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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. 2. RIG-I interacts with incoming influenza virus nucleocapsids and is activated in a 5’ppp-dsRNA-dependent manner.

Fig. 2

(A to C) CHX / LMB-treated A549 cells were infected with A/PR/8/34 (MOI 1) for 1 h. (A) Cells analyzed for RIG-I and FLUAV by 3D GSD superresolution immunofluorescence microscopy. Scale bar, 1 μm. Insets are digitally magnified and shown below the main image (taken from one individual cell). (B) Co-immunoprecipitation. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against p21 (negative control), RIG-I, or FLUAV NP, and analyzed by immunoblot. Input control: 2% of the lysate. Asterisks (*) indicate unspecific bands. (C) Co-sedimentation assay. Cell lysates were separated by a discontinuous CsCl gradient (2% lysate as input control), and fractions analyzed by immunoblotting. (D) Conditions for activation of RIG-I by nucleocapsids in vitro. Dialyzed lysate of RIG-I-expressing S2 cells was mixed with nucleocapsids of strain A/PR/8/34 (RNPs) or a control preparation (CTRL) and supplemented with 1 mM ATP. The nucleocapsids had either been pretreated with 5 μg RNase A (A), 1 U RNase III (III), or 2 U Shrimp Alkaline Phosphatase (SAP), or left untreated (−), for 1 h at 37°C. RIG-I conformational switch was assayed after 1 h of nucleocapsid co-incubation at 37°C. See also Figures S2A-S2K.