FIGURE 3.

Signal transduction by MDA5 and RIG-I proteins and RIG-I RNA binding. Constitutive and inducible activation of the IFNβ promoter luciferase reporter gene by expression of the wild type and mutant MDA5 and RIG-I proteins. HEK293T (A and C) and 2fTGH (B and D) cells were transfected with luciferase reporter gene plasmids and expression vectors for helicase proteins MDA5 (A and B) or RIG-I (C and D). Parallel samples were left unstimulated (white) or were transfected with 5 μg/ml poly(I-C) (gray), 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 unstimulated wild type protein activity for each experiment. Representative experiments of four independent experiments are shown. Error bars depict standard deviation of triplicate samples. E–G, RNA binding by RIG-I wild type and mutants. E and F, RIG-I interaction with short RNA molecules. RIG-I wild type and mutant proteins were purified from HEK293T cells by immunoprecipitation with FLAG M2 affinity beads. Immobilized proteins were incubated with radioactively labeled 80-bp dsRNA (E) or 79-nt ssRNA (F) molecules and washed extensively. GFP, green fluorescent protein. Retained radioactivity was measured by scintillation counting, and counts/min are displayed as percent of wild type normalized to the total protein in each sample. Values are averaged from two independent experiments. G, HEK293T cell lysate expressing RIG-I wild type or mutant protein were incubated with poly(I-C)-coated agarose beads and analyzed by immunoblot with FLAG tag-specific antiserum. Ve, vector control; wt, wild type.