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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. 6. Effect of the PB2 627 signature on protein-protein interactions.

Fig. 6

(A) NP immunoprecipitation. Cells were CHX / LMB treated and infected with A/WSN/33 strains carrying PB2-627K or 627E as described for 1A. Lysates were immunoprecipitated 1 h later with anti-NP and immunoblotted as indicated. Normalized quantifications of the immunoprecipitated proteins are shown below. Note that amounts of viral input proteins are too low to be detected in the total extracts. (B) RIG-I immunoprecipitations from infected cells. HEK293 cells were infected with A/WSN/33 PB2 variants as described for 1A. Immunoprecipations with anti-RIG-I and immunoblotting were performed as indicated for 2B. (C) RIG-I immunoprecipitations of recombinant nucleocapsids. HEK293 cells were transfected with A/WSN/33 NP combined with GFP or the PB2 variants (left panel), or with all A/WSN/33 minireplicon plasmids (right panel). Immunoprecipations with anti-RIG-I were performed as indicated for (B). (D) Polymerase destabilization. A549 cells were infected with PB2 variants of strain A/WSN/33 (MOI 1), treated with peptides Borna-X-Tat (CTRL) or PB11-15 T6Y-Tat (PB1-T6Y), and tested for RIG-I conformational switch 1 h post-infection. See also Figures S6A-S6C.