IRF3, but not TBK1 and IKKε, is sequestered into viral inclusion bodies (IBs) upon EBOV trVLPs infection.

(A), HepG2 cells infected with the EBOV trVLPs were immunostained with anti-IRF3 (red) and anti-VP35 (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. White arrows: IRF3 in IBs. (B), The left panel shows a magnified image of the IBs boxed in the merged panel of (A). The graphs (right panel) show the fluorescent intensity profiles along the indicated white lines drawn across one or more IBs. (C and E), HepG2 cells infected with the EBOV trVLPs were immunostained with anti-TBK1 (red in (C)) or anti-IKKε (red in (E)) and anti-VP35 (green in (C and E)) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. (D and F), The left panel shows a magnified image of the IBs boxed in the merged panel shown in (C) and (E). The graphs (right panel) show the fluorescent intensity profiles along the indicated white lines drawn across one or more IBs.

EBOV trVLPs induce the recruitment of IRF3 into intracytoplasmic IBs.

(A), HepG2 cells were infected with EBOV trVLPs. At the indicated time points after infection, cells were fixed and immunostained with anti-IRF3 (red) and anti-VP35 (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. The data from two independent replicates are presented. (B), The percentage of IRF3 distribution in IBs at different time points in cells infected with EBOV trVLPs (A) was analyzed using the R programming language. The intensity of IRF3 in 8 cells from two independent assays is presented as the mean ± SEM (n=8; ***PL<L0.001). (C), The left panel shows a magnified image of the IBs boxed in the merged panel shown in (A). The graphs (right panel) show the fluorescent intensity profiles along the indicated white lines drawn across one or more IBs. (D), IRF3 levels in HepG2 cells infected with EBOV trVLPs were analyzed by immunoblotting with an anti-IRF3 antibody at the indicated hours post infection (hpi). (E), The IRF3 intensity in cells infected with or without EBOV trVLPs for 48 h (the lower panel of (A)) was analyzed using ImageJ software. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The intensity of IRF3 in 5 cells from two independent assays is presented as the mean ± SEM (n = 5; ns, not significant).

EBOV trVLPs inhibit IRF3 activation.

(A), HepG2 cells were infected with or without the EBOV trVLPs. Thirty-six hours after infection, the cells were treated with or without 5 μg/ml poly(I:C) for 12 h and then subjected to in situ PLA with anti-TBK1 and anti-IRF3 antibodies and immunostaining with an anti-NP antibody (green). Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Arrows: white arrows indicate TBK1-IRF3 complexes in trVLPs-infected cells, and yellow and green arrows indicate TBK1-IRF3 complexes in uninfected and infected cells with small IBs, respectively. Scale bar, 10 μm. (B), The signal for the PLA complex in each cell in (A) was counted from at least 12 cells and is presented as the mean ± SEM. (C), Lysates of HEK293 cells cotransfected with or without the EBOV minigenome (p0) and the indicated plasmids were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. (D), HEK293 cells were cotransfected with or without the EBOV minigenome (p0) and Myc-IRF3 plasmids. Thirty-six hours after transfection, the cells were infected with SeV at an MOI of 2 for 12 h, and the phosphorylation of IRF3 was analyzed by immunoblotting with an anti-IRF3-S396 antibody.

EBOV trVLPs inhibit nuclear translocation of IRF3.

(A), HepG2 cells were infected with or without the EBOV trVLPs for 36 h, and the cells were infected with or without SeV at an MOI of 2 for another 12 h. The cells were then fixed and immunostained with anti-IRF3 (red) and anti-VP35 (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. (B), The percentage of IRF3 nuclear distribution in (A) was analyzed using ImageJ software. The ratio of IRF3 distribution in ten cells from two independent assays is presented as the mean ± SEM (ns, not significant, ***P < 0.001). (C), HepG2 cells infected with live EBOV (MOI=10) for 72 h were immunostained with anti-IRF3 (red) and anti-NP (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm.

EBOV NP and VP35 play an important role in sequestering IRF3 into IBs.

(A), HepG2 cells were transfected with the indicated plasmids for 36 h, and the cells were treated with 5 μg/ml poly(I:C) for another 12 h. Then, the cells were fixed and immunostained with anti-IRF3 (red) and anti-NP (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. (B), The nuclear/cytoplasmic distribution of IRF3 in (A) was analyzed by ImageJ software. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The ratio of IRF3 distribution in at least 5 cells from two independent assays is presented as the mean ± SEM (n=5; **P < 0.01, ***P < 0.001).

EBOV trVLPs recruit IRF3 into viral IBs via STING.

(A), Lysates of HEK293 cells transfected with the indicated plasmids were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. (B) HepG2 cells were transfected with the EBOV minigenome (p0). Forty-eight hours after infection, the cells were fixed and immunostained with anti-STING (red) and anti-VP35 (green) antibodies. White arrows: STING in IBs. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. (C) The left panel shows a magnified image of the IBs boxed in the merged panel of (B). The graphs (right panel) show the fluorescent intensity profiles along the indicated white lines drawn across one or more IBs. (D) HepG2 cells were infected with the EBOV trVLPs. At the indicated hours post infection (hpi), cells were fixed and immunostained with anti-STING (red) and anti-VP35 (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. The data from two independent replicates are presented. (E) The left panel shows a magnified image of the IBs boxed in the merged panel of (D). The graphs (right panel) show fluorescent intensity profiles along the indicated white lines drawn across one or more IBs. (F and G) HepG2 cells were transfected with STING siRNA (STING si) or scrambled siRNA (Scr si) for 6 h. The cells were then infected with the EBOV trVLPs for 36 h and then immunostained with Fluor 488-conjugated-anti-IRF3 (green), anti-VP35 (red) and anti-STING (purple) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. The silencing efficiency of STING siRNA was determined by immunoblotting (G).

The hijacking of IRF3 by viral IBs inhibits IFN-β production.

(A), HEK293 cells were transfected with the indicated plasmids for 24 h, and the cells were infected with or without SeV at an MOI of 2 for another 12 h. The mRNA level of IFN-β was quantified by qRT-PCR. Differences between the two groups were evaluated by a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (*P☐<☐0.05, **P☐<☐0.01, ***P☐<☐0.001). (B), HEK293 cells were cotransfected with the firefly luciferase reporter plasmid pGL3-IFN-β-Luc, the Renilla luciferase control plasmid pRL-TK, and viral protein expression plasmids (0.0625 μg of pCAGGS-NP, 0.0625 μg of pCAGGS-VP35, 0.0375 μg of pCAGGS-VP30 and 0.5 μg of pCAGGS-L) for 24 h, and the cells were infected with or without SeV at an MOI of 2 for another 12 h. The luciferase activities were then analyzed. The data were analyzed to determine the fold induction by normalizing the firefly luciferase activity to the Renilla luciferase activity. Empty plasmid without SeV infection was used as a control, and the corresponding data point was set to 100%. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (ns, not significant, *P☐<☐0.05, ***P☐<☐0.001). (C), Wild-type (WT) and IRF3-depleted (IRF3-/-) HeLa cells were transfected with or without pCASSG-NP, pCASSG-VP35, pCASSG-VP30 and pCASSG-L plasmids for 36 h and then treated with or without 5 μg/ml poly(I:C) for 12 h. The mRNA level of IFN-β was quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (ns, not significant, *P☐<☐0.05). (D-F), Wild-type (WT) and IRF3-depleted (IRF3-/-) HeLa cells were transfected with or without pCAGGS-VP35 or pCASSG-NP, pCASSG-VP35, pCASSG-VP30 and pCASSG-L plasmids for 36 h, and the cells were infected with or without SeV at an MOI of 5 for another 12 h. The mRNA level of CXCL10 (D), ISG15 (E) and ISG56 (F) were quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (*P☐<☐0.05, **P☐<☐0.01, ***P☐<☐0.001). (G), Wild-type (WT) and IRF3 knockout (IRF3-/-) HeLa cells were transfected with the EBOV minigenome (p0), pGL3-promoter and Myc-vector, Myc-IRF3 or Myc-IRF3/5D plasmids for 96 h. The amounts of trVLPs were determined by a luciferase activity assay (left panel). Differences between the two groups were evaluated by a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (ns, not significant, ***P☐<☐0.001). (H). Wild-type (WT) and IRF3-knockout (IRF3-/-) HeLa cells were infected with live EBOV (MOI = 0.1). The cell culture supernatants were collected on the indicated days post infection (dpi), and the viral titers were quantified as TCID50 by a plaque assay. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The data are presented as the means☐±☐SEM (ns, not significant).

Model of the molecular mechanism by which EBOV hijacks IRF3 into viral IBs through VP35-STING to comprehensively disrupt IFN-I production.

VP35 sequesters IRF3 to EBOV IBs, which in turn spatially segregates IRF3 from TBK1 and IKKε, blocks RLR signaling and inhibits IFN-I production.

Transmission electron microscopy and immunofluorescence detection of EBOV trVLPs and IBs.

(A), HepG2 cells infected with or without EBOV trVLPs were fixed and observed with a HITACHI H-7650 transmission electron microscope at an accelerating voltage of 80 kV. The IBs and viral particles (right panel) are marked with bold arrows and regular arrows, respectively. Scale bar, 500 nm. (B), HepG2 cells infected with the EBOV trVLPs were immunostained with anti-NP (red) and anti-VP35 (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm.

Neither VP35 nor NP interacts directly with IRF3 in cells.

(A and B) Lysates of HEK293 cells transfected with the indicated plasmids were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. The data from two independent replicates are presented.

Neither VP24 nor VP30 plays an important role in sequestering IRF3 into IBs.

(A), HepG2 cells were transfected with the indicated plasmids for 36 h, and the cells were infected with SeV at an MOI of 2 for another 12 h. The cells were then fixed and immunostained with anti-IRF3 (red) and anti-NP (green) antibodies. Nuclei were stained with DAPI (blue), and images were obtained using a Zeiss LSM 800 Meta confocal microscope. Scale bar, 10 μm. (B), The nuclear/cytoplasmic distribution of IRF3 in (A) was analyzed using ImageJ software. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The ratio of IRF3 distribution in at least 5 cells from two independent assays is presented as the mean ± SEM (n = 5; ns, not significant, ***P < 0.001).

EBOV trVLPs recruit STING into viral IBs.

The percentage of STING distribution in IBs at different time points in cells infected with EBOV trVLPs in Fig. 6D was analyzed with R programming language. The intensity of STING in 6 cells from two independent assays is presented as the mean ± SEM (n=6; ***P☐<☐0.001).

The expression of IRF3 and its mutants were detected by immunoblotting.

(A) Lysates of WT and IRF3-/- HeLa cells were analyzed by immunoblotting with an anti-IRF3 antibody. (B) Lysates of WT and IRF3-/- HeLa cells transfected with Myc-vector, Myc-IRF3 or Myc-IRF3/5D were analyzed by immunoblotting with the indicated antibodies.