Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7
RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its in...
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Elsevier
2025-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S216225312500006X |
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| author | Deepa Singh Tushar Kushwaha Rajkumar Kulandaisamy Vikas Kumar Kamal Baswal Saras H. Tiwari Arkadyuti Ghorai Manoj Kumar Saroj Kumar Soumya De Aparoy Polamarasetty Deepak Sehgal Madhumohan R. Katika Suresh Gadde Marceline Côté Sarala R. Kayampeta Mohan Babu Appaiahgari Krishna K. Inampudi |
| author_facet | Deepa Singh Tushar Kushwaha Rajkumar Kulandaisamy Vikas Kumar Kamal Baswal Saras H. Tiwari Arkadyuti Ghorai Manoj Kumar Saroj Kumar Soumya De Aparoy Polamarasetty Deepak Sehgal Madhumohan R. Katika Suresh Gadde Marceline Côté Sarala R. Kayampeta Mohan Babu Appaiahgari Krishna K. Inampudi |
| author_sort | Deepa Singh |
| collection | DOAJ |
| description | RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication. |
| format | Article |
| id | doaj-art-3d9c0632dcb542908910f303cd194fec |
| institution | Kabale University |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-3d9c0632dcb542908910f303cd194fec2025-01-30T05:14:01ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-03-01361102452Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7Deepa Singh0Tushar Kushwaha1Rajkumar Kulandaisamy2Vikas Kumar3Kamal Baswal4Saras H. Tiwari5Arkadyuti Ghorai6Manoj Kumar7Saroj Kumar8Soumya De9Aparoy Polamarasetty10Deepak Sehgal11Madhumohan R. Katika12Suresh Gadde13Marceline Côté14Sarala R. Kayampeta15Mohan Babu Appaiahgari16Krishna K. Inampudi17Department of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaDepartment of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, IndiaSchool of Bioscience, Indian Institute of Technology Kharagpur, Kharagpur 721302, IndiaFaculty of Biology, Indian Institute of Petroleum & Energy, Visakhapatnam, Andhra Pradesh 530003, IndiaVirology Lab, Department of Life Sciences, Shiv Nadar University, Greater Noida, Uttar Pradesh 201314, IndiaESIC Medical College and Hospital, Sanath Nagar, Hyderabad, Telangana 500038, IndiaDepartment of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, CanadaDepartment of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, CanadaR&D Division, Srikara Biologicals Private Limited, Tirupati, Andhra Pradesh 517507, IndiaR&D Division, Srikara Biologicals Private Limited, Tirupati, Andhra Pradesh 517507, India; Yenepoya (deemed to be) University, University Road, Deralakatte, Mangalore, Karnataka 575018, India; Corresponding author: Dr. Mohan Babu Appaiahgari, PhD, Associate Professor, Virology Laboratory, Yenepoya (Deemed to be University), Deralakatte, Mangaluru, Karnataka 575018, India.Department of Biophysics, All India Institute of Medical Sciences, New Delhi, Delhi 110029, India; Corresponding author: Dr. Krishna K. Inampudi, PhD, Additional Professor, Dept. of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, Delhi 110029, India.RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication.http://www.sciencedirect.com/science/article/pii/S216225312500006XMT: Oligonucleotides: Therapies and ApplicationsSARS-CoV-2replication-transcription complexRdRpholoenzymeNSP12 |
| spellingShingle | Deepa Singh Tushar Kushwaha Rajkumar Kulandaisamy Vikas Kumar Kamal Baswal Saras H. Tiwari Arkadyuti Ghorai Manoj Kumar Saroj Kumar Soumya De Aparoy Polamarasetty Deepak Sehgal Madhumohan R. Katika Suresh Gadde Marceline Côté Sarala R. Kayampeta Mohan Babu Appaiahgari Krishna K. Inampudi Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Therapies and Applications SARS-CoV-2 replication-transcription complex RdRp holoenzyme NSP12 |
| title | Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 |
| title_full | Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 |
| title_fullStr | Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 |
| title_full_unstemmed | Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 |
| title_short | Redefining NSP12 activity in SARS-CoV-2 and its regulation by NSP8 and NSP7 |
| title_sort | redefining nsp12 activity in sars cov 2 and its regulation by nsp8 and nsp7 |
| topic | MT: Oligonucleotides: Therapies and Applications SARS-CoV-2 replication-transcription complex RdRp holoenzyme NSP12 |
| url | http://www.sciencedirect.com/science/article/pii/S216225312500006X |
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