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ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Virology
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1560251
This article is part of the Research Topic Research Advances and Challenges in Emerging and Re-Emerging Viral Diseases View all 16 articles
Development of a Highly Sensitive Luciferase Assay for Intracellular Evaluation of Coronavirus Mpro Activity
Provisionally accepted
Bao Dong 1
Yuehong Chen 2
Jing Li 2
Sen Zhang 2
Xiaoping Kang 2 Yuchang Li 2 Biao Li 1 Liangning Liao 3 Zhengwei Zhang 3 Jiaqi Xiong 1 Lele Shao 4
Shenghai Huang 1* Ye Feng 2*
Tao Jiang 1,2,3,4*- 1 School of Basic Medical Sciences, Anhui Medical University, Hefei, China
- 2 State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Science, Beijing, Beijing Municipality, China
- 3 School of Public Health Mudanjiang Medical University, Mudanjiang, China
- 4 College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, China
COVID-19, caused by SARS-CoV-2 virus, has emerged as a global threat to human health. The main protease (Mpro) of SARS-CoV-2 is an excellent target for the development of antiviral drugs against COVID-19, and various protease biosensors have been developed to evaluate anti-coronavirus drugs. However, the application of these protease biosensors was limited due to high background fluorescence, poor signal-to-noise ratios, and constraints in enzyme activity thresholds for accessing live viruses. In this study, we rationally designed a highly conserved Mpro cleavage site sequence among different coronaviruses (CoVs) with high proteolytic activity, and described an intracellular coronavirus Mpro proteolytic (ICMP) reporter system that takes advantage of virus-encoded Mpro expressed in infected cells to reform the NanoBiT fluorescent protein. The system can be used to visualize and identify cells infected with coronavirus, and demonstrated high compatibility with various Mpro proteins from 13 different mammalian coronaviruses (covering α, β, γ, and δ CoVs), exhibiting at least a 1,030-fold increase in luminescence. Stronger Nluc signals were detectable with CoV 229E virus infection at a MOI of 0.001. Additionally, the system proved suitable for evaluating and screening of antiviral compounds, including lufotrelvir, GC376, Nirmatrelvir, X77, MG-101, and the potential inhibitor Cynaroside.The ICMP system is not only an invaluable tool for the detection of live coronaviruses, but also for the discovery of antivirals against current and future pandemic coronaviruses.
Keywords: Coronavirus, main protease, protease activity, Reporter system, NanoBit, protease inhibitor
Received: 14 Jan 2025; Accepted: 06 Mar 2025.
Copyright: © 2025 Dong, Chen, Li, Zhang, Kang, Li, Li, Liao, Zhang, Xiong, Shao, Huang, Feng and Jiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Shenghai Huang, School of Basic Medical Sciences, Anhui Medical University, Hefei, China
Ye Feng, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Science, Beijing, Beijing Municipality, China
Tao Jiang, School of Basic Medical Sciences, Anhui Medical University, Hefei, China
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