Dengue Virus (DenV), considered now an emerging viral pathogen in the US, is responsible for causing Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome, which are often fatal. Currently the only treatment for DenV infection is supportive care. This form of treatment is non-specific and has no direct effect on the virus. Therefore, there is an increasing need for safe and accurate assays to search for novel inhibitors that specifically target DenV. DenV belongs to the Flaviviridae family. It is a positive sense, single-stranded RNA virus that is targeted to, translated, and anchored in the Endoplasmic Reticulum (ER) membrane of the infected cell. Once translated into a single polyprotein that weaves in and out of the ER membrane, proteolytic processing must occur by both cellular and virally encoded proteases. The N terminal one third of the Non-Structural (NS) Protein 3, and the central hydrophilic region of the NS2B cofactor comprise the domains for protease activity of the viral protease. Cleavage of viral targets by NS2B/NS3 occurs in the cytosolic side of the ER membrane and is essential for the maturation of new virions, making the viral protease an ideal target for DenV antivirals. The goal of this project is to develop a cell-based assay that: a) monitors protease activity in the natural cellular compartment: the cytosol, and b) investigates the ER anchoring properties of viral proteins and their domains. The intricate topology of DenV and its complex protease activity will be exploited for the engineering of the assay. The assay is based on the engineering of a fusion protein comprised of an anchoring domain of viral origin fused to the yeast transcription factor Gal4 through a sequence that serves as a putative protease substrate. In the laboratory, we routinely utilize the DNA binding and transcription activation domains of Gal4 to drive Gal4-dependent green fluorescent protein expression. The assay was developed to ensure that the viral protease, which is supplied in cis, acts not only in a cellular milieu of infection, but also in the natural compartment. Flow cytometry and microscopy-based techniques are used to show protein localization with or without cleavage. This assay will be further developed as a platform for high-throughput screening of novel protease substrates as well as for drug discovery of novel protease inhibitors.