The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans

Author summary Coronaviral genomes code for several proteins, with the large 1a/1ab being expressed directly from genomic (g)RNA. For the expression of other viral proteins, a set of subgenomic mRNAs is produced during replication. It includes mRNAs for structural (S-E-M-N) and accessory proteins. While the function of structural proteins is well described, the function of the latter ones is under debate. Some of them are required for replication, while others are dispensable in vitro but essential in vivo. Initially, 10 open reading frames (ORFs) were annotated in the SARS-CoV-2 genome, amongst which ORF10 is the most peculiar, as it does not share sequence homology with any known protein. Shortly after the genomic sequences became available, speculations on this protein's role in pathogenesis and innate immunity breaching started. Here, we identified two patients infected with SARS-CoV-2 variants with the ORF10 gene prematurely terminated. The disease was not attenuated, and the transmissibility was maintained. The in vitro study showed that the ORF10 is also not essential for replication. Consequently, ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended. SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.