared and analyzed in accordance with the procedures in the Materials and Methods section. The monomer ATM was marked with a black triangle. β-actin served as a loading control. (F) MRN complex activity was required for intracellular NDV replication in a dose-dependent manner. A549 cells were pretreated at a working concentration of Mirin at 50, 75, and 100 μm for 1 h prior to NDV infection and mock-infected and NDV-infected (MOI = 5) for 24 h. (G) MRN complex activity was required for NDV extracellular replication in a time-dependent manner. A549 cells were mock-infected, NDV-infected (MOI = 5) for 24 h. At 24 h.p.i., the cell culture supernatants were collected and the extracellular NDV titers were determined on DF-1 cells as TCID50 based on the Reed-Muench method. Significance was analyzed using a two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (H) MRN complex activity was required for NDV extracellular replication in a dose-dependent manner. A549 cells were mock-infected and NDV-infected (MOI = 5) for 18 h and 24 h. At 18 and 24 h.p.i., the cell culture supernatants were collected and the extracellular NDV titers were determined on DF-1 cells. (I) Statistical analysis of the effect of the MRN inhibitor Mirin on syncytium formation in A549 cells. A549 cells were pretreated at a working concentration of Mirin at 100 μm for 1 h prior to co-transfection, and then co-transfected with both Flag-F and HA-HN plasmids in the absence or presence of Mirin. Syncytium formation was observed on the number of nuclei. Numerical data were the average number of syncytia in different microscope fields in at least three independent experiments. Significance was analyzed using a two-tailed Student’s t-test. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (J) Western blot analysis of the effect of Mirin on syncytium formation in A549 cells. A549 cells were pretreated at a working concentration of Mirin at 100 μm for 1 h prior to transfection, and then transfected with both Flag-F and HA-HN plasmids. Western blot samples corresponding to the marked timepoints were collected and analyzed in accordance with the procedures in the Materials and Methods section. β-actin served as a loading control.
Fig 8.
Schematic of the ATM-mediated DNA double-strand breaks (DSBs) triggered by virulent NDV infection and membrane fusion.
Virulent oncolytic NDV infects target tumor cells through viral HN-mediated attachment. It then triggers F protein conformational changes and releases fusion peptides to fuse the viral and cellular membranes [29, 30]. Meanwhile, after transfection, the inactive precursor of F protein (F0) is proteolytically cleaved by the host-cell protease at the cleavage site and forms a biologically active protein comprising the disulfide-linked F1+F2 heterodimer [29–31]. After a series of proteolytic processing and conformational changes, the fusogenically active F1 protein and HN form an F+HN complex via the interaction between a stalk and an HRB domain [29, 30]. Once triggered, HRB completely refolds around HRA, thereby forming stable 6 HB and a fusion pore [30] and consequently inducing membrane fusion and syncytium formation. The virulent oncolytic NDV infection and F-HN co-expression induce DSBs in tumor cells, leading to the recruitment of the MRN complex and the separation of the dimeric, inactive form of ATM into a monomeric, phosphorylated form, which is further activated by the MRN complex through directly binding ATM at DSB sites [10]. ATM activation by DSBs occurs in two steps [2, 14]. Activated ATM initially triggers a rapid cascade of phosphorylation events involving H2AX, 53BP1, and Chk2. Mediators and signal transducers are then recruited to amplify the signal, which finally reaches effector proteins that trigger the cellular response to DNA damage [1, 6, 18]. Eventually, ATM-mediated DSBs facilitate oncolytic NDV replication and promote syncytium formation. The solid arrows indicate the stimulation and protein requirements for accumulation.