Dengue virus (DENV) and other flaviviruses are prevented from replicating in mosquitoes by Wolbachia. To date, several reports have appeared that highlight multiple molecular and cellular pathways involved in the blocking mechanism, which underlines the complicated nature of the mechanism. Here, we developed a hypothesis on whether Wolbachia proteins interact with pro-viral host proteins by using a unique approach to study the antiviral mechanism based on Wolbachia-host protein-protein interaction. We selected Wolbachia surface protein (WSP) for co-immunoprecipitation because of its abundance and possible secretion. We first confirmed WSP's secretion in mosquito cells and found two host proteins, Ae. aegypti serine-threonine kinase (STK) and synaptic vesicle membrane (SVM) protein VAT-1, and one Wolbachia protein (wGroEL) interacting with WSP. We examined the role of STK and SVM genes in relation to DENV replication in Ae. aegypti mosquitoes and mosquito cell lines with and without Wolbachia. In DENV-infected Aag2 cells, the expression of SVM and STK was significantly increased. However, although these genes were induced in Wolbachia-infected Aag2 cells, they were downregulated after DENV infection. Silencing of STK, but not SVM, reduced DENV replication in Aag2 cells and mosquitoes. Conversely, RNA activation of STK, by utilizing promoter induction via short activating oligos, resulted in higher DENV replication in Wolbachia-infected and uninfected cell lines. Overall, our findings suggest that STK is a pro-viral gene, and Wolbachia WSP binds to STK, possibly making it less accessible for DENV replication.
Importance: Wolbachia is an endosymbiotic bacterium that blocks the replication of arboviruses in transinfected Aedes aegypti mosquitoes. In this study, we focused on identifying the potential interaction of Wolbachia proteins with the host pro-viral proteins. For this, we embarked on identifying the interacting proteins with a major Wolbachia protein, WSP, which is both structural and also secreted into the host cells. An Ae. aegypti STK was identified, which is induced in DENV and Wolbachia-infected cells. Silencing or induction of the gene led to reduced and increased DENV replication in vitro. Consistently, knocking down the gene in mosquitoes resulted in decreased virus replication. We hypothesize that WSP may sequester STK, which is pro-viral, contributing to Wolbachia virus blocking.
Keywords: Aedes aegypti; Wolbachia; dengue virus; mosquito; serine threonine kinase.