Non-ligand-binding TLR20.2 and dsRNA-binding TLR20.3 form heterodimer for synergistic antiviral response in grass carp

TLR20 is a teleost-specific TLR member. However, its species distribution, biological function, and underlying mechanism remain largely unknown. In this study, we systematically retrieved the TLR20 species distribution and found only a few teleosts contain different TLR20 variants, especially in Cyprinidae. Subsequently, we employed an economically important model, grass carp (Ctenopharyngodon idella) with three TLR20 variants, to investigate the functional characterizations and mechanisms of TLR20. Ci-TLR20.1 localizes to both early endosomes and lysosomes, whereas Ci-TLR20.2/3 exclusively localize to lysosomes. Further, we found that Ci-TLR20.1 cannot form homodimer and heterodimers with Ci-TLR20.2/3, but Ci-TLR20.2/3 can form homodimers and heterodimer. Ci-TLR20.3 can directly bind to the dsRNA analog poly (I:C) via key residues F27, Q630, and T631. Although Ci-TLR20.2 lacks this ability, the reverse mutation I26L+R630Q confers dsRNA binding ability. Interestingly, Ci-TLR20.2/3 can also respond to PGN stimulation. However, they cannot directly bind PGN. Ci-TLR20.2/3 recruit MyD88 as an adaptor independent of the conserved proline residue in BB loop, significantly activating IFN, NF-κB and AP-1 pathways. Functionally, overexpression of Ci-TLR20/3, or their combination significantly inhibits grass carp reovirus (GCRV) replication. Correspondingly, knockdown of these receptors facilitates viral infection. In vivo knockdown of Ci-TLR20.2/3 reduces the survival rate of grass carp following GCRV infection, exacerbates virus-induced tissue damage, and suppresses complement C3, SOD, IFN1, and antiviral effector Mx2. We also found that Ci-TLR20.2 and Ci-TLR20.3 synergistically defend against viral infection in vitro and in vivo. These findings provide novel insights into the functional divergence and synergistic effects of teleost-specific TLR20 in antiviral defense.