The biochemical composition of coronavirus Nucleocapsid proteins drives the bicistronic properties of their sub-genomic mRNAs
Year: 2026
Type: preprint
Abstract: The nucleocapsid (N) gene of coronaviruses (CoVs) encodes a structural RNA-binding protein required for viral assembly. In SARS- and MERS-CoV viruses, reading frame 2 of N contains an overlapping open reading frame (ORF) encoding an innate immune antagonist accessory protein. However, the extent of this dual coding capacity across the CoV family, and the genetic features driving overlapping ORF formation, remain unexamined. Here, we developed an in-silico tool (ORFan) to screen structural gene sub-genomic mRNA sequences from 152 species of coronavirus for the presence of overlapping accessory gene candidates. We found that frame 2 of the N gene in Alpha-, Beta- and Delta-CoVs frequently contains a potential accessory ORF (AcORF), conserved at the subgenus level, unlike the spike, envelope and membrane genes. We also show that CoV nucleotide biases in conjunction with codon biases integral to N protein function drive the relatively high abundances of start codons in frame two and stop codons in frame three, explaining the tendency towards frame 2 AcORFs. We further suggest that viral nucleotide biases and the codon composition required for RNA-binding function facilitate overlapping ORF formation in other virus families
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: Wellcome Trust, Government of the United Kingdom, UK Research and Innovation, Department for Environment, Food and Rural Affairs, UK Government, National Research Foundation, South African Medical Research Council, Directorate for Biological Sciences, Medical Research Council, Biotechnology and Biological Sciences Research Council
Awards: G4735283310, G5523275428, Coronaviridae’s ‘Utility Belt’: Identification, Characterization and Evolutionary Analysis of the Accessory Proteome of SARS-CoV-2 and other Coronaviruses, Coronaviridae’s ‘Utility Belt’: Identification, Characterization and Evolutionary Analysis of the Accessory Proteome of SARS-CoV-2 and other Coronaviruses, G8672903764, SE0569: Coronavirus Prevalence in GB Bats (Active and Passive Surveillance)
OpenAlex ID: W7225379865