{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,13]],"date-time":"2025-12-13T05:22:57Z","timestamp":1765603377216,"version":"3.48.0"},"reference-count":95,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T00:00:00Z","timestamp":1765411200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001656","name":"Helmholtz Association","doi-asserted-by":"publisher","award":["VH-NG-1347"],"award-info":[{"award-number":["VH-NG-1347"]}],"id":[{"id":"10.13039\/501100001656","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001656","name":"Helmholtz Association","doi-asserted-by":"publisher","award":["VH-NG-128"],"award-info":[{"award-number":["VH-NG-128"]}],"id":[{"id":"10.13039\/501100001656","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001665","name":"Thematic Institute IMCBio+","doi-asserted-by":"publisher","award":["ANR-10-IDEX-0002"],"award-info":[{"award-number":["ANR-10-IDEX-0002"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001665","name":"Thematic Institute IMCBio+","doi-asserted-by":"publisher","award":["ANR 20-SFRI-0012"],"award-info":[{"award-number":["ANR 20-SFRI-0012"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001665","name":"Thematic Institute IMCBio+","doi-asserted-by":"publisher","award":["ANR-17-EURE-0023"],"award-info":[{"award-number":["ANR-17-EURE-0023"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Helmholtz Institute for RNA-based Infection Research (HIRI) Graduate Program"},{"name":"ERC Starting Grant","award":["101040914"],"award-info":[{"award-number":["101040914"]}]},{"DOI":"10.13039\/100004410","name":"European Molecular Biology Organization","doi-asserted-by":"crossref","award":["ALTF 1260-2020"],"award-info":[{"award-number":["ALTF 1260-2020"]}],"id":[{"id":"10.13039\/100004410","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host\u2013virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5\u2032-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5\u2032-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development.<\/jats:p>","DOI":"10.3390\/v17121604","type":"journal-article","created":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T14:30:17Z","timestamp":1765463417000},"page":"1604","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["High-Fidelity and Cost-Effective Engineering of SARS-CoV-2"],"prefix":"10.3390","volume":"17","author":[{"given":"Marco","family":"Olguin-Nava","sequence":"first","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Institute of Virology, Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Hennig","sequence":"additional","affiliation":[{"name":"Institute of Virology, Hannover Medical School, 30625 Hannover, Germany"},{"name":"Institute of Virology and Immunology, University of W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlene","family":"B\u00f6rtlein","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8515-2067","authenticated-orcid":false,"given":"Patrick","family":"Bohn","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7266-1324","authenticated-orcid":false,"given":"Uddhav B.","family":"Ambi","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8064-3289","authenticated-orcid":false,"given":"Alexander","family":"Gabel","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Institute of Medical Virology, Goethe-University Frankfurt, 60596 Frankfurt am Main, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1344-8844","authenticated-orcid":false,"given":"Lina M.","family":"G\u00fcnter","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anne-Sophie","family":"Gribling-Burrer","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Architecture et R\u00e9activit\u00e9 de l\u2032ARN, Institute of Molecular and Cellular Biology, CNRS, University of Strasbourg, 67084 Strasbourg, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3232-3491","authenticated-orcid":false,"given":"Nora","family":"Schmidt","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Institute of Virology, Hannover Medical School, 30625 Hannover, Germany"},{"name":"Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neva","family":"Caliskan","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Faculty of Medicine, University of W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"},{"name":"Faculty of Biology and Preclinical Medicine, University of Regensburg, 93053 Regensburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lars","family":"D\u00f6lken","sequence":"additional","affiliation":[{"name":"Institute of Virology, Hannover Medical School, 30625 Hannover, Germany"},{"name":"Institute of Virology and Immunology, University of W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"},{"name":"Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mathias","family":"Munschauer","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Institute of Medical Virology, Goethe-University Frankfurt, 60596 Frankfurt am Main, Germany"},{"name":"Heidelberg University, Medical Faculty Heidelberg, Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Disease Research, 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1580-0671","authenticated-orcid":false,"given":"Redmond P.","family":"Smyth","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-Based Infection Research, Helmholtz Centre for Infection Research, 97080 W\u00fcrzburg, Germany"},{"name":"Architecture et R\u00e9activit\u00e9 de l\u2032ARN, Institute of 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