IFN-β mRNA expression in H358 cells and knockdown clones after MV infection or transfection with MV leader RNA. (A) The parental H358 cells, the clone expressing a nontargeting luciferase siRNA (Luc), MDA5 knockdown clones (MDA5 KD: M1, M2 and M3) and RIG-I knockdown clones (RIG-I KD: R1, R2 and R3) were infected with wt MV at a multiplicity of infection (MOI) of 0.5. H358 cells were also infected with UV-inactivated wt MV (UV), treated with an FBP before wt MV infection (FBP), or mock infected (mock). Total RNAs were extracted from the cells at 48 h after infection, and the IFN-β mRNA levels were quantified by RT-qPCR. (B) H358 cells, the clone expressing a luciferase siRNA, and MDA5 and RIG-I knockdown clones were transfected with in vitro-transcribed MV leader RNA. H358 cells were also mock transfected (mock). Total RNAs were extracted from the cells at 6 h posttransfection, and the IFN-β mRNA levels were quantified by RT-qPCR. All data were normalized by the corresponding β-actin mRNA levels in the respective cells. The mean value in the parental H358 cells infected with wt MV (A) or transfected with MV leader RNA (B) was set to 100%. The data represent the means ± the standard deviations of triplicate samples. *, P < 0.05; **, P < 0.01 (significant differences based on a t test).

Inductions of IFN-β mRNA and ISG56 and dimerization of IRF3 in MVΔV-infected cells. (A) The parental H358 cells, MDA5 knockdown clones (MDA5 KD: M1, M2, and M3) and RIG-I knockdown clones (RIG-I KD: R1, R2, and R3) were infected with MVΔV at an MOI of 0.5. H358 cells were also infected with UV-inactivated MVΔV (UV), treated with an FBP before MVΔV infection (FBP), or mock infected (mock). Total RNAs were extracted from the cells at 24 h after infection, and the IFN-β mRNA levels were quantified by RT-qPCR. The data were normalized by the β-actin mRNA levels, and the mean value in MVΔV-infected H358 cells was set to 100%. *, P < 0.05 (significant difference based on a t test). N.S., not significant. (B and C) H358 cells and MDA5 and RIG-I knockdown clones were infected with MVΔV at an MOI of 0.5. H358 cells were also mock infected. At 24 h after infection, the monomeric and dimeric forms of IRF3 in the cells were examined by native PAGE and Western blot analysis (B), and the ISG56 levels in the cells were detected by SDS-PAGE and Western blot analysis (C). β-Actin was examined as an internal control.

Expression of MDA5 and RIG-I in H358 cells and knockdown clones. The expression levels of MDA5 and RIG-I in the parental H358 cells, MDA5 knockdown clones (MDA5 KD: M1, M2, and M3), and RIG-I knockdown clones (RIG-I KD: R1, R2, and R3) were examined by Western blot analysis at 24 h after treatment with IFN-αA/D. β-actin was evaluated as an internal control. Indicated blow each band are the signal intensities of MDA5 and RIG-I, which were normalized by that of β-actin. The value of the parental H358 cells was set to 100%.

Generation and properties of the V protein-deficient MV (MVΔV). (A) Diagram of the MV genome indicating the locations of the introduced mutations to generate MVΔV. Underlines indicate the mutated nucleotides. The predicted amino acids are shown below the trinucleotide codons. nt, nucleotide. (B) Protein synthesis in MV-infected cells. B95a cells were infected with wt MV or MVΔV at an MOI of 0.01. At 36 h after infection, viral proteins (N, P, V, and C) in the infected cells were detected by Western blot analysis. (C) IFN-β mRNA levels in H358 cells infected with wt MV or MVΔV at an MOI of 0.5. At 12, 24, and 48 h after infection, total RNA was extracted from the MV-infected cells, and the IFN-β mRNA levels were quantified by RT-qPCR. The data were normalized by the β-actin mRNA levels, and the mean value in wt MV-infected cells at 48 h after infection was set to 100%. The data represent the means ± the standard deviations of triplicate samples. The IFN-β mRNA level in uninfected cells is also shown (0 h). **, P < 0.01 (significant difference based on a t test). (D) Induction of ISG56 in MV-infected cells. H358 cells were mock infected or infected with wt MV or MVΔV at an MOI of 0.5. At 24 h after infection, the ISG56 levels in the infected cells were detected by Western blot analysis. β-Actin was analyzed as an internal control. (E) N mRNA levels in H358 cells infected with wt MV or MVΔV at an MOI of 0.5. At 12, 24, and 48 h after infection, total RNA was extracted from MV-infected cells, and the N mRNA levels were quantified by RT-qPCR. The data were normalized by the β-actin mRNA levels, and the mean value in wt MV-infected cells at 48 h after infection was set to 100%. *, P < 0.05 (significant difference based on a t test).

Growth kinetics of wt MV and MVΔV in H358 cells and knockdown clones. The parental H358 cells, an MDA5 knockdown clone (M3) and an RIG-I knockdown clone (R1) were infected with wt MV or MVΔV at an MOI of 0.5. At 24 and 48 h after infection, the cells were harvested, together with the culture media, and the virus titers were determined by plaque assays. The data represent the means ± the standard deviations of triplicate samples. *, P < 0.05; **, P < 0.01 (significant differences based on a t test).