{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T06:03:34Z","timestamp":1771049014764,"version":"3.50.1"},"reference-count":39,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T00:00:00Z","timestamp":1770854400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["ITAC grant"],"award-info":[{"award-number":["ITAC grant"]}]},{"name":"NIH","award":["Intramural research"],"award-info":[{"award-number":["Intramural research"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Coronaviruses pose a global pandemic threat, making development of a pan-coronavirus inhibitor crucial for preparedness and containment in the event of a new coronavirus outbreak. The 3C-like protease (3CLpro) is a key target for antiviral development, as it is essential for viral replication and conserved across human coronaviruses. We previously developed an assay to monitor SARS-CoV-2 3CLpro activity in cells. This assay uses a single vector that coexpresses the 3CLpro enzyme and the reporter, which consists of two luciferase fragments linked by a 3CLpro cleavage site. Cleavage of this site by 3CLpro decreases luciferase activity, whereas inhibition of 3CLpro increases the luciferase activity. Here, we adapted this assay to examine 3CLpro activity from six other human coronaviruses: SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. We further determined the effects of different cleavage sites to improve the signal-to-background ratio. The Nsp4-Nsp5 site and super-active substrate (SAS) resulted in the largest dynamic range for most coronaviruses in our assay. Using the broad-spectrum 3CLpro inhibitor GC376, we observed increased reporter activity, indicating the assay\u2019s efficacy for identifying inhibitors across multiple coronaviruses. The adaptation and improvement of the assay can facilitate the development of inhibitors against 3CLpro from multiple or novel coronaviruses.<\/jats:p>","DOI":"10.3390\/v18020234","type":"journal-article","created":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T17:42:13Z","timestamp":1770918133000},"page":"234","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Complementation Assay to Monitor Pan-Coronavirus 3C-like Protease Activity"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1944-6726","authenticated-orcid":false,"given":"Akhil","family":"Chameettachal","sequence":"first","affiliation":[{"name":"Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9297-6291","authenticated-orcid":false,"given":"Alice","family":"Duchon","sequence":"additional","affiliation":[{"name":"Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew A.","family":"Brown","sequence":"additional","affiliation":[{"name":"Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan M. O.","family":"Rawson","sequence":"additional","affiliation":[{"name":"Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vinay K.","family":"Pathak","sequence":"additional","affiliation":[{"name":"Viral Mutation Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei-Shau","family":"Hu","sequence":"additional","affiliation":[{"name":"Viral Recombination Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1038\/s41564-020-0695-z","article-title":"The Species Severe Acute Respiratory Syndrome-Related Coronavirus: Classifying 2019-nCoV and Naming It SARS-CoV-2","volume":"5","author":"Gorbalenya","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.1056\/NEJMoa030781","article-title":"A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome","volume":"348","author":"Ksiazek","year":"2003","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1814","DOI":"10.1056\/NEJMoa1211721","article-title":"Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia","volume":"367","author":"Zaki","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A Pneumonia Outbreak Associated with a New Coronavirus of Probable Bat Origin","volume":"579","author":"Zhou","year":"2020","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A Novel Coronavirus from Patients with Pneumonia in China, 2019","volume":"382","author":"Zhu","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_6","unstructured":"(2025, March 24). COVID-19 Cases|WHO COVID-19 Dashboard. Available online: https:\/\/data.who.int\/dashboards\/covid19\/cases."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1038\/s41392-020-0190-2","article-title":"Overview of Lethal Human Coronaviruses","volume":"5","author":"Chen","year":"2020","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.3201\/eid3006.231285","article-title":"Estimates of SARS-CoV-2 Hospitalization and Fatality Rates in the Prevaccination Period, United States","volume":"30","author":"Griffin","year":"2024","journal-title":"Emerg. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2940","DOI":"10.1128\/JCM.00636-10","article-title":"Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method","volume":"48","author":"Gaunt","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1038\/s41579-024-01036-y","article-title":"COVID-19 Drug Discovery and Treatment Options","volume":"22","author":"Chan","year":"2024","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1038\/s41564-023-01356-4","article-title":"Therapeutics for COVID-19","volume":"8","author":"Toussi","year":"2023","journal-title":"Nat. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/s41579-020-00468-6","article-title":"Coronavirus Biology and Replication: Implications for SARS-CoV-2","volume":"19","author":"Kratzel","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4085","DOI":"10.1111\/febs.12936","article-title":"From SARS to MERS: Crystallographic Studies on Coronaviral Proteases Enable Antiviral Drug Design","volume":"281","author":"Hilgenfeld","year":"2014","journal-title":"FEBS J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.1056\/NEJMoa2118542","article-title":"Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with COVID-19","volume":"386","author":"Hammond","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1763","DOI":"10.1126\/science.1085658","article-title":"Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs","volume":"300","author":"Anand","year":"2003","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"127377","DOI":"10.1016\/j.bmcl.2020.127377","article-title":"The SARS-CoV-2 Main Protease as Drug Target","volume":"30","author":"Ullrich","year":"2020","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Cihlova, B., Huskova, A., B\u00f6serle, J., Nencka, R., Boura, E., and Silhan, J. (2021). High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses. Molecules, 26.","DOI":"10.3390\/molecules26133792"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"101739","DOI":"10.1016\/j.jbc.2022.101739","article-title":"Improved SARS-CoV-2 Main Protease High-Throughput Screening Assay Using a 5-Carboxyfluorescein Substrate","volume":"298","author":"Legare","year":"2022","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1038\/s42003-020-01577-x","article-title":"Identification of 3-Chymotrypsin like Protease (3CLPro) Inhibitors as Potential Anti-SARS-CoV-2 Agents","volume":"4","author":"Mody","year":"2021","journal-title":"Commun. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1186\/s13578-021-00720-3","article-title":"Development of a Simple and Miniaturized Sandwich-like Fluorescence Polarization Assay for Rapid Screening of SARS-CoV-2 Main Protease Inhibitors","volume":"11","author":"Yan","year":"2021","journal-title":"Cell Biosci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e01265-20","DOI":"10.1128\/JVI.01265-20","article-title":"Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CLpro Reporter Assay","volume":"94","author":"Froggatt","year":"2020","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1038\/s42003-022-03090-9","article-title":"Identification of SARS-CoV-2 Inhibitors Targeting Mpro and PLpro Using in-Cell-Protease Assay","volume":"5","author":"Narayanan","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1007\/s12033-021-00299-7","article-title":"Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-CoV-2 Main Protease (3CLpro)","volume":"63","author":"Rothan","year":"2021","journal-title":"Mol. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"20697","DOI":"10.1038\/s41598-024-71305-6","article-title":"A Gaussia Luciferase Reporter Assay for the Evaluation of Coronavirus Nsp5\/3CLpro Activity","volume":"14","author":"Vlachou","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6786","DOI":"10.1038\/s41467-021-27072-3","article-title":"Design of Modular Autoproteolytic Gene Switches Responsive to Anti-Coronavirus Drug Candidates","volume":"12","author":"Franko","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1038\/s42003-022-03277-0","article-title":"A VSV-Based Assay Quantifies Coronavirus Mpro\/3CLpro\/Nsp5 Main Protease Activity and Chemical Inhibition","volume":"5","author":"Heilmann","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2211688","DOI":"10.1080\/22221751.2023.2211688","article-title":"Orthogonal Dual Reporter-Based Gain-of-Signal Assay for Probing SARS-CoV-2 3CL Protease Activity in Living Cells: Inhibitor Identification and Mutation Investigation","volume":"12","author":"Bei","year":"2023","journal-title":"Emerg. Microbes Infect."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"105183","DOI":"10.1016\/j.antiviral.2021.105183","article-title":"A Fluorescence-Based, Gain-of-Signal, Live Cell System to Evaluate SARS-CoV-2 Main Protease Inhibition","volume":"195","author":"Smith","year":"2021","journal-title":"Antivir. Res."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Rawson, J.M.O., Duchon, A., Nikolaitchik, O.A., Pathak, V.K., and Hu, W.-S. (2021). Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors. Viruses, 13.","DOI":"10.3390\/v13020173"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Chuck, C.-P., Chow, H.-F., Wan, D.C.-C., and Wong, K.-B. (2011). Profiling of Substrate Specificities of 3C-like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0027228"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1021\/acschembio.5b00753","article-title":"NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells","volume":"11","author":"Dixon","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4417","DOI":"10.1038\/s41467-020-18233-x","article-title":"Both Boceprevir and GC376 Efficaciously Inhibit SARS-CoV-2 by Targeting Its Main Protease","volume":"11","author":"Fu","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11754","DOI":"10.1128\/JVI.01348-12","article-title":"Broad-Spectrum Antivirals against 3C or 3C-Like Proteases of Picornaviruses, Noroviruses, and Coronaviruses","volume":"86","author":"Kim","year":"2012","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4282","DOI":"10.1038\/s41467-020-18096-2","article-title":"Feline Coronavirus Drug Inhibits the Main Protease of SARS-CoV-2 and Blocks Virus Replication","volume":"11","author":"Vuong","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e02374-20","DOI":"10.1128\/JVI.02374-20","article-title":"Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity","volume":"95","author":"Resnick","year":"2021","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e00784-22","DOI":"10.1128\/mbio.00784-22","article-title":"Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro\/3CLpro in Living Cells","volume":"13","author":"Moghadasi","year":"2022","journal-title":"mBio"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1637","DOI":"10.1074\/jbc.M310875200","article-title":"Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase","volume":"279","author":"Fan","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.virusres.2015.05.018","article-title":"Prediction and Biochemical Analysis of Putative Cleavage Sites of the 3C-like Protease of Middle East Respiratory Syndrome Coronavirus","volume":"208","author":"Wu","year":"2015","journal-title":"Virus Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1586","DOI":"10.1126\/science.abl4784","article-title":"An Oral SARS-CoV-2 Mpro Inhibitor Clinical Candidate for the Treatment of COVID-19","volume":"374","author":"Owen","year":"2021","journal-title":"Science"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/2\/234\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T05:14:57Z","timestamp":1771046097000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/2\/234"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,12]]},"references-count":39,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["v18020234"],"URL":"https:\/\/doi.org\/10.3390\/v18020234","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,12]]}}}