Fig. 5. ORF10 is not protein-coding.

a Alignment of Sarbecovirus genomes at ORF10, including 30nt on each side. Most substitutions are radical (red) or conservative (dark green) amino-acid-changing, with only two synonymously changing positions (light green), indicating this is not a conserved protein-coding ORF. Nearly all strains show an earlier stop codon (cyan), further reducing the length of this already-short ORF from 38 codons to 25, and another strain includes a frame-shifting deletion (orange). Putative partial transcription-regulatory sequence (TRS) present in SARS-CoV-2 and Bat CoV RaTG13 is not present in other strains. The surrounding region shows high nucleotide-level conservation, spanning ORF10 and extending beyond its boundaries in both directions, indicating this region is functionally important even though it does not encode protein (indeed, it is part of a known RNA structure). b Ribosome footprints previously used to suggest that ORF10 might be translated²⁰ in fact localize either in an upstream ORF (uORF, green) or in an internal ORF (green, “final predictions” track²⁰), but density in the unique portion of ORF10 (dashed black box) is no greater than after the stop codon (red box), indicating they are less likely to reflect the functional translation of ORF10, and more likely to represent incidental translation initiation events. The internal ORF is only 18 codons long in 4 strains, and 5 in the other strains, given the early stop codon (purple box), and unlikely to be functional. Footprint tracks show elongating ribosome footprints in cells treated with cycloheximide (blue, CHX), and footprints enriched for initiating ribosomes using harringtonine (Harr, red), and lactimidomycin (LTM, green). “mRNA-seq” track shows RNA-seq reads. c Alignment of six closely related strains (SARS-CoV-2, three bat viruses, two pangolin viruses) previously used to argue that high dN/dS ratio in ORF10 indicated positive selection for protein-coding-like rapid evolution¹⁵. A frameshifting deletion (orange/gray) in one bat virus militates against conserved protein-coding function. Even ignoring that strain, the evidence is not statistically significant: the alignment includes only 9 substitutions, including 1 synonymous. In a neutrally evolving region with 9 substitutions, we would expect 2–3 synonymous changes, and a depletion to only 1 is not statistically significant even without multiple-hypothesis correction (P > 0.18).