RNA structure-altering mutations underlying positive selection on Spike protein reveal novel putative signatures to trace crossing host-species barriers in Betacoronavirus

Similar to other RNA viruses, the emergence of Betacoronavirus relies on cross-species viral transmission, which requires careful health surveillance monitoring of protein-coding information as well as genome-wide analysis. Although the evolutionary jump from natural reservoirs to humans may be mainly traced-back by studying the effect that hotspot mutations have on viral proteins, it is largely unexplored if other impacts might emerge on the structured RNA genome of Betacoronavirus. In this survey, the protein-coding and viral genome architecture were simultaneously studied to uncover novel insights into cross-species horizontal transmission events. We analysed 1,252,952 viral genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 distributed across the world in bats, intermediate animals, and humans to build a new landscape of changes in the RNA viral genome. Phylogenetic analyses suggest that bat viruses are the most closely related to the time of most recent common ancestor of Betacoronavirus, and missense mutations in viral proteins, mainly in the S protein S1 subunit: SARS-CoV (G > T; A577S); MERS-CoV (C > T; S746R and C > T; N762A); and SARS-CoV-2 (A > G; D614G) appear to have driven viral diversification. We also found that codon sites under positive selection on S protein overlap with non-compensatory mutations that disrupt secondary RNA structures in the RNA genome complement. These findings provide pivotal factors that might be underlying the eventual jumping the species barrier from bats to intermediate hosts. Lastly, we discovered that nearly half of the Betacoronavirus genomes carry highly conserved RNA structures, and more than 90% of these RNA structures show negative selection signals, suggesting essential functions in the biology of Betacoronavirus that have not been investigated to date. Further research is needed on negatively selected RNA structures to scan for emerging functions like the potential of coding virus-derived small RNAs and to develop new candidate antiviral therapeutic strategies.