Fig 1: Riok3 promotes TRIM40-mediated ubiquitination and degradation of RIG-I and MDA5(A) TRIM40 interacts with Riok3. The primary macrophages were infected with VSV at MOI = 1 for 4 h, followed by immunoprecipitation with indicated antibodies. IgG was used as negative control.(B) VSV infection promotes TRIM40 expression. The primary macrophages were infected with VSV at MOI = 1 for 4 h, followed by western blot (WB) with indicated antibodies.(C) TRIM40 promotes K27- and K48-linked ubiquitination of RIG-I and MDA5. The HEK293T cells were transfected with 10 nM siTRIM40 for 24 h, then infected with VSV for 4 h. Cell lysates were immunoprecipitated with anti-RIG-I (or anti-MDA5) antibody, followed by WB with the indicated antibodies.(D) Riok3 promotes TRIM40-mediated degradation of RIG-I and MDA5 in a dose-dependent manner. The HEK293T cells were transfected with plasmids encoding FLAG-RIG-I (or FLAG-MDA5), HA-ubiquitin, Myc-TRIM40, and varying doses of a plasmid encoding His-Riok3 (0.5 and 1 μg). Half of each cell aliquot was treated with ΜG132 (10 μM). The level of RIG-I and MDA5 proteins was examined in cells harvested 24 h later.(E) Riok3 interacts with TRIM40. The primary macrophages were infected with VSV at MOI = 1 for indicated times, followed by immunoprecipitation using indicated antibodies.(F) Riok3 forms a complex with TRIM40 and RIG-I (or MDA5). The primary macrophages were infected with VSV at MOI = 1 for 4 h, followed by immunoprecipitation using indicated antibodies.(G) Riok3 is necessary for this complex formation. WT and Riok3 knockout PMs were infected with VSV at MOI = 1 for 4 and 8 h, followed by immunoprecipitation using indicated antibodies.(H) Schematic diagram of TRIM40 and its truncate mutants.(I) TRIM40 interacts with Riok3 via its internal CC domain. Myc-tagged TRIM40 or its mutants and FLAG-Riok3 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Myc antibody and then immunoblotted with the indicated antibodies.(J) Schematic diagram of Riok3 and its truncate mutants.(K) Riok3 interacts with TRIM40 via its RIO domain. FLAG-tagged Riok3 or its mutants and Myc-TRIM40 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with the indicated antibodies.(L) The deficiency of TRIM40 increases the IFN-β transcription. WT and Riok3 knockout PMs were transfected with 50 nM siTRIM40 or control siRNA to knock down TRIM40 before VSV infection for indicated time points. qRT-PCR was performed to evaluate the IFN-β mRNA expression.B, B box; CC, coiled-coil region; R, RING domain.