Heterodimerization with signaling-inert TLR8b converts TLR8a from proviral to antiviral sensor in Ctenopharyngodon idella

Toll-like Receptor 8 (TLR8) is crucial for detecting viral and Bacterial ssRNA in mammals. However, its functional characteristics in early vertebrates remain largely unclear. Here, we employed the economically important fish Ctenopharyngodon idella, which harbors two TLR8 homologs (CiTLR8a/b), as a representative model. CiTLR8a/b are trafficked to early endosomes and lysosomes by UNC93B1, where they dynamically form homodimers and heterodimer, and bind ssRNA and dsRNA. Upon ligand binding, they first recruit MyD88, then TIRAP as adaptors. Unexpectedly, CiTLR8a enhances viral replication by suppressing downstream IFN, NF-κB, and AP-1 pathways in vitro and in vivo. In contrast, CiTLR8b is signaling-defective despite ligand binding and adaptor recruitment. Most strikingly, heterodimerization with signaling-inert CiTLR8b switches CiTLR8a into an Antiviral sensor by remodeling intracellular TIR-domain conformation through extracellular LRR-ligand interaction. Further, structural and mutagenesis analyses identify critical functional determinants: specific residues (CiTLR8a-V116/S164; CiTLR8b-S43/N790) required for dsRNA binding, and key N-glycosylation sites (N138/N189) and cysteines (C3/C4/C6/C7) in CiTLR8a responsible for its proviral functions, with Cys6/Cys7 being critical for CiTLR8a/b proper localization. TLR8 evolution follows a stepwise pattern: absent in cyclostomes, it emerges in jawed vertebrates and expands via lineage-specific duplications (whole-genome in teleosts; tandem in amphibians/reptiles), is lost in avians, and persists as a single copy in mammals. Notably, Cyprinid TLR8 can bind dsRNA besides ssRNA. Collectively, our findings elucidate the functions, adaptive mechanisms, and evolutionary trajectory of TLR8 in lower vertebrates, uncovering a unique regulatory system involving its two homologs. These results offer a new perspective on how TLR evolution shapes vertebrate immune system.