Analysis of RIG-I oligomerization <i>in cellula</i> as determined by co-immunoprecipitation 18 hours after stimulation by a cognate RNA ligand.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108770#pone-0108770-g002" target="_blank">figure 2</a> 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).</p

Lack of RNA induced RIG-I oligomerization <i>in cellula</i> as detected using PCA.

<p>(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.</p

RIG-I binding to synthetic RNA and activation of IFN-β promoter.

<p>(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 <i>in cellula</i>. Synthetic RNA were transfected in Huh7.5 cells previously transfected or not with Flag-RIG-I or Flag-RIG-I^ko 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.</p

Oligomeric state of RIG-I:RNA complexes produced <i>in vitro</i> as determined by SEC MALLS (A) and SAXS (B).

<p>(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 (R_g) was determined from the Guinier plot. P(R) functions of the scattering curves that were fitted to attain the experimental R_g show both a maximal intramolecular distance of 150 Å.</p

Model of viruses carrying α-peptide tagged proteins and visual selection of recombinant viruses and plaque growth by α-complementation.

<p>(<b>a–c</b>) α-peptide is shown as blue squares. (<b>a</b>) Model of MHV-αN and western blot analysis of N protein in purified virus stock. (<b>b</b>) Model of MHV-Sα and western blot analysis of S protein in purified virus stock. (<b>c</b>) Model of VSVΔG-Gα* pseudovirus and western blot analysis of VSV structural proteins in purified virus stock. (<b>d</b>) Serial dilution plaque assay of recombinant MHV-αN on LR7ΔM15 cell monolayers. After inoculation cells were covered for 2 days with a X-Gal containing agar-medium overlay. (<b>e</b>) Visualization of plaque growth of MHV-αN in LR7ΔM15 cell monolayers after 16, 30 or 48 h incubation (from left to right). Size bar corresponds to 1 mm.</p

Fusion assay.

<p>(<b>a</b>) Virus-cell fusion measured by flow cytometry. Sorting of MHV-αN infected cells by flow cytometry showed increasing fluorescence at increasing MOI. Cells were treated as described in <b>b</b>. (<b>b</b>) Increase of fusion signal relative to MOI. Increasing amounts of MHV-αN, MHV-Sα, and VSVΔG-Gα* were bound to ΔM15 expressing cells on ice. 40 min (VSV) or 100 min (MHV) post warming to 37°C fusion was assayed by measuring β-galactosidase activity using FDG substrate and flow cytometry. Inlay highlights β-galactosidase activity at low MOI. Error bars represent 1 SEM, n = 3. (<b>c, d</b>) Kinetics of internalized α-peptide tagged protein in comparison to β-galactosidase activity. MHV-αN (MOI = 100) was bound to cells on ice. Unbound virus was removed, and samples shifted to 37°C with (<b>c</b>) or without addition of cycloheximide (<b>d</b>). At the indicated time points, cells were washed and trypsinized on ice, removing surface bound virus. Virus-cell fusion was measured by β-galactosidase activity using flow cytometry or cells were lysed and immunoblotted against N for quantification the internalized α-peptide proteins. (<b>e</b>) Fluorescence microscopy image of β-galactosidase activity in infected cells. MHV-αN was bound to LR7ΔM15 cells on ice. Inoculum was washed off and cultures shifted to 37°C for the indicated time periods. β-galactosidase activity was visualized by fluorescein production using fluorescence microscopy. Size bar corresponds to 250 µm.</p

Effects of drugs on binding, internalization, and fusion of MHV and VSV.

