Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity
Abstract Targeting RNA-dependent RNA polymerase (RdRp), a highly conserved enzyme essential for SARS coronavirus 2 (SARS-CoV-2) replication and transcription, represents a promising antiviral strategy due to its lower mutation rate than structural proteins such as Spike. This study introduces a cell...
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Nature Portfolio
2025-05-01
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| Online Access: | https://doi.org/10.1038/s41598-025-03813-y |
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| author | Eunjeong Kang Haelim Yoon Junho Lee JinAh Lee Seungtaek Kim Inseong Jo Soo Bong Han Dae Gwin Jeong Sayeon Cho |
| author_facet | Eunjeong Kang Haelim Yoon Junho Lee JinAh Lee Seungtaek Kim Inseong Jo Soo Bong Han Dae Gwin Jeong Sayeon Cho |
| author_sort | Eunjeong Kang |
| collection | DOAJ |
| description | Abstract Targeting RNA-dependent RNA polymerase (RdRp), a highly conserved enzyme essential for SARS coronavirus 2 (SARS-CoV-2) replication and transcription, represents a promising antiviral strategy due to its lower mutation rate than structural proteins such as Spike. This study introduces a cell-based assay system for screening potential SARS-CoV-2 RdRp inhibitors, contributing to ongoing efforts to identify effective antiviral agents. The assay utilizes a reporter vector containing the 3′ untranslated region (UTR), luciferase reporter gene, and 5’ UTR gene, sequentially arranged in reverse under the control of the cytomegalovirus promoter in the pcDNA3.1 vector. Co-transfection with SARS-CoV-2 RdRp resulted an increase in luminescence-based quantification of RdRp activity, achieving a Z-factor of 0.605, indicative of high reproducibility and reliability for high-throughput screening. Established RdRp inhibitors, including remdesivir, molnupiravir, tenofovir, and sofosbuvir, significantly reduced reporter activity, with remdesivir exhibiting the strongest inhibition. A newly identified RdRp inhibitor was further validated through primer extension polymerase and NMPylation assays, along with virus-based experiments, confirming its inhibitory mechanism. These results highlight the utility of this screening system in identifying effective RdRp-targeting antivirals, reinforcing the strategic importance of RdRp inhibition in combating SARS-CoV-2 and emerging variants. |
| format | Article |
| id | doaj-art-8a2d283bedc642c498fcdd6d0919eadd |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
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| spelling | doaj-art-8a2d283bedc642c498fcdd6d0919eadd2025-08-20T02:03:31ZengNature PortfolioScientific Reports2045-23222025-05-0115111410.1038/s41598-025-03813-yConstruction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activityEunjeong Kang0Haelim Yoon1Junho Lee2JinAh Lee3Seungtaek Kim4Inseong Jo5Soo Bong Han6Dae Gwin Jeong7Sayeon Cho8Laboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang UniversityLaboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang UniversityLaboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang UniversityZoonotic Virus Laboratory, Institut Pasteur KoreaZoonotic Virus Laboratory, Institut Pasteur KoreaInfectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical TechnologyInfectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical TechnologyBionanotechnology Research Center, Korea Research Institute of Bioscience and BiotechnologyLaboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang UniversityAbstract Targeting RNA-dependent RNA polymerase (RdRp), a highly conserved enzyme essential for SARS coronavirus 2 (SARS-CoV-2) replication and transcription, represents a promising antiviral strategy due to its lower mutation rate than structural proteins such as Spike. This study introduces a cell-based assay system for screening potential SARS-CoV-2 RdRp inhibitors, contributing to ongoing efforts to identify effective antiviral agents. The assay utilizes a reporter vector containing the 3′ untranslated region (UTR), luciferase reporter gene, and 5’ UTR gene, sequentially arranged in reverse under the control of the cytomegalovirus promoter in the pcDNA3.1 vector. Co-transfection with SARS-CoV-2 RdRp resulted an increase in luminescence-based quantification of RdRp activity, achieving a Z-factor of 0.605, indicative of high reproducibility and reliability for high-throughput screening. Established RdRp inhibitors, including remdesivir, molnupiravir, tenofovir, and sofosbuvir, significantly reduced reporter activity, with remdesivir exhibiting the strongest inhibition. A newly identified RdRp inhibitor was further validated through primer extension polymerase and NMPylation assays, along with virus-based experiments, confirming its inhibitory mechanism. These results highlight the utility of this screening system in identifying effective RdRp-targeting antivirals, reinforcing the strategic importance of RdRp inhibition in combating SARS-CoV-2 and emerging variants.https://doi.org/10.1038/s41598-025-03813-ySARS-CoV-2RdRpHigh-throughput screeningCell-based reporter assaySARS-CoV-2 RdRp inhibitor |
| spellingShingle | Eunjeong Kang Haelim Yoon Junho Lee JinAh Lee Seungtaek Kim Inseong Jo Soo Bong Han Dae Gwin Jeong Sayeon Cho Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity Scientific Reports SARS-CoV-2 RdRp High-throughput screening Cell-based reporter assay SARS-CoV-2 RdRp inhibitor |
| title | Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity |
| title_full | Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity |
| title_fullStr | Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity |
| title_full_unstemmed | Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity |
| title_short | Construction and validation of a cell based reporter assay for identifying inhibitors of SARS coronavirus 2 RNA dependent RNA polymerase activity |
| title_sort | construction and validation of a cell based reporter assay for identifying inhibitors of sars coronavirus 2 rna dependent rna polymerase activity |
| topic | SARS-CoV-2 RdRp High-throughput screening Cell-based reporter assay SARS-CoV-2 RdRp inhibitor |
| url | https://doi.org/10.1038/s41598-025-03813-y |
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