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: Mpro‐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