SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Abstract

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. 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.

SARS-CoV-2 Accessory Proteins in Viral Pathogenesis: Knowns and Unknowns

“…ORF3c (originally known as iORF1) is a 41-codon long protein recently described as an accessory protein ( Figure 1 ) ( 26 , 27 ). ORF3c is encoded by a frame overlapping ORF3a (close to the 5’ end) and is conserved across Sarbecoviruses .…”

Prediction of two novel overlapping ORFs in the genome of SARS-CoV-2

“…Analysis of the 3’ genome region of SARS-CoV-2 by the present method can be compared to that performed by Jungreis et al (2021b) using PhyloCSF, a computational tool to detect evolutionary signatures of protein-coding regions ( Lin et al, 2011 ). By analysis of 44 Sarbecovirus genomes, Jungreis et al (2021b) found strong protein-coding signatures for the overlapping ORFs ORF3c and ORF9b, but not for the overlapping ORFs ORF2b, ORF3b, ORF3d and its isoform ORF3d-2, and ORF9c. CodScr + SeqComp differed from PhyloCSF because it predicted as candidate also ORF3d, in addition to ORF3c and ORF9b ( Table 3 ), and because it detected ORF-Sh and ORF-Mh.…”

The Evolving Faces of the SARS-CoV-2 Genome

“…The percentage of SNVs in the S-gene (coding the S-glycoprotein) is nearly twice as large in spot A (and thus PAT A) compared with spot D reflecting an increase of the relative mutational load in this gene from PATs D, and EF towards PATs A-C paralleled by the decrease of the mutational load in ORF1a,b (Figure 6b and middle and Figure 6d for comparison with the respective percentages across all SNV and nucleotides of the SARS-CoV-2 genome). The percentage of SNVs of the N gene is large in PAT A, B and D indicating subtle shifts between the different genes as a result of evolutionary adaptation [39]. SNVs in N involve a B-cell epitope, suggesting immune-avoidance selection [39,40].…”

“…The percentage of SNVs of the N gene is large in PAT A, B and D indicating subtle shifts between the different genes as a result of evolutionary adaptation [39]. SNVs in N involve a B-cell epitope, suggesting immune-avoidance selection [39,40]. The S-genes divides into different parts, namely, S1 coding the 'spike' (pointing towards the host, see scheme in Figure 6e) and including the RBD (receptor binding domain), as well as the S2 region anchoring the protein in the virus membrane.…”