Figure 4.

Interferon-β (IFN-β) induction was suppressed by the N-terminus of pdm/09 polymerase acid protein (PA). (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 N-terminal PA fragment (PAN), pdm/09 C-terminal PA fragment (PAC), or an empty vector, together with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV or were left uninfected for 8 h and then they were lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 1 µg of Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV for 8 h and subsequently harvested, and total RNA was then extracted for detection of IFN-β and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 12-well plates were transfected with HA-tagged PAN and PAC in increasing quantities and co-transfected with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV for 8 h and then lysed for use in the luciferase assay. (D) 293T cells in 6-well plates were transfected with 2 µg of HA-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or an empty vector, and Flag-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and IPs were analyzed by Western blotting using anti-Flag and anti-HA antibodies. (E) 293T cells were seeded onto coverslips and placed into 12-well plates and were transfected with Flag-tagged pdm/09 PA, pdm/09 PAN, pdm/09 PAC, or empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h and were fixed for IFA, with endogenous IRF3 (red) and nuclei (blue) shown with anti-IRF3 antibody and DAPI via confocal microscopy. The bars represent the SEs of the means, based on three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 [as determined by Student’s t-test (A,B) or by one-way ANOVA (C)].