(A) ATP hydrolysis activity of RIG-I, the RIG-I Singleton-Merten syndrome (SMS) variant C268F and the RIG-I motif I and II mutants K270I and E373Q. RIG-I proteins were incubated with [γ-32P]-ATP in the presence or absence of a 12mer dsRNA for 15 min at room temperature and free phosphate was separated from ATP by thin layer chromatography. (B) Affinity of RIG-I, RIG-I C268F and the RIG-I motif I and II mutants to MANT-ATP or MANT-ATPγS measured by tryptophan fluorescence Förster resonance energy transfer to the MANT-nucleotide. Proteins were incubated with increasing amounts of nucleotides in the presence or absence of a 14mer dsRNA. MANT fluorescence was recorded minus a MANT-nucleotide-only control. n = 4, error bars represent mean values ± SD. (C) Fold change of interferon (IFN)-β promoter-driven luciferase activity in uninfected HEK293T RIG-I KO cells or in cells stimulated with a 19mer 5’-triphosphate (ppp)-dsRNA upon overexpression of different RIG-I mutants. Cells were co-transfected with RIG-I expression vectors and p-125luc/pGL4.74 reporter plasmids, and stimulated with ppp-dsRNA 6 hr post transfection. Firefly luciferase activities were determined in respect to Renilla luciferase activities 16 hr after RNA stimulation. All ratios were normalized to an empty vector control. n = 4–12, error bars represent mean values + SEM.