<p>(<b>a–f</b>) Cells were pretreated with cycloheximide (CHX), ammonium chloride (NH₄Cl), bafilomycin A1 (BafA1), dynasore (Dyn), chlorpromazine (Chlopro), monensin (Mon), or latrunculin A (LatA), as well as with solvents dimethyl sulfoxide (DMSO) and methanol (MeOH) for 30 min. MHV and VSV viruses without α-peptide were included as background controls (inf wt). Error bars represent 1SEM, n = 3. (<b>a, d</b>) MHV-αN or VSV-Gα* were bound to ΔM15 expressing cells in presence of compounds on ice for 90 min. Cells were washed, lysed and assayed with Beta-Glo substrate as described in <b>4a</b>. Binding was determined relative to the complementation luminescence signal generated by virus bound to ΔM15 cells, treated without compound added (untr inf). (<b>b,e</b>) After binding as described in <b>a</b>, MHV-αN and VSV-Gα* were allowed to internalize at 37°C in presence of compounds for 40 and 30 min, respectively. Internalization was determined relative to the complementation luminescence signal of virus internalized into ΔM15 cells, treated without compound added (untr inf). (<b>c,f</b>) After binding as described in <b>a</b>, MHV-αN or VSV-Gα* were allowed to internalize and fuse at 37°C in presence of compounds for 100 and 40 min, respectively. MHV fusion inhibitor HR2 peptide (HR2) was included as control. Fusion was determined relative to the number of positive cells showing complementation fluorescein signal of virus fused in ΔM15 cells, treated without compound added (untr inf).</p

Design of the virus entry assays.

<p>Schematic overview of binding- (left), internalization- (middle), and fusion assay (right). 1 - Binding of virus to cell membrane; 2 – Lysis of cells and surface-bound virus; 3 – Complementation of ΔM15 by intravirion α-peptide, substrate conversion yielding luminescent readout; 4 – Invagination and 5 – Budding of endosomal vesicles containing virus particles; 6- Lysis of cell, intracellular compartment, and virion (after removal of cell surface-bound virions by protease treatment); 7 - Complementation of ΔM15 by intravirion α-peptide, substrate conversion yielding luminescent readout; 8 – Fusion of virion with endosomal membrane, exposure of intravirion α-peptide to the cytosol; 9 – Complementation of intracellular ΔM15 by virion α-peptide in intact cells, substrate conversion yielding fluorescent readout.</p

Binding and internalization assay.

<p>(<b>a</b>) Luminescent signal after virus binding at various MOI. Increasing amounts of MHV-αN were bound to LR7ΔM15 cells on ice for 90 min before removing the inoculum and washing-off of unbound virus with ice-cold PBS. Cells and bound viruses were lysed and binding was determined by measuring the β-galactosidase activity using Beta-Glo substrate conversion to a luminescent product. (<b>b</b>) Internalization signal relative to MOI. Increasing amounts of MHV-αN were bound to LR7ΔM15 cells on ice for 90 min. Inoculum was removed and samples transferred to 37°C for 40 min. Cell-surface bound virus was removed by trypsinization. Cells and intracellular viruses were lysed and internalization determined by measuring β-galactosidase activity using Beta-Glo substrate conversion to a luminescent product. (<b>c</b>) Controls of binding and internalization assay. Samples were treated as described in <b>a</b> (binding) and <b>b</b> (internalization). After binding, attached virus was removed by trypsin treatment (trypsin). Binding and internalization were inhibited by incubation of cells with MHV receptor CC1a blocking anti-CC1a antibody (anti-CC1a) 30 min prior to and during inoculation. Error bars in <b>a</b> - <b>c</b> represent 1 SEM, n = 3.</p

Binding parameters of the N_TAIL variants towards XD as obtained by ITC.

<p>Data are representative of at least two independent experiments. The derived equilibrium dissociation constants (K_D), the stoichiometry number (n), the binding enthalpy ΔH (kcal mol^-1), and the binding entropy ΔS (cal mol^-1 deg^-1) are shown. Shown are the curves obtained using the following concentrations of N_TAIL in the microcalorimeter cell and of XD in the microsyringe: wt N_TAIL/XD: 50 μM/500 μM; N_TAIL R489Q/XD: 50 μM/500 μM; N_TAIL R490S/XD: 150 μM/854 μM; N_TAIL S491L/XD: 160 μM/800 μM; N_TAIL A492T/XD: 50 μM/500 μM; N_TAIL D493G/XD: 25 μM/600 μM; N_TAIL R497G/XD: 150 μM/955 μM. Graphs shown at the bottom of each panel correspond to integrated and corrected ITC data fitted to a single set of sites model. Note that for the binding reactions characterized by K_D values in the tens of micromolar range, it is difficult to obtain the first plateau because the necessary concentrations are too high. Consequently, it should be kept in mind that the actual errors may be larger than those estimated by the fit.</p