Figure 1.
Oligomeric state of RIG-I:RNA complexes produced in vitro as determined by SEC MALLS (A) and SAXS (B).
(A) 37 mM RIG-I and RIG-I:RNA complexes formed by incubation with 40 mM of RNA and 2 mM ATP analogue were analysed by size-exclusion chromatography on a S200 column coupled to multi-angle laser light scattering. Free RIG-I as well as the RIG-I:RNA complex elutes as monomers or 1∶1 complexes, respectively, with indicated apparent molecular weights. Theoretical values are 106 kDa for RIG-I and 11.8 kDa for the RNA. (B) Scattering data was collected for different protein concentrations of RIG-I or RIG-I:RNA complex and from the merged curves. shPH RNA is an influenza virus derived short pan-handle RNA. The radius of gyration (Rg) was determined from the Guinier plot. P(R) functions of the scattering curves that were fitted to attain the experimental Rg show both a maximal intramolecular distance of 150 Å.
Figure 2.
RIG-I binding to synthetic RNA and activation of IFN-β promoter.
(A) Expression of Flag-RIG-I in Huh7.5 cells two days after transfection and analysed by western blot as revealed with Flag-specific antibody. (B) Luciferase expression driven under the control of the IFN-β promoter measured 24 h after transfection with 20 ng of synthetic RNA in Huh7.5 cells expressing or not Flag-RIG-I. (C, D) Immunoprecipitation of RIG-I:RNA complexes formed in cellula. Synthetic RNA were transfected in Huh7.5 cells previously transfected or not with Flag-RIG-I or Flag-RIG-Iko 24 h before. Cells were harvested 6 hours after RNA transfection and RIG-I:RNA complexes were eluted from anti-Flag antibody immobilized on beads with a Flag peptide. (C) Specific immunoprecipitation of Flag-RIG-I as analysed by western blot. (D) RNA immunoprecipitated with Flag-RIG-I and analysed by RT-PCR.
Figure 3.
Analysis of RIG-I oligomerization in cellula as determined by co-immunoprecipitation 18 hours after stimulation by a cognate RNA ligand.
(A) Similar expression of Flag-RIG-I and cl25-RIG-I constructs in 293T cells as revealed by western blot. (B) Efficiency of Flag-RIG-I and Cl25-RIG-I to activate the IFN-β promoter after Poly(I:C) transfection. See figure 2 legend for details. (C, D) Lack of co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I after their co-transfection in Huh7.5 cells and stimulation with Poly(I:C), 5′pppssRNA(62-mer) or 5′pppdsRNA(62-mer) (C) or MeV infection (MOI 1) (D) as detected by western blot. (E) Nonsensical co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I expressed in 293T cells and after transfection of 1 µg or 50 ng of 5′pppds(or ss)RNA(62-mer) or MeV infection (MOI 0.5).
Figure 4.
Analysis of RIG-I oligomerization in Huh7.5 (A) and 293T (B) cells determined by co-immunoprecipitation 4 hours after stimulation with Poly(I:C) (A, B), 5′pppssRNA(62-mer), 5′pppdsRNA(62-mer) (B) or MeV infection (MOI 1, A, B) of cells expressing Flag-RIG-I and Cl25-RIG-I.
Figure 5.
Lack of RNA induced RIG-I oligomerization in cellula as detected using PCA.
(A) Ability of RIG-I/glu/gcn4 constructs to self-associate in the absence or presence of Poly(I:C) determined by PCA. Luciferase activity was measured 18 hours after transfection or not with Poly(I:C) in 293T cells transfected one day before with RIG-I/glu1/2/gcn4 constructs. (A, inset) Expression of chimeric RIG-I/glu1/2 constructs tagged with Cl25 or HA peptides in Huh7.5 cells two days after transfection as detected by western blot (note that the third sample (Glu1-RIG-I-GCN4 was overloaded, hence the overexposure of this protein and GAPDH). (B) Ability of RIG-I/glu/gcn4 chimeric proteins (left panel) and glu-gcn4 protein (right panel) for self-binding determined by western blot 24 hours post-transfection of 293T cells with glu1 or glu2 constructs alone or in combination. Lysates were separated without prior heat denaturation on SDS-PAGE before western blot analysis.