Detecting SARS-CoV-2 3CLpro expression and activity using a polyclonal antiserum and a luciferase-based biosensor

• Published in: Virology (New York, N.Y.) Vol. 556; pp. 73 - 78
• Main Authors: O'Brien, Amornrat, Chen, Da-Yuan, Hackbart, Matthew, Close, Brianna J., O'Brien, Timothy E., Saeed, Mohsan, Baker, Susan C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2021
• Subjects: 3CLpro; Animals; antiserum; antiviral agents; Antiviral Agents - pharmacology; assays; Biosensing Techniques - methods; biosensors; Coronavirus 3C Proteases - antagonists & inhibitors; Coronavirus 3C Proteases - genetics; Coronavirus 3C Proteases - immunology; Coronavirus 3C Proteases - metabolism; Coronavirus infections; Cross Reactions; drug development; enzyme activity; Immune Sera - immunology; Infectious Disease; Luciferases - genetics; Luciferases - metabolism; Main protease; Mpro; nsp5; pathogenesis; pGlo biosensor; polyclonal antibodies; polyproteins; Protease inhibitors; Protease Inhibitors - pharmacology; proteinase inhibitors; proteinases; Rabbits; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; SARS-CoV-2; SARS-CoV-2 - drug effects; SARS-CoV-2 - genetics; SARS-CoV-2 - immunology; SARS-CoV-2 - metabolism; Severe acute respiratory syndrome coronavirus; Severe acute respiratory syndrome-related coronavirus - immunology; Severe acute respiratory syndrome-related coronavirus - metabolism; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - immunology; Viral Nonstructural Proteins - metabolism; virology; Virus Replication - drug effects; SARS-CoV-2; Main protease; Mpro; Protease inhibitors; pGlo biosensor; nsp5; 3CLpro
• ISSN: 0042-6822; 1096-0341; 1096-0341
• DOI: 10.1016/j.virol.2021.01.010

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors. •Coronavirus 3CLpro activity is efficiently measured using a luciferase-based biosensor.•Small molecule inhibitors of 3CLpro block activation of the biosensor.•Polyclonal antiserum raised against SARS-CoV 3CLpro recognizes SARS-CoV-2 3CLpro in WB and IF assays.