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Description
We have previously developed an assay for HIV-1 Protease (PR) in a Gal4 fusion context in T-cells. The previous assay exploits the autocatalytic properties of PR, and Gal4, a transcription factor that is only active when the terminal DNA binding and trans-activating domain (DBD, TAD) are linked. When the protease within the fusion is inhibited, the Gal4/protease fusion remains uncleaved activating the reporter Green Fluorescent Protein (GFP). When protease is active, the Gal4/protease fusion is cleaved and will not induce the expression of GFP, allowing the monitoring of protease activity based on expression of GFP. Like HIV, West Nile Virus (WNV), and other members of the Flaviviridae, rely on autocatalytic cleavage of their proteome, and inhibition of this step in the viral life-cycle prevents infection, making inhibition of protease an attractive target for drug discovery. This assay adapted for WNV Protease provides a platform for the high-throughput screening for novel inhibitors of WNV Protease utilizing flow cytometry and/or plate reader based technologies. In addition, this adaptation provides the proof of concept for the utility of the Gal4/protease fusion system for protease of Flaviviridae, which in contrast to HIV-1, requires a cofactor for full activity. Moreover, the assay was developed in adherent cells rather than in T-cells, providing a more natural milieu to mimic flaviviral infection. A processive RNA-dependent RNA polymerase prone to errors, the emergence of resistant strains, and lack of vaccines, highlight the need for novel antivirals and innovative methods to facilitate their discovery. While the main purpose of the assay is to drastically facilitate drug discovery, it can also be used to study the interaction of protease, cofactor, and other host or viral factors, required for protease activity