Figure 1. Viral Infection Induces the Formation of Large MAVS Particles That Activate IRF3.

(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.