Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling.

Toll-like receptor 3 (TLR3), an innate immune sensor for double-stranded RNA (dsRNA), traffics from the endoplasmic reticulum (ER) after synthesis to endolysosomes for proteolytic cleavage and activation. However, the molecular mechanisms governing TLR3 trafficking remain largely unclear. Here, we identify the ER-resident E3 ligase HMG-CoA reductase degradation protein 1 (HRD1), a core component of ER-associated degradation (ERAD), as a key regulator that promotes TLR3 trafficking and downstream signaling. HRD1 deficiency in macrophages significantly impairs poly(I:C)-induced TLR3 signaling and inflammatory responses in vitro and in vivo, caused by a marked reduction in TLR3 transport into endolysosomes and subsequent proteolytic processing. Mechanistically, HRD1 mediates ubiquitination of ER-localized TLR3 at lysine 813, which is required for its recognition and sorting by the endosomal sorting complex required for transport (ESCRT) machinery. This HRD1 function is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α). Hence, our study identifies a previously unrecognized mechanism controlling TLR3 signaling and links HRD1-mediated ubiquitination to immune sensor trafficking during innate immune responses.