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. Author manuscript; available in PMC: 2016 Mar 11.
Published in final edited form as: Cell Host Microbe. 2015 Feb 19;17(3):309–319. doi: 10.1016/j.chom.2015.01.005

Fig. 3. Adaptive mutations in PB2 influence the activation of RIG-I by FLUAV nucleocapsids.

Fig. 3

(A) RIG-I activation by viruses with different PB2-627 signatures. Cells were infected with strains of A/quail/Shantou/2061/00 (H9N2), A/Thai/KAN-1/04 (H5N1), A/Hamburg/05/2009 (pH1N1), or A/WSN/33 (H1N1) containing avian-signature E or mammalian-signature K at PB2-627. Infections, CHX/LMB treatment and RIG-I conformational switch testing were performed as described for 1 and 2. (B) Quantification of virus RNAs by RT-qPCR for genomic segment 7. Input represents RNA amounts harvested after the 1-h infection period. (C) Co-sedimentation assay. Lysates of cells infected with PB2 variants of A/WSN/33 (H1N1) using our standard 1 h-protocol were separated by a CsCl gradient and analyzed by immunoblotting. (D) RIG-I-dependent IFN induction by incoming nucleocapsids. A549 cells were transfected with the indicated siRNAs or a negative control siRNA (CTRL). A549 cells siRNA-depleted of RIG-I or MDA5 were pretreated with CHX and LMB, and infected with FLUAV strains (MOI 1) for 16 h. IFN-β mRNA levels were determined by real-time RTPCR. See also Figures S3A-S3H.