Viral tolerance in Aedes aegypti relies on the negative cooperativity between Toll5A and the cytokine ligand Spz1C

A. aegypti has evolved to become an efficient vector of Dengue viruses among other arboviruses despite Toll-regulated infection levels. Interestingly, both Toll and its ligand Spaetzle (Spz) have undergone gene duplication in A. aegypti raising the possibility of neofunctionalization and mutualism to develop between arboviruses and mosquitoes. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A-Spz1C complexes that display transient but specific interactions. Binding of the first ligand alters receptor-receptor interactions and promotes asymmetric contacts in the vicinity of the Z-loop in Toll5A. This conformation then restricts binding of a second ligand, while bridging the C termini that promote signalling. In contrast, increased receptor concentrations promote inactivating head-to-head receptor assemblies. We also found that Spz1C differs from orthologous and paralogous cytokines in their transcriptional responses upon A. aegypti Aag2 cell stimulation. Interestingly, Spz1C uniquely controls genes involved in innate immunity, lipid metabolism and tissue regeneration. Given the remarkable DENV-induced expression patterns of these proteins, our data rationalises how Spz1C upregulation might promote innate immunity in the midgut, and Toll5A upregulation, viral tolerance in the salivary glands.