COVID-19 E protein biochemical studies

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

This is a new project undertaken this year based on identified knowledge gaps in the roles served by E protein in coronavirus infections. Because our laboratory has the required technologies, we have initiated the following specific aims within this project. AIM 1: Use electrophysiology to determine essential transport properties of SARS-CoV-2 E protein. We are using recombinant E protein in electrophysiology experiments to define the essential transport properties of this protein encoded by the SARS-CoV-2 coronavirus. Our experiments are designed to directly establish whether purified E protein can form ion channels, to use single-channel studies to measure ion flux rates and the pores preference for specific ions. These selectivity measurements are essential for defining the role and mechanism of viroporin action in SARS-CoV-2 replication and disease pathogenesis. AIM 2: Develop and miniaturize a mammalian cell-based, target-specific reporter assay for viroporin activity. We are developing a cell-based assay for E protein activity suitable for measurements in individual wells of a 384-well microplate. AIM 3: Execute a high-throughput screen for small molecule inhibitors. A chemical screen will be executed using the above cell-based assay to find potent inhibitors of the E protein channel. AIM 4: Evaluate inhibitor-mediated block of viral replication in cultured mammalian cells via the NIAID SARS-CoV-2 virology core (SVC). As inhibitors are identified, we will collaborate with the SARS-CoV-2 Virology Core (SVC) for BSL-3 studies to evaluate in vitro and in vivo inhibition of viral replication by viroporin inhibitors. These studies will guide the selection of compounds for advancement into hit-to-lead chemistry and the antiviral drug development pipeline.