Stepwise evolution and exceptional conservation of ORF1a/b overlap in coronaviruses

The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for propagation of coronaviruses. A combination of genomic features that make up PFE—the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements—puts severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess evolutionary dynamics of PFE in great detail. Here we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well defined phylogenetic specificity. We further examined sequencing data from over 350,000 complete genomes and 55,000 raw read datasets to demonstrate exceptional conservation of the PFE region.