Fig 2: RIG-I G731R is a loss-of-function variant with a dominant negative effect.(A) 5’ppp ds39 RNA induced IFN-β reporter activities after transfection with empty vector (EV), WT RIG-I, and G731R plasmids (50 ng) in HEK293T RIG-I−/− cells (Star Methods) (technical replicates, n=3).(B) hPIV-induced IFN-β reporter luciferase activities for G731R. HEK293T cells were transfected with either the WT or G731R construct or EV along with the IFN-β-Luc reporter and a constitutively expressed Renilla reporter for 6 hours. Cells were then infected with hPIV (MOI 3) for an additional 20 hours before measuring luciferase activity. IFN-β-Luc reporter activity in each condition was first normalized to Renilla and then presented relative to WT RIG-I. Data show means ± SD from 3 independent experiments. Statistical analysis was performed using one-sample t-test. ****, P<0.0001.(C) IFN-β promoter response measured by dual luciferase reporter assay in HEK293T RIG-I−/− cells transfected with a constant amount of WT RIG-I plasmid (50 ng), increasing amount of G731R plasmid (0-500 ng), and 5 nM 5’ppp ds39 RNA. The error bars represent the standard error of the mean (technical replicates, n=3), and the curve represents fitting of the mean data to equation 1 to obtain the half maximal inhibitory concentration IC50.(D) hPIV-induced IFN-β reporter luciferase activities in cells co-transfected with WT RIG-I (5 ng) and increasing doses (5 to 20 ng) of G731R or EV plasmids. IFN-β-Luc reporter activity in each condition was first normalized to Renilla and then shown relative to EV. Data show means ± SD from 3 independent experiments. Statistical analysis was performed using one-sample t-test. ***, P<0.001.

Fig 3: HDX-MS analysis of G731R reveals CARDs-CHL protection.(A) Linear mapping of the HDX differential data of WT and G731R with and without 5’ppp HP RNA. In the colorimetric HDX scheme, shifting towards red indicates a higher degree of solvent exchange, while blue indicates less. If there is no significant difference in the solvent exchange of two samples analyzed side-by-side, the HDX differential remains gray. The white/black areas indicate that the peptides were not resolved for mass spectrometry.(B) The differential HDX pattern of G731R apo vs WT RIG-I apo mapped onto a 3D composite model of G731R apo (4A2W, 3TBK, 4AY2, & computationally modeled CH-Linker).(C) The differential HDX pattern of WT RIG-I ± 5’ppp HP RNA mapped onto a 3D composite model of RNA-bound WT RIG-I with computationally modeled CHL and CARDs in the exposed state (PDB: 5E3H, 4P4H, 4AY2). (Ci) Deuterium uptake plots of select RIG-I peptides represented as percent deuterium uptake over time.(D) The differential HDX pattern of G731R ± 5’ppp 10bp HP RNA mapped onto a composite model of RNA-bound G731R with computationally modeled CHL and CARDs sequestered by Hel2i (PDB: 4A2W, 3TBK, 4AY2). (Di) Deuterium uptake plots of select RIG-I peptides represented as percent deuterium uptake over time.

Fig 4: The CHL regulates IFN-response and ATPase activities of G731R.(A) Amino acid sequence of the intrinsically disordered CHL of RIG-I. Negatively charged residues are colored red and underlined; positive charges are colored blue. The Δ190-200 segment is indicated.(B) IFN-β reporter activities of WT RIG-I, Δ190-200 WT, G731R, and Δ190-200 G731R RIG-I in the absence and presence of 5 nM 5’ppp ds39 RNA (technical replicates, n=3). The fold-change from the 10 aa deletion in WT RIG-I and G731R is shown.(C) Partial rescue of the ATPase function of G731R upon deletion of the CHL-CARDs domains. The ATPase activities of full-length and HelCTD constructs of WT RIG-I and G731R were measured at 1 μM protein in the presence of 5 nM 5’ppp ds39 RNA. Mean rates of hydrolysis (Moles Pi / Moles RNA / sec) are reported (technical replicates, n=3).

Fig 5: G731R RIG-I mutant is trapped in the “CTD-mode” RNA-bound conformation.(A) RIG-I forms kinetically distinguishable “CTD-mode” and “Helicase-mode” conformations on 5’ppp RNA.(B) Experimental setup to measure the off-rate of RIG-I from 5’ppp RNA using a rapid stopped-flow instrument.(C) Biphasic kinetics of WT RIG-I and G731R dissociation from 5’ppp ds27 RNA with and without ATP. The time axis is shown on a log scale to clearly show the two phases. The dissociation kinetics (average of n=3) best fit to the sum of two exponentials (equation 4), providing slow and fast off-rates and the corresponding amplitudes plotted in panel D.(D) The “CTD-mode” and “Helicase-mode” phase amplitudes from data in panel C in the absence and presence of ATP are shown as percent population from three biological replicates with SEM.(E) The lifetimes (1/off-rate) in seconds of the “CTD-mode” and “Helicase-mode” phases are shown in the absence and presence of ATP from three biological replicates with SEM.