The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal-the Urmetazoan-used PIWI proteins both to generate piRNA guides and to execute piRNA function. TUDOR-domain containing proteins facilitate PIWI interacting (pi)RNA biogenesis in Drosophila melanogaster and other model organisms. In Aedes aegypti mosquitoes, a somatically active piRNA pathway generates piRNAs from viral RNA during acute infection with cytoplasmic RNA viruses. Viral piRNA biogenesis requires ping-pong amplification by the PIWI proteins Ago3 and Piwi5. We hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all Ae. Knockdown of several Tudor genes resulted in reduced viral piRNA levels, with silencing of AAEL012437 having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts directly with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from exogenous viral RNA. In animals, piRNAs guide PIWI-proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. PiRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts.

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