FIGURE 4.

Differential activity of RIG-I motif III mutant reflects endogenous RIG-I signaling. A, luciferase reporter gene assay assessing RIG-I motif III mutant (MIII) reporter gene signaling activity in HeLa, Vero, 2fTGH, HEK293T, and Huh 7.5 cells. Cells were transfected with IFNβ promotor luciferase reporter gene plasmids and expression vectors for RIG-I (wt) or mutant. B and C, titration of RIG-I wild type and RIG-I motif III mutant. HEK293T cells were transfected with IFNβ reporter gene plasmids, 0.5 μg of RIG-I wild type expression vector, and increasing amounts of RIG-I motif III mutant expression vector (B). 0.5 μg of RIG-I motif III mutant expression vector and increasing amounts of RIG-I wild type expression vector were used in a similar assay (C). Each transfection reaction contained 1.25 μg of total DNA, using empty vector plasmid DNA for all assays. Parallel samples were left unstimulated (white) or infected with 3 × 10⁶ PFU Sendai virus (black), strain Cantell, for 6 h prior to lysis and luciferase assay. Values are normalized to the stimulated wild type sample. Representative data for at least two independent experiments are shown. Error bars depict standard deviation of triplicate samples. Ve, vector control; wt, wild type. D, model scheme summarizing the apparent cross-talk between RIG-I (wt) and RIG-I motif III mutant (MIII) signaling activity when expressed alone (left) in absence (basal) or presence of Sendai virus (SeV), or when co-expressed in a titration experiment (right). Triangles indicate increasing abundance. For further explanation see text.