Despite its clinical importance, the SARS-CoV-2 gene set remains unresolved, hindering dissection of COVID-19 biology. We use comparative genomics to provide a high-confidence protein-coding gene set, characterize evolutionary constraint, and prioritize functional mutations. We select 44 Sarbecovirus genomes at ideally-suited evolutionary distances, and quantify protein-coding evolutionary signatures and overlapping constraint. We find strong protein-coding signatures for ORFs 3a, 6, 7a, 7b, 8, 9b, and a novel alternate-frame gene, ORF3c, whereas ORFs 2b, 3d/3d-2, 3b, 9c, and 10 lack protein-coding signatures or convincing experimental evidence of protein-coding function. Furthermore, we show no other conserved protein-coding genes remain to be discovered. Mutation analysis suggests ORF8 contributes to within-individual fitness but not person-to-person transmission.
Fig: a Coronavirus-wide (black font) and species-specific or candidate (blue font) SARS-CoV-2 genes, with confirmed protein-coding (green), rejected (red), or novel protein-coding (purple) classification, using evolutionary and experimental evidence. b Phylogenetic Codon Substitution Frequencies (PhyloCSF) scores distinguish protein-coding (left) vs. non-coding (right) using evolutionary signatures, including distinct frequencies of amino-acid-preserving (green) vs. amino-acid-disruptive (red) substitutions, and stop codons (cyan/magenta/yellow) in frame-specific alignments, and additional features. c PhyloCSF score (x-axis) for all confirmed (green) and rejected (red) ORFs, showing annotated/candidate/novel (labeled) and all AUG-initiated ≥25-codons-long locally maximal ORFs (unlabeled). Novel ORF3c (purple) clusters with protein-coding.
Cross-strain and within-strain evolutionary pressures agree, except for fewer-than-expected within-strain mutations in nsp3 and S1, and more-than-expected in nucleocapsid, which shows a cluster of mutations in a predicted B-cell epitope, suggesting immune-avoidance selection. Evolutionary histories of residues disrupted by spike-protein substitutions D614G, N501Y, E484K, and K417N/T provide clues about their biology, and we catalog likely-functional co-inherited mutations. Previously reported RNA-modification sites show no enrichment for conservation. Here we report a high-confidence gene set and evolutionary-history annotations providing valuable resources and insights on SARS-CoV-2 biology, mutations, and evolution.
Jungreis, I., Sealfon, R. & Kellis, M. SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes.Nat Commun 12, 2642 (2021). https://doi.org/10.1038/s41467-021-22905-7