Figure 5.

The binding activity of pdm/09 polymerase acid protein (PA) to interferon regulatory factor 3 (IRF3) is dependent on Asp108. (A) 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or empty vector, together with 0.3 µg of interferon-β (IFN-β)-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with Sendai virus (SEV) or were left uninfected for 8 h and were then lysed for the luciferase assay. (B) A549 cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector. After 24 h, cells were infected with SEV or were left uninfected for 8 h. The cells were harvested, and total RNA was extracted for detection of IFN-β, CXCL-10, ISG-15, and ISG-56 expression levels by real-time q-PCR. (C) 293T cells in 6-well plates were transfected with 2 µg of Flag-tagged pdm/09 PA, pdm/09 PA-D108A mutant or an empty vector, and HA-tagged IRF3. After 24 h, cells were lysed and precipitated with anti-Flag antibody. The cell lysates and immunoprecipitates (IPs) were analyzed by Western blotting using anti-Flag and anti-HA antibodies. 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).