Fig. 9. ORF3d is not protein-coding.

Sarbecovirus alignment of 57-codon ORF3d (referred to by some authors as ORF3b) overlapping ORF3a shows mostly function-altering radical amino-acid substitutions (red columns), and repeated interruption by one or more premature stop codons in all other strains (red ovals), unambiguously indicating that ORF3d is not a conserved protein-coding gene. A substantial fraction of SARS-CoV-2 isolates have stop-introducing mutations, and ribosome profiling did not identify ORF3d as a translated ORF²⁰, indicating that it is not a recently evolved strain-specific gene either. There is ribosome profiling and other evidence of translation of ORF3d-2, beginning at a downstream AUG and thus avoiding the stop-introducing mutations. However, ORF3d-2 is not conserved, is only 33 codons long, and lacks evidence that its translation product contributes to viral fitness.