Ancestral sequence reconstruction analysis indicates the S1/S2 insertion in the SARS-CoV-2 spike gene likely interrupted a codon

The genome of the SARS-CoV-2 virus has a 12-nucleotide insertion that produced a furin cleavage site at the S1/S2 junction of its spike protein. It has been noted that compared to related coronavirus genomes, the insertion aligns best in the middle of a codon. However, a precise placement of the insertion would require an understanding of the S1/S2 junction sequence in the ancestor of SARS-CoV-2 in which the insertion occurred, whose genome sequence we do not have. Leveraging ancestral sequence reconstruction, we find that the genome of the most recent common ancestor of SARS-CoV-2 and other published coronaviruses very likely shared its S1/S2 junction sequence with coronaviruses like RaTG13 and BANAL-20-52. The insertion occurred in a descendant of this ancestor; we considered whether mutations could have occurred along the SARS-CoV-2 lineage that would have allowed the insertion to fall cleanly between codons, and found that such mutations were unlikely. Thus, the insertion likely interrupted a codon.