GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE328nnn/GSE328556/primaryOK200TranscriptomicsSevere acute respiratory syndrome coronavirus 2Expression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE328556GEOGSEfalseRecovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viabilityCoronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3′-to-5′ 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.2026/06/16GSE328556GSM9685249GSM9685248GSM9685247GSM9685246GSM9685245GSM9685244GSM9685243GSM9685242GSM9685241GSM9685240GSM9685260GSM9685239GSM9685238GSM9685259GSM9685237GSM9685258GSM9685236GSM9685257GSM9685235GSM9685256GSM9685234GSM9685233GSM9685255GSM9685254GSM9685232GSM9685253GSM9685231GSM9685230GSM9685252GSM9685251GSM968525029240328556Severe acute respiratory syndrome coronavirus 2