Abstract

Two Cellular Compartments for the Screening of Antivirals Against Two Processing-related Targets

Roland Wolkowicz, Associate Professor, San Diego State University

Viral life cycles are complex and intertwined with the host cellular machineries. Processing of the viral proteome, for example, relies on both viral and host proteolytic enzymes and viruses have thus evolved to contain sites which are recognized and cleaved in different cellular compartments. For instance, viruses like HIV-1 and the Flaviviridae, including Dengue, Hepatitis C, Yellow Fever and Wet Nile viruses, rely on their own protease, active in a cytosolic environment, and on enzymes that reside in the luminal compartment of the Endoplasmic Reticulum/Golgi/TransGolgi Network of the classical secretory pathway. In order to monitor the activity of enzymes involved in the processing of the viral proteome, we have developed two distinct cell-based assays. The first monitors the activity of the viral protease in the cytosolic environment and relies on a Gal4-fusion protein that travels to the nucleus and activates the green fluorescent protein gene. The second monitors the activity of the cellular enzymes within the secretory pathway and relies on a two-tag system that travels to the cell surface and is assessed by antibody staining. The two independent assays were proven to be robust and suitable for the screening of antivirals targeting proteome processing of important human viral pathogens.