Genetic Evidence Indicates the Evolutionary Importance of the SARS-CoV-2 ORF9b protein

Abstract

All known betacoronaviruses possess an overlapping alternate-frame gene within the nucleocapsid gene. In SARS-CoV-2, the gene for this "internal protein" is ORF9b. The WHO Variants of Concern (VOC) Alpha, Delta, and Omicron all possess noncoding mutations that increase ORF9b expression. We show that with two exceptions, every major variant of the VOC era has had similar noncoding mutations to increase ORF9b expression and that these mutations are also frequently seen in long-branch, anachronistic, posited chronic-infection (PCI) sequences. Furthermore, we show that the amino acid substitution rate in ORF9b is higher than for any other SARS-CoV-2 gene, both in high-quality circulating sequences and in PCI sequences. This suggests that, as immunity to SARS-CoV-2 has grown in the population, increased ORF9b expression has conferred an evolutionary advantage, likely due to its ability to antagonize the antiviral type-I interferon response. We also show that PCI sequences are marked by distinct mutational patterns in ORF9b, some of which later became prominent in major variants. This evidence points to the importance of ORF9b for both viral transmission and within-host persistence.