Dicer-2-Dependent Activation of Culex Vago Occurs via the TRAF-Rel2 Signaling Pathway

Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-kappa B-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-kappa B ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago. Author Summary Viruses like West Nile, dengue and Japanese encephalitis are responsible for large number of human and livestock diseases worldwide. These viruses, transmitted by female mosquitoes via saliva during blood-feeding, elicit an immune response in these mosquitoes. The details of this immune response are still being investigated. Dicer2, which has previously been shown to be involved in RNAi mediated antiviral activity in mosquitoes, contains helicase domain, which leads to activation of antiviral protein, Vago. Vago is functionally similar to mammalian interferon and after secretion activates Jak-STAT pathway in neighboring cells leading to antiviral effect. Here we demonstrate that sensing of viral RNA by Dicer2 leads to activation of TNF receptor-associated factor (TRAF), which in turn leads to cleavage and release of amino-terminal domain of Rel2, NF-kappa B ortholog. Rel2 binds to a conserved NF-kappa B binding site on Vago promoter region leading to its induction. This proposed mechanism of Vago activation is similar to mammalian interferon activation after viral infection. The identification of this novel and evolutionarily conserved pathway downstream of Dicer2 provides new insight into the immune signalling in mosquitoes and other invertebrates.