Prediction of two novel overlapping ORFs in the genome of SARS-CoV-2

Abstract

Six candidate overlapping genes have been detected in SARS-CoV-2, yet current methods struggle to detect overlapping genes that recently originated. However, such genes might encode proteins beneficial to the virus, and provide a model system to understand gene birth. To complement existing detection methods, I first demonstrated that selection pressure to avoid stop codons in alternative reading frames is a driving force in the origin and retention of overlapping genes. I then built a detection method, CodScr, based on this selection pressure. Finally, I combined CodScr with methods that detect other properties of overlapping genes, such as a biased nucleotide and amino acid composition. I detected two novel ORFs (ORF-Sh and ORF-Mh), overlapping the spike and membrane genes respectively, which are under selection pressure and may be beneficial to SARS-CoV-2. ORF-Sh and ORF-Mh are present, as ORF uninterrupted by stop codons, in 100% and 95% of the SARS-CoV-2 genomes, respectively.

Keywords: Codon usage; Membrane protein; Multivariate statistics; Overlapping reading frame; Selection pressure; Spike protein; Virus evolution.

Fig. 1

Location of the eight overlapping ORFs detected in the 3′ genome region of SARS-CoV-2.

Fig. 2

Example workflow for CodScr + SeqComp analysis. As input data, CodScr + SeqComp requires the nucleotide sequence of a protein coding region (the ancestral reading frame) which contains an overlapping ORF shifted one nucleotide 3’ (+1 overlapping ORF) or an overlapping ORF shifted two nucleotides 3’ (+2 overlapping ORF). ORF indicates a contiguous stretch of codons, beginning with a start AUG codon, ending with a stop codon, not interrupted by premature stop codons, and having a length ≥ 90 nt. A detailed example of calculation of the five prediction scores (P-value, PLS-DA score, LDA score, LDA-ancestral score, and LDA-novel score) is shown in Supplementary File S1.

Fig. 3

Nucleotide and amino acid sequence of the two predicted overlapping ORFs in the 3′ genome region of SARS-CoV-2. (A) Overlapping ORF-Sh: the nucleotide sequence (from nt 24,050 to 24,172) encodes the region of protein S spanning residues 830–868, while the +1 overlapping ORF-Sh (from nt 24,051 to 24,170) encodes a predicted protein of 39 aa (underlined characters). Bold characters indicate a predicted transmembrane helix. (B) Overlapping ORF-Mh: the nucleotide sequence (from nt 26,691 to 26,873) encodes the region of protein M spanning residues 57–116, while the +2 overlapping ORF-Mh (from nt 26,693 to 26,872) encodes a predicted protein of 59 aa (underlined characters). Bold characters indicate two predicted transmembrane helices.