• ¹State Key Laboratory of Virology, Medical Research Institute,College of Life Sciences Wuhan University, Wuhan, China.
• ²State Key Laboratory of Virology, Medical Research Institute,College of Life Sciences Wuhan University, Wuhan, China yuliu@whu.edu.cn.

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs exceptTLR3recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereasTLR3and internalized TLR4 use TIR-domain-containing adaptorTRIFfor the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing proteinWDFY1as a crucial adaptor protein in theTLR3/4signalingpathway. Overexpression ofWDFY1potentiatesTLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines.WDFY1depletion has the opposite effect.WDFY1interacts withTLR3and TLR4 andmediatesthe recruitment ofTRIFto these receptors. Our findings suggest a crucial role forWDFY1in bridging the TLR-TRIFinteraction, which is necessary for TLRsignaling.