Fig. 3

Interaction of M with MAVS. a, b The M protein interacts with MAVS in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids for 20 h. Coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies. c Association of the M protein with endogenous MAVS. Flag-tagged M-expressing HEK293 cells were infected with SeV (MOI = 1) for the indicated times. Coimmunoprecipitation and immunoblot analyses were performed with the indicated antibodies. d The M protein directly binds to MAVS. Purified recombinant GST-M was bound to glutathione agarose beads and incubated with recombinant His-MAVS for 3 h. The bead-bound proteins were analyzed by immunoblotting with the indicated antibodies. e Domain mapping of the M-MAVS association. HEK293 cells were transfected with the indicated M and MAVS truncation mutants for 20 h before coimmunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. f Effects of M truncations on the SeV-induced activation of the IFNβ promoter. HEK293 cells were transfected with the IFNβ promoter luciferase plasmid and the indicated expression plasmids for 20 h. The cells were then infected with SeV (MOI = 1) or left uninfected for 12 h before luciferase assays were performed. g Effects of M truncations on the MAVS-mediated transcription of downstream antiviral genes. HEK293 cells were transfected with the indicated plasmids for 15 h before qPCR analysis was performed. Graphs show the mean ± SD; n = 3; ns not significant; *p < 0.05, **p < 0.01 (Student’s unpaired t test)