Figure 3. Viral RNA is required for formation of functional SG to activate RIG-I/IPS-1 signaling pathway.

(A) 293T cells were transfected with empty vector (empty) or the HA-PKR expression vector (HA-PKR) for 24 h, or treated with NaAsO₂ for 1 h and stained with anti-RIG-I, anti-HA (PKR) and anti-TIAR antibodies and DAPI. The zoomed images correspond to the boxed regions. (B) 293T cells were transfected with empty vector (empty), or the HA-PKR expression vector for 48 h, or treated with NaAsO₂ for 1 h, or infected with IAVÄNS1 for 12 h. Relative mRNA levels of endogenous IFN-â gene were determined by quantitative PCR (qPCR). Data are represented as the mean standard ± error of the mean (SEM). (C and D) HeLa cells were mock-treated or infected with IAVÄNS1 for 12 h. Viral RNA (vRNA) was detected by the FISH method using an RNA probe complementary to the segment 1 of the IAV, and NP (C) and RIG-I (D) were detected using anti-NP and anti-RIG-I antibodies (97.1%, and 98.2% colocalization of vRNA with NP and RIG-I, respectively). TO-PRO-3 was used for staining of nuclear DNA (DNA). (E) HeLa cell lines stably expressing FLAG-tagged IPS-1 were mock-treated or infected with IAV or IAVÄNS1 for 10 h. The cells were stained with anti-PMP70, anti-FLAG, and anti-TIAR antibodies. The white arrowheads indicated the contacts between FLAG-IPS-1 and TIAR. 67.8% of IAVÄNS1 infected cells exhibited contacts, whereas IAV infected cells hardly exhibited the contact (2.7%). The zoomed images of PMP70 (Green) and FLAG (Red), PMP70 (Green) and TIAR (Red), and FLAG (Green) and TIAR (Red) in IAVÄNS1-infected cells were shown in the bottom panel.