{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:28:56Z","timestamp":1787059736851,"version":"build-2736575974"},"reference-count":48,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T00:00:00Z","timestamp":1787011200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T00:00:00Z","timestamp":1787011200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["U19AI171421"],"award-info":[{"award-number":["U19AI171421"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI161841"],"award-info":[{"award-number":["R01AI161841"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Virol"],"published-print":{"date-parts":[[2026,8,18]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3\u2032-to-5\u2032 exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A\/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A\/E92A mutant was recovered only after an extended delay, and K9E could not be rescued despite repeated attempts. The D90A\/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A\/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Coronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened because earlier studies suggested that loss of the enzyme\u2019s catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00809-26","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T14:49:59Z","timestamp":1785336599000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Recovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viability"],"prefix":"10.1128","volume":"100","author":[{"given":"Li","family":"He","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02f6dcw23","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio","place":["San Antonio, 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