{"database":"GEO","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE328nnn/GSE328556/"]},"type":"primary"},"statusCodeValue":200,"statusCode":"OK"}],"scores":null,"additional":{"omics_type":["Transcriptomics"],"species":["Severe acute respiratory syndrome coronavirus 2"],"gds_type":["Expression profiling by high throughput sequencing"],"full_dataset_link":["https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE328556"],"repository":["GEO"],"entry_type":["GSE"],"additional_accession":[]},"is_claimable":false,"name":"Recovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viability","description":"Coronaviruses (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.","dates":{"publication":"2026/06/16"},"accession":"GSE328556","cross_references":{"GSM":["GSM9685249","GSM9685248","GSM9685247","GSM9685246","GSM9685245","GSM9685244","GSM9685243","GSM9685242","GSM9685241","GSM9685240","GSM9685260","GSM9685239","GSM9685238","GSM9685259","GSM9685237","GSM9685258","GSM9685236","GSM9685257","GSM9685235","GSM9685256","GSM9685234","GSM9685233","GSM9685255","GSM9685254","GSM9685232","GSM9685253","GSM9685231","GSM9685230","GSM9685252","GSM9685251","GSM9685250"],"GPL":["29240"],"GSE":["328556"],"taxon":["Severe acute respiratory syndrome coronavirus 2"]}}