Most viruses rely on the processing of their proteome by viral and/or host proteases (PRs). This is the case of Zika Virus (ZIKV), a member of the Flaviviridae family of viruses, a widespread arthropod-borne viral infection of humans. ZIKV currently has no effective vaccines or antiviral drug treatments available. Developing antiviral drugs that target ZIKV NS2B/NS3 PR could be one of the most successful strategies in combating ZIKV pathogenesis and spread. The primary goal of this project is to develop an assay that will provide a robust, cell-based, high throughput method to quickly identify novel ZIKV PR inhibitors Hilton and Wolkowicz developed a cell-based assay to monitor the catalytic activity of Human Immunodeficiency Virus-1 (HIV-1) PR in T-cells. The assay relies on three components: a Gal4/PR transcription factor fusion, a reverse tetracycline transactivator (rtTA) off/on system for inducible activation in the presence of doxycycline (Dox), and the green fluorescent protein (GFP) under control by the Gal4 promoter. This project will adapt the assay from HIV-1 PR to the NS2B/NS3 complex of ZIKV and monitor the proteolytic activity via a similar GFP reporter system. In this system, an active PR can cleave and disrupt the Gal4/PR fusion, which results in loss of GFP expression. On the contrary, when an active PR is inhibited or mutated, the Gal4 fusion remains intact, leading to GFP expression. In addition, all the necessary components of the reporter system and the ZIKV Gal4/PR fusion will be stably expressed in a non-adherent T-cell line. The final version of the assay will be a cell-based, high throughput system designed to efficiently screen hundreds of thousands of candidate inhibitors of ZIKV PR activity. In addition, the assay can be utilized to further establish the cellular localization of various NS2B transmembrane deletion mutants, how these mutants effect NS2B/NS3 activity ex vivo, and possibly discover novel cleavage targets of NS2B/NS3.