The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes

Biogenesis of PIWI interacting RNAs (piRNAs) in Drosophila melanogaster and other model organisms relies on a myriad of co-factors, many of which belong to the Tudor protein family. In the vector mosquito Aedes aegypti , a somatically active piRNA pathway generates piRNAs de novo from cytoplasmic RNA viruses during an acute infection. In the Ae. aegypti -derived Aag2 cell line, piRNA biogenesis from alphaviruses and bunyaviruses requires ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Thus far, the protein complex that mediates somatic piRNA biogenesis from exogenous, parasitic RNA remains unknown. We hypothesized that TUDOR-domain containing (Tudor) proteins are involved in vpiRNA production and thus performed a knockdown screen targeting all Ae. aegypti Tudor genes. Depletion of several Tudor proteins resulted in reduced vpiRNA production, with silencing of AAEL012441 having the most prominent and robust effect. This Tudor protein, which we named Veneno (Ven), interacts directly with Ago3 and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila . Analyses of the Ven interactome reveals a network of additional co-factors including orthologues of the Drosophila piRNA pathway components Vasa (AAEL004978) and Yb (AAEL001939). Yb in turn interacts directly with Piwi5 and we propose that this multi-protein complex provides a molecular scaffold to allow efficient ping-pong amplification of vpiRNAs.