Figure 3.

dsRNA length-dependent oligomerization is a mechanism for the high immunostimulatory activity of SeV DI RNA. (A) 5 ng and five-fold dilutions of WT, 46- and 25-bp stem RNAs were transfected or not into 293T-IFNβ-FF-Luc cells and 24 h later IFNβ promoter-driven luciferase activity was measured by a luciferase assay. (B) 500 fmol and three-fold dilutions of Biotin-UTP-labelled WT, 46- and 25-bp stem RNAs were immobilized onto NeutrAvidin-coated wells and incubated with lysates from HA-RIG-I expressing 293T cells. The levels of bound RIG-I were determined by measuring the absorbance/HRP activity of HA-HRP antibody. (C) 100 and 50 fmol of RNAs was incubated with 0.5 μg of RIG-I in the presence of 0.5 mM ATP and 2.5 mM Mg²⁺ at 37 °C for 25 min. Released phosphates were measured by a colorimetric ATPase assay at absorbance 620 nm. (D) Lysates from cells expressing eYFP-RIG-I or HA-RIG-I were mixed and incubated with 1.25 and 5 pmol of WT, 46- and 25-bp stem RNA or no RNA, eYFP-RIG-I was immunoprecipitated with GFP antibody and eYFP-RIG-I and HA-RIG-I levels were assessed by SDS–PAGE and immunoblotting with GFP or HA antibodies. Levels of input HA-RIG-I in WCL were determined by SDS–PAGE and immunoblotting with HA antibody. (E) 1 μg of RIG-I was incubated with 1.25 or 0.625 pmol of WT, 46- and 25-bp stem RNAs or no RNA in the presence of 0.5 mM ATP and 2.5 mM Mg²⁺ for 25 min at 37 °C and RIG-I complexes were analysed by NativePAGE and immunoblotting. Data are representative of at least three independent experiments and error bars indicate mean±s.d. Log₁₀ in Fig 3A refers to the scale on the x-axis. DI, defective interfering; dsRNA, double-stranded RNA; GFP, green fluorescent protein; HRP, horseradish peroxidase; SeV, Sendai virus; WCL, whole cell lysates; WT, wild type.

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