(A–C) HeLa cells were mock-treated or infected with IAV (A) or IAVÄNS1 (B) for the indicated period, fixed, stained, and analyzed with confocal microscopy. The cells were stained with anti-RIG-I (RIG-I), anti-IAV nucleocapsid protein (NP), and anti-TIAR (TIAR) antibodies. Nuclei were stained with DAPI. At 9 h and 12 h after infection, the percentage of speckle-like distribution of RIG-I was 0.5% and 0.0% in IAV-infected cells, and 62.4% and 83.6% in IAVÄNS1-infected cells, respectively. The zoomed images correspond to the boxed region in each panel. The cells at 9 h post infection were stained with anti-RIG-I and anti-IRF-3 antibodies (C). (D and E) HeLa cells were infected with IAVÄNS1 for 9 h, and stained with anti-G3BP (G3BP), together with anti-RIG-I (RIG-I) (D) or anti-eIF3 (eIF3) (E). The zoomed images correspond to the boxed regions. (F) HeLa cells were mock-treated or infected with IAV or IAVÄNS1 for 12 h. Cell extracts were prepared and immunoprecipitated with anti-RIG-I antibody. The precipitates were analyzed by immunoblotting (IP:RIG-I) using antibody against G3BP, HuR, TIAR and RIG-I. Input: 1/50 of the extracts used for immunoprecipitation were analyzed similarly by immunoblotting.