Evolution at Spike protein position 519 in SARS-CoV-2 facilitated adaptation to humans

As the COVID-19 pandemic enters its fourth year, the pursuit of identifying a progenitor virus to SARS-CoV-2 and understanding the mechanism of its emergence persists, albeit against the backdrop of intensified efforts to monitor the ongoing evolution of the virus and the influx of new mutations. Surprisingly, few residues hypothesized to be essential for SARS-CoV-2 emergence and adaptation to humans have been validated experimentally, despite the importance that these mutations could contribute to the development of effective antivirals. To remedy this, we searched for genomic regions in the SARS-CoV-2 genome that show evidence of past selection around residues unique to SARS-CoV-2 compared with closely related coronaviruses. In doing so, we identified a residue at position 519 in Spike within the receptor binding domain that holds a static histidine in human-derived SARS-CoV-2 sequences but an asparagine in SARS-related coronaviruses from bats and pangolins. In experimental validation, the SARS-CoV-2 Spike protein mutant carrying the putatively ancestral H519N substitution showed reduced replication in human lung cells, suggesting that the histidine residue contributes to viral fitness in the human host. Structural analyses revealed a potential role of Spike residue 519 in mediating conformational transitions necessary for Spike prior to binding with ACE2. Pseudotyped viruses bearing the putatively ancestral N519 also demonstrated significantly reduced infectivity in cells expressing the human ACE2 receptor compared to H519. ELISA data corroborated that H519 enhances Spike binding affinity to the human ACE2 receptor compared to the putatively ancestral N519. Collectively, these findings suggest that the evolutionary transition at position 519 of the Spike protein played a critical role in SARS-CoV-2 emergence and adaptation to the human host. Additionally, this residue presents as a potential drug target for designing small molecule inhibitors tailored to this site.