Abstract
The COVID-19 pandemic offered an unprecedented glimpse into the evolution of its causative virus, SARS-CoV-2. It has been estimated that since its outbreak in late 2019, the virus has explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain. Spike protein of the virus exhibits the largest sequence variation in particular, with many individual mutations impacting target recognition, cellular entry, and endosomal escape of the virus. Moreover, recent studies unveiled a significant increase in the total charge on the spike protein during the evolution of the virus in the initial period of the pandemic. While this trend has recently come to a halt, we perform a sequence-based analysis of the spike protein of 2665 SARS-CoV-2 variants which shows that mutations in ionizable amino acids continue to occur with the newly emerging variants, with notable differences between lineages from different clades. What is more, we show that within mutations of amino acids which can acquire positive charge, the spike protein of SARS-CoV-2 exhibits a prominent preference for lysine residues over arginine residues. This lysine-to-arginine ratio increased at several points during spike protein evolution, most recently with BA.2.86 and its sublineages, including the recently dominant JN.1, KP.3, and XEC variants. The increased ratio is a consequence of mutations in different structural regions of the spike protein and is now among the highest among viral species in the Coronaviridae family. The impact of high lysine-to-arginine ratio in the spike proteins of BA.2.86 and its daughter lineages on viral fitness remains unclear; we discuss several potential mechanisms that could play a role and that can serve as a starting point for further studies.