Aedes aegypti Piwi4 is a non-canonical PIWI protein involved in antiviral responses. Aedes aegypti aegypti

The siRNA pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5 and Piwi6) in an Aedes aegypti derived cell line Aag2. Analysis of small RNAs captured by Piwi4 reveals that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus infected cells and with viral piRNAs only to a lesser extent. By using a Dcr2 knockout cell line we showed directly that Ago2 lost its antiviral activity as it is no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathway in Ae. aegypti and identify Piwi4 as a non-canonical PIWI protein that interacts with members of siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway.