Figure 3.

Activation of the IRF3 pathway by candidate oncolytic VACV

(A and B) Deletion of viral genes interfering in the IRF3 pathway leads to IRF3 phosphorylation in human cells. HeLa (A) and THP-1 (B) cells were infected at an MOI of 10 and, 5 h after infection, cells were lysed and western blot analysis was performed using a monoclonal antibody against p-IRF3. The non-replicating VACV MVA (modified vaccinia virus Ankara) served as a positive control and GAPDH-specific immunoblotting served as a loading control. (C and D) Detection of IFN-β mRNA by RT-PCR in human cells. HeLa (C) and THP-1 (D) cells were infected at an MOI of 5. At 6 h after infection, total RNA was obtained and mRNAs of indicated genes were amplified by RT-PCR. The VACV E3L mRNA was used as an infection control, and GAPDH mRNA was used as a loading control. (E) Relative expression of IFN-β measured by qRT-PCR. Indicated cells were infected with WR/TK− or WR/TK−/3Δ, and total RNA was obtained as indicated in (C) and (D). mRNA expression levels of IFN-β were determined via qRT-PCR and normalized to that of the GAPDH gene by the 2^−ΔΔCt. ∗∗p < 0.01, ∗∗∗p < 0.001.