(A) A protocol for purification of active Flag-MAVS particles. (B) Silver staining of Flag-MAVS purified from HEK293T cells infected with Sendai virus or not infected. (C) IRF3 dimerization assay using purified Flag-MAVS shown in (B). MAVS was also analyzed by immunoblotting following SDD-AGE and SDS-PAGE.

(A) Sumo-MAVS in Peaks I and II from Superdex-200 was digested with proteinase K (PK) for the indicated time at 22°C, then analyzed by Coomassie blue staining. (B) Sumo-MAVS was treated with or without proteinase K for 1 hour and then fractionated on Superdex-200 (2.4 ml column). The fractions were analyzed by Coomassie blue staining. Mass spectrometry showed that the protease-resistant fragments in Peak I contained the N-terminus of MAVS including the intact CARD domain (PK-MAVS; Figure S4A), and that the protease-resistant protein in Peak II was Hsp70. (C) The fraction containing PK-MAVS shown in B (lane 9) and the prion PrP were imaged by electron microscopy using negative staining. (D) PK-MAVS and PrP at the indicated amounts were incubated with the mitochondria from HEK293T cells at 22°C for 30 min (total volume: 10 μl). The mitochondria were subsequently incubated with ³⁵S-IRF3 and cytosolic extracts to measure IRF3 dimerization. Mitochondrial extracts were also analyzed by SDD-AGE and SDS-PAGE using a MAVS antibody, which reacts very weakly with PK-MAVS due to the removal of the epitopes. (E) The CARD domain of MAVS is required for its conversion into aggregated forms by PK-MAVS. Full-length MAVS and MAVSΔCARD were expressed in Mavs^−/− MEF cells by retroviral transduction. Mitochondria (P5) from these cells were incubated with or without PK-MAVS for 30 minutes before mitochondrial proteins were separated by sucrose gradient ultracentrifugation. (F) The MAVS CARD domain was expressed in HEK293T cells as a Flag fusion protein and affinity purified. The purified protein was analyzed by silver staining, IRF3 dimerization assay and electron microscopy.

(A) Full-length RIG-I was incubated with 5′-pppRNA and/or ubiquitin chains as indicated at 22°C for 10 min. The mixtures were then incubated with mitochondria from HEK293T cells for 30 min before mitochondrial extracts were analyzed by SDD-AGE and SDS-PAGE. 0.25 μg of ubiquitin chains or mono-Ub was analyzed by silver staining (right panel). (B) GST-RIG-I(N) was incubated with different ubiquitin chains at 22°C for 10 min in the presence or absence of ATP or EDTA as indicated. The mixture was then incubated with mitochondria from HEK293T cells for the indicated time, followed by analysis of the mitochondrial extracts using SDD-AGE and SDS-PAGE. (C) Similar to (B) except that varying amounts of GST-RIG-I(N) and K63-Ub4 were used, and their incubation time with mitochondria were kept at 30 min. (D) GST-RIG-I(N) was incubated with K63-Ub4 and then with mitochondria for the indicated time, followed by analysis of the mitochondrial extracts using SDD-AGE and SDS-PAGE. (E) GST-RIG-I(N) was incubated with K63-Ub4 before incubation with mitochondria in the presence or absence of DTT. The mitochondrial extracts were analyzed by SDD-AGE, SDS-PAGE and IRF3 dimerization assays. Mitochondria from Sendai virus-infected cells were used as a positive control. (F) Mitochondrial extracts from (E) were fractionated by sucrose gradient ultracentrifugation followed by SDS-PAGE and immunoblotting with a MAVS antibody.

(A) Crude mitochondria isolated from HEK293T cells infected with Sendai virus for 14 hours (+SeV) or uninfected (−SeV) were solubilized in a buffer containing 1% DDM, then subjected to sucrose gradient ultracentrifugation. Aliquots of the fractions were immunoblotted with a MAVS antibody, or incubated with ³⁵S-IRF3 and cytosolic extracts in the presence of ATP at 30°C for 60 min, followed by native gel electrophoresis and autoradiography. Arrows indicate the positions of proteins used as molecular size markers, including 20S and 26S proteasome. ΔMAVS denotes a truncated MAVS lacking the N-terminus. (B) YFP-MAVS was expressed in Mavs^−/− MEF cells by retroviral transduction, and the cells expressing low levels of YFP-MAVS were sorted by FACS. These cells were infected with Sendai virus for 13 hours, stained with Mitotracker, and then visualized by confocal fluorescent microscopy. The images are representative of >50 % of the cells under examination. (C) Crude mitochondrial extracts were prepared from HEK293T cells infected with Sendai virus for the indicated time, then aliquots of the extracts were analyzed by SDD-AGE, SDS-PAGE, or IRF3 dimerization assays. (D) Crude mitochondrial extracts were treated with or without β-mercaptoethanol (BME at 0.35, 3.5 and 35 mM), followed by SDD-AGE. (E) HEK293T cells were treated with geldanamycin or 17-AAG at the indicated concentrations for 1 hour before Sendai virus infection. 9 hours later, the activation of endogenous IRF3 in the cytosolic extracts were analyzed by native gel electrophoresis. Mitochondria (P5) were prepared and analyzed by SDD-AGE or SDS-PAGE using a MAVS antibody. An aliquot of P5 was incubated with cytosolic extracts, ³⁵S-IRF3 and ATP followed by native gel electrophoresis to measure MAVS activity.

(A) Sumo-MAVS lacking the proline-rich and TM domains was expressed and purified from E. coli as described in Experimental Procedures, then further fractionated by gel filtration on Superdex-200. Each fraction was analyzed by Coomassie blue staining and IRF3 dimerization assay. (B) Two peaks of the Sumo-MAVS protein as shown in (A) were imaged by electron microscopy using negative staining. (C) Mouse MAVS lacking the TM domain was expressed as a His₆-tagged protein in E. coli and affinity purified. The purified protein was analyzed by IRF3 dimerization assay and electron microscopy. (D) Peak I and II of Sumo-MAVS as shown in (A) was diluted 5 fold serially as indicated, and then incubated with mitochondria from HEK293T cells (total volume: 10 μl). The mitochondria were solubilized in a buffer containing 1% DDM, and then analyzed by SDD-AGE and SDS-PAGE with a MAVS antibody. (E) The mitochondria incubated with Sumo-MAVS as described in (D) were subsequently incubated with ³⁵S-IRF3 and cytosolic extracts to measure IRF3 dimerization.

(A) Crude mitochondria (P5) were isolated from cells infected with Sendai virus or not infected, solubilized in 1% DDM and then separated by sucrose gradient ultracentrifugation. Fractions were analyzed by immunoblotting with the indicated antibodies. Aliquots of the fractions were incubated with cytosolic extracts, ³⁵S-IRF3 and ATP followed by native gel electrophoresis. (B) Similar to (A), except that cells were transfected with siRNA against MAVS or GFP (control) before virus infection.

Following sequential binding of RIG-I to viral RNA (5′-ppp RNA) and K63 ubiquitin chains, the CARD domains of RIG-I interact with the CARD domain of MAVS. This interaction induces a conformational change of the MAVS CARD (depicted by the change in color and shape of the CARD), which in turn converts other MAVS on the mitochondrial outer membrane into prion-like aggregates. These aggregates activate cytosolic signaling cascades to turn on NF-κB and IRF3, leading to induction of type-I interferons and other antiviral molecules. MAVS on the mitochondrial membrane of uninfected cells may be a multimer but is depicted as a monomer for simplicity. Also not depicted is the possibility that MAVS on the surface of two adjacent mitochondria could aggregate through the N-terminal CARD domains, thereby propagating the antiviral signal between mitochondria.