Natural selection pressure exerted on ?Silent? mutations during the evolution of SARS-CoV-2: Evidence from codon usage and RNA structure

From the first emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) till now, multiple mutations that caused synonymous and nonsynonymous substitutions have accumulated. Among them, synon-ymous substitutions were regarded as "silent" mutations that received less attention than nonsynonymous substitutions that cause amino acid variations. However, the importance of synonymous substitutions can not be neglected. This research focuses on synonymous substitutions on SARS-CoV-2 and proves that synonymous substitutions were under purifying selection in its evolution. The evidence of purifying selection is provided by comparing the mutation number per site in coding and non-coding regions. We then study the two forces of purifying selection: synonymous codon usage and RNA secondary structure. Results show that the codon usage optimization leads to an adapted codon usage towards humans. Furthermore, our results show that the main-tenance of RNA secondary structure causes the purifying of synonymous substitutions in the structural region. These results explain the selection pressure on synonymous substitutions during the evolution of SARS-CoV-2.