Defining the mechanism of action of the broadly-active antiviral mosquito protein aBravo - Project Summary/Abstract Understanding virus-mosquito interactions is crucial for the development of new transmission control strategies for mosquito-borne viruses. However, few broadly active antiviral mechanisms have been identified and characterized in much detail in mosquitoes. The RNA interference (RNAi) response is one of the few known broadly active antiviral mechanisms in mosquitoes. However, many flaviviruses are not strongly affected by RNAi, possibly due to their ability to suppress the RNAi response. Specifically, in Culex quinquefasciatus mosquito cells, no significant antiviral effect on the flavivirus Usutu virus (USUV) following gene silencing of RNAi pathway components was observed. Similarly, the protein Piwi4 is antiviral against a large set of arboviruses but did not impact USUV replication in Cx. quinquefasciatus mosquito cells. In contrast, the aedine broadly active antiviral protein (aBravo) is antiviral against all viruses tested in Aedes aegypti and, based on preliminary data described here, in Cx. quinquefasciatus mosquito cells. The proposed project is aimed at improving the understanding of the exact mechanisms of Bravo in both Aedes aegypti (aBravo) and Culex quinquefasciatus (CqBravo) mosquitoes. Bravo is one of the few genes with broad antiviral activity against all tested arboviruses across two different mosquito vector species. It is also mosquito specific, possibly indicating a previously undescribed mechanism of mosquito antiviral response. Overall, it is a promising candidate for future transmission control strategies, but it is crucial to fully understand its role in cellular responses to virus infection. Here, we will generate new tools to study aBravo, determine which protein residues are required to mediate antiviral activity, and perform CLIP-seq and proteomic analysis to study what RNA molecules and proteins Bravo binds to. We will perform our experiments initially in the context of flavivirus infection of Cx. quinquefasciatus cells with the aim to then validate our findings in vivo, and to compare these findings to other viruses and virus infection of Ae. aegypti cells. The outcomes of the proposed project would significantly advance our understanding of mosquito Bravo and allow further investigation as a candidate for future control strategies.
