Characterization of key spike RBD residues influencing SARS-CoV-2 variant adaptation to avian ACE2.

Introduction: The beta-coronavirus SