Vesicular Stomatitis Virus as a Platform for Protease Activity Measurements

Protease inhibitors are among the most powerful antiviral drugs. They have been used successfully against viruses, such as the human immunodeficiency virus (HIV), hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protease inhibitor screening tools are therefore important to identify inhibitors that have the potential to become antiviral drugs. In this article, we describe newly developed cell- and virus replicon-based platforms to screen inhibitors. We developed the methods presented here by genetically modifying vesicular stomatitis virus, a model virus from the family Rhabdoviridae. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: M^pro-On and -Off assay Alternate Protocol 1: Virus production with transient P- and L TransIT transfection Alternate Protocol 2: Virus production with transient P- and L Ca₂PO₄ transfection Alternate Protocol 3: Luciferase-based variation of the On assay Alternate Protocol 4: Screening assay with fluorescence-activated cell sorting readout Support Protocol: Performing kinetic measurements with Off assay.