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Here, we explored a dual approach combining structure-guided peptides predicted to interfere with the viral replication complex with lethal mutagenesis to limit SARS-CoV-2 replication. Using the crystallographic interfaces of nsp10 with nsp14 and nsp16, we designed short inhibitory peptides predicted to interact with the viral proofreading and RNA-capping machinery. In parallel, the mutagenic analog 5-fluorouracil was evaluated to determine its effect on SARS-CoV-2 in Vero E6 cells. Peptides P1 and P6 exhibited potent antiviral activity with minimal cytotoxicity, whereas 5-FU reduced specific infectivity without impairing genome replication. Combined treatment with 5-FU and peptide P1 resulted in &gt;10\n                      <jats:sup>4<\/jats:sup>\n                      -fold reduction in infectious virus, achieving near-complete loss of infectivity at non-cytotoxic concentrations. Next-generation sequencing revealed that dual treatment increased mutation frequency, altered mutant spectra, and decreased genome stability, consistent with progression toward error catastrophe. Principal component analysis confirmed that combined treatment generated mutant spectra distinct from either monotherapy. These findings are consistent with a dual antiviral strategy in which structure-guided peptides designed to interact with components of the SARS-CoV-2 replication complex act in combination with lethal mutagenesis to produce a synergistic interaction between these two complementary processes. This integrated approach suggests a potential broad-spectrum antiviral strategy with applicability to other coronaviruses.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aac.01885-25","type":"journal-article","created":{"date-parts":[[2026,4,27]],"date-time":"2026-04-27T09:07:37Z","timestamp":1777280857000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Synergistic antiviral effects of structure-guided peptides and a mutagenic base analog on SARS-CoV-2 replication"],"prefix":"10.1128","volume":"70","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3975-6951","authenticated-orcid":true,"given":"Sergio","family":"Ortega Del Campo","sequence":"first","affiliation":[{"name":"\u00c1rea de Gen\u00e9tica, Facultad de Ciencias, Campus de Teatinos, Universidad de M\u00e1laga","place":["M\u00e1laga, 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