(A) Activity of A/WSN/33-based minireplicon systems containing PB2-627K, −627E, or no PB2 (−) in HEK293 wt, ΔRIG-I or ΔMDA5 cells. (B) Reporter activities in HEK293 cells producing VLPs containing nucleocapsids with the indicated PB2 signatures. (C) Reporter activities in wt and deletion cells infected with VLPs. Cells had been pretransfected with PB1, PA, NP, and matching PB2. (D) Multicycle virus kinetics. 293 wt or ΔRIG-I cells were infected with the indicated PB2 variants of strain A/Thai/KAN-1/04 (H5N1) at an MOI 0.0001. Virus yields were determined 24 h later by plaque assay. In all cases, mean and SDs from 3 independent experiments are shown. (E) Single-cycle virus kinetics. Cells were infected at an MOI of 1 and monitored for NP expression over time. See also Figures S4A-S4D.