Figure 5: dsRNA binding ability of TRBP but not that of PACT influences RIG-I induced IFN production. A. Schematic of dsRNA-binding mutations in M1 and M2 domains of PACT and TRBP.

Residues mutated from lysine to alanine based on previously established research on residues important for dsRNA-binding. B. dsRNA-binding assay. dsRNA-binding ability of wt and mutant PACT and TRBP was measured by polyI:C-agarose binding assay with in vitro translated ³⁵S-labeled proteins. T, total input; B, protein bound to polyI:C agarose. C. Quantification of data in 5 B. Radioactivity in both total input and bound protein bands was quantified, and % bound was calculated by (bound protein/total input) *100. The error bars represent standard deviation calculated from 3 experiments. The p-values are as indicated. D. dsRNA-binding ability of PACT does not affect RIG-I signaling. HEK293Ts were transfected with IFN-β-Luc reporter, RIG-I expression construct, and 50ng of expression construct for either wt PACT or dsRNA-binding mutants K84A or K177A as indicated, then treated with polyI:C 24 h after transfection. pRLnull plasmid was co-transfected for normalization of transfections. Cell extracts were assayed for dual luciferase activity 8 h after treatments. The p-values calculated from 2 independent experiments with each sample in triplicates are indicated. E. dsRNA-binding ability of TRBP is essential for inhibition of RIG-I signaling. HEK293Ts were transfected with IFN-β-Luc reporter, RIG-I expression construct, and 50ng of expression construct of either wt TRBP or dsRNA-binding mutants K59A or K189A as indicated, then treated with polyI:C 24 h after transfection. pRLnull plasmid was co-transfected for normalization of transfections. Cell extracts were assayed for dual luciferase activity 8 h after treatments. The p-values calculated from 2 independent experiments with each sample in triplicates are indicated.