Figure 7. The hijacking of interferon regulatory factor 3 (IRF3) by viral inclusion bodies (IBs) inhibits IFN-β production.

(A) HEK293 cells were transfected with the indicated plasmids for 24 hr, and the cells were infected with or without Sendai virus (SeV) at an MOI of 2 for another 12 hr. The mRNA level of IFN-β was quantified by quantitative RT-PCR (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  ± standard error of the mean (SEM; n=3; *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 hr, and the cells were infected with or without SeV at an MOI of 2 for another 12 hr. 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 (n=3; 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 hr and then treated with or without 5 μg/ml poly(I:C) for 12 hr. 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 (n=3; 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 hr, and the cells were infected with or without SeV at an MOI of 5 for another 12 hr. The mRNA level of CXCL10 (D), ISG15 (E), and ISG56 (F) 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 (n=3; *p < 0.05, **p < 0.01, ***p < 0.001). (G) Wild-type (WT) and IRF3-knockout (IRF3^−/−) HeLa cells were transfected with the Ebola virus (EBOV) minigenome (p0), pGL3-promoter and Myc-vector, Myc-IRF3 or Myc-IRF3/5D plasmids for 96 hr. The amounts of transcription- and replication-competent virus-like particles (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 (n=3; 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 (n=3; ns, not significant).

Figure 7—figure supplement 1. The expression of interferon regulatory factor 3 (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 HeLa cells transfected with Myc-vector, Myc-IRF3 or Myc-IRF3/5D were analyzed by immunoblotting with the indicated antibodies.