{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T07:12:58Z","timestamp":1778656378762,"version":"3.51.4"},"posted":{"date-parts":[[2026,5,12]]},"group-title":"Microbiology","reference-count":0,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T00:00:00Z","timestamp":1778544000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,12]]},"abstract":"<jats:p>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 in vitro. 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.<\/jats:p>","DOI":"10.64898\/2026.05.12.724615","type":"posted-content","created":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T06:40:16Z","timestamp":1778654416000},"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":"10.64898","author":[{"given":"Li","family":"He","sequence":"first","affiliation":[{"name":"The University of Texas Health Science Center at San Antonio Joe R and Teresa Lozano Long School of Medicine;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuan-Wei Norman","family":"Su","sequence":"additional","affiliation":[{"name":"The University of North Carolina at Chapel Hill School of Medicine;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fushun","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Texas Health Science Center at San Antonio;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ibrahim","family":"Moustafa Abdelrady","sequence":"additional","affiliation":[{"name":"Pennsylvania State University;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David W","family":"Gohara","sequence":"additional","affiliation":[{"name":"Research Computing Consultants, LLC;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1934-9494","authenticated-orcid":false,"given":"Chengjin","family":"Ye","sequence":"additional","affiliation":[{"name":"Texas Biomedical Research Institute;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7084-0804","authenticated-orcid":false,"given":"Luis","family":"Martinez-Sobrido","sequence":"additional","affiliation":[{"name":"Texas Biomedical Research Institute;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jamie J","family":"Arnold","sequence":"additional","affiliation":[{"name":"The University of North Carolina at Chapel Hill School of Medicine;"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7564-5642","authenticated-orcid":false,"given":"Craig E.","family":"Cameron","sequence":"additional","affiliation":[{"name":"University of North Carolina at Chapel Hill"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7633-1629","authenticated-orcid":false,"given":"Yan","family":"Xiang","sequence":"additional","affiliation":[{"name":"The University of Texas Health Science Center at San Antonio Joe R and Teresa Lozano Long School of Medicine;"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.12.724615","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T06:40:16Z","timestamp":1778654416000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.12.724615"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,12]]},"references-count":0,"URL":"https:\/\/doi.org\/10.64898\/2026.05.12.724615","relation":{},"subject":[],"published":{"date-parts":[[2026,5,12]]},"subtype":"preprint"}}