MORC3 promotes degradation of IRF3.

(A) The subcellular distribution of MORC3 in EPC cells was examined by immunofluorescence. Scale bars = 10 μm. (B) Immunoprecipitation analysis of the interaction between MORC3 and IRF3 in HEK293 cells transfected with MORC3-Myc and IRF3-Flag plasmids under poly(I:C) treatment. (C) MORC3-Myc were co-transfected with IRF3-Flag into MsbC cells. Immunofluorescence (IF) was performed to determine MORC3/IRF3 colocalization under SCRV infection. Scale bar = 10 μm. (D) Immunoprecipitation and immunoblot analysis of MORC3-Flag in MsbC cells. (E) Schematic diagram of the wild type (WT) and mutants of MORC3. (F) Schematic diagram of the wild type (WT) and mutants of IRF3. (G) Western blot analysis of lysates in HEK293 cells transfected with control vector, MORC3-Flag, IRF3-HA, and three mutants of IRF3. (H) Immunoblot analysis of IRF3 protein level in MsbC cells transfected with MORC3-Flag, three mutants of MORC3 or pcDNA3.1. (I) Effects of MORC3 and its truncations on poly(I:C)-induced IRF3 promoter activation. (J) The IRF3 protein level in MsbC cells transfected with either control vector or MORC3-Flag, followed with SCRV or poly(I:C) stimulation. (K) MsbC cells were transfected with the indicated plasmids for 24 h and then infected with SCRV for 24 h. Immunoblot assays were performed with the indicated antibodies. (L) The proposed working model of circMORC3 in the regulation of antiviral innate immune response. Upon virus infection, the TLR3 sensors undergo conformational change after binding to virus RNA, which promotes the activation of type I IFN expression mediated by TRIF. Furthermore, MORC3 not only directly interacts with IRF3 protein but also suppresses its expression. To evade antiviral immune responses, the expression of circular MORC3 (circMORC3) is induced during viral infection. circMORC3 encodes the MORC3-84aa protein, which enhances the ubiquitination of TRIF at the K6 site. This prompts the activation of the autophagy pathway in TRIF, followed by fusion with lysosomes, ultimately resulting in TRIF protein degradation. As a consequence, the antiviral innate immunity mediated by TRIF is inhibited. All data presented as the means ± SE from at least three independent triplicated experiments. **, p < 0.01; *, p < 0.05 versus the controls.