Abstract Zika virus (ZIKV) represents a new threat to global health, with particular relevance to the developing brain and risk of congenital microcephaly. The basis by which ZIKV leads to neuronal death in the brain of the fetus is unknown, but presumably relates to effects of ZIKV proteins on cellular health. ZIKV is an RNA Flavivirus encoding 10 proteins including the NS2B/NS3 heterodimer that forms an active serine protease involved in cleavage of the ZIKV polypeptide into individual proteins. Here we propose to study the molecular basis of ZIKV-induced microcephaly. We hypothesize that the ZIKV protease can function to cleave cytosolic host cellular proteins in neural precursor cells, leading to a range of cellular defects, contributing to toxicity. This application brings together three experienced research groups with a track record of collaboration on ZIKV. Our published work has focused on tropism of ZIKV for neural precursor cells, interaction of ZIKV with the immune system, small molecule inhibitors of ZIKV, and effect of ZIKV on neuronal survival. We have also published on neural stem cell biology, recessive human genetic microcephaly mutations, and flavivirus molecular pathology. Our preliminary data demonstrates that ZIKV NS2B/NS3 misexpression can mediate cell death, in a manner similar to ZIKV-induced cell death. Cell death can be abrogated with a protease-inactive mutation or by inhibition of protease activity through ZIKV protease inhibitors (ZPIs). Proteomic analysis identified a series of proteins bound to the ZIKV protease, which we term the ZIKV Protease-Ome (ZPO), at least one of which is a direct cleavage target of ZIKV NS2B/NS3, and leads to blocked cytokinesis and cell death when inactivated, in a manner similar to ZIKV-induced cell death. The goal of this application is to discover the molecular, cellular and genetic basis by which ZIKV proteins mediate neuronal cell death, particularly in the context of mammalian cerebral cortical development. We combine mass spectrometry analysis, protein interaction networks, advanced bioinformatics, neuronal culture models, analysis of cell cycle dynamics, and in vivo modeling. Importantly, we will focus on differences between toxicity observed with ZIKV proteins and other sequence-similar flaviviruses that do not mediate neuronal death, and will study the mechanisms of the particular neuronal vulnerability to cell death following ZIKV exposure. We will: 1] Test ZPO constituents to determine which are direct protease targets of ZIKV NS2B/NS3. 2] Test ZPO to determine mechanisms by which ZIKV NS2B/NS3-mediated cleavage leads to neuronal death. 3...