Cyprinid-specific duplicated membrane TLR5 senses dsRNA as functional homodimeric receptors

Membrane-embedded Toll-like receptor 5 (TLR5) functions as a homodimer to detect bacterial flagellin. Cyprinid grass carp (Ctenopharyngodon idella) encodes two TLR5 genes, CiTLR5a and CiTLR5b. Here, we show that cyprinid TLR5a and TLR5b homodimers unexpectedly bind the dsRNA analog poly(I:C) and regulate interferon (IFN) response in early endosomes and lysosomes. Although TLR5 homodimers also bind flagellin, an immune response to flagellin is only triggered by TLR5a/b heterodimer. Moreover, we demonstrate that two TLR5 paralogs have opposite effects on antiviral response: CiTLR5a slightly promotes and powerfully maintains, whereas CiTLR5b remarkably inhibits virus replication. We show that the ectodomain of CiTLR5 is required for dsRNA-induced IFN signaling, and we map the key poly(I:C) binding sites to G240 for CiTLR5a and to N547 for CiTLR5b. Furthermore, we reveal that differential N-glycosylation of CiTLR5a/b affects dsRNA-IFN signaling but has no role in flagellin-mediated NF-κB induction, with paralog-specific roles for CiTLR5a-T101 and corresponding CiTLR5b-I99. Moreover, we provide evidence that the ability to sense dsRNA represents a neofunctionalization specific for membrane-bound TLR5 in cyprinid, bridging viral and bacterial immune responses.