Potassium channel regulation of the antiviral immune system and viral replication - Immune responses can be evoked through diverse inducible pathways and several genes in these pathways are under investigation as putative therapeutic targets, yet it is likely that additional regulators of immune responses are present and remain undescribed. Recent data has shown potassium (K+) efflux through ATP-gated inward rectifier potassium (KATP) channels reduces virus replication and virus-mediated mortality in mammals, fruit flies, honey bees, and mosquitoes. These data raise the intriguing question of how ion channels and K+ efflux can regulate the antiviral immune response, which are two seemingly disconnected physiological systems, and is the focus of this proposal. KATP channels act as molecular sensors of the cell by coupling cell metabolism to the electrical activity of the cell via the cell membrane potential. Thus, the premise of this proposal is that KATP channels are functionally coupled to antiviral immunity by K+ efflux controlling neurosecretion, bioavailability of ATP, and kinase activity that are essential for circulatory homeostasis, function of the RNAi machinery, and reactive oxygen species generation, respectively. We will directly test the hypothesis that KATP channels are essential to antiviral immune responses in the vector by regulating 1) circulatory homeostasis 2) antiviral RNAi machinery, and 3) products of aerobic respiration that regulates antiviral immune pathways. The experiments outlined in this proposal will systematically test this hypothesis and delineate the involvement of each physiological system to KATP-mediated viral immunity in model organisms that will yield insights to regulation of antiviral immune responses in humans. In Specific Aim 1, we will test the influence of viral infection and modulation of K+ efflux through KATP channels to dorsal vessel contraction dynamics and circulatory homeostasis through gene silencing and transgenic approaches. The data collected in SA1 will bolster our understanding of physiological systems driving virus infection in a competent vector. In Specific Aim 2, we will expand our preliminary results that clearly indicate KATP channels interact with the antiviral RNAi pathway, but do not define the point of interaction. We will perform small RNA sequencing from transgenic mosquitoes to determine whether the defect is in dicer complex or in downstream steps, such as assembly of RISC complexes. The data collected in SA2 will address the mechanism of how ion channels can alter function of antiviral RNAi machinery, which is currently unresolved. In Specific Aim 3, we will test if reduced DENV-2 replication after KATP channel activation is due to ROS/antioxidant-mediated regulation of antiviral immune pathways. The data collected in SA3 will yield mechanistic insights regarding the functional linkage between KATP channels and antiviral RNAi pathways that can be used to inform downstream therapeutic development. Combined, the data generated in this study will fill fundamental gaps in our knowledge of how antiviral immune responses are triggered and will identify putative intervention points to interrupt viral replication.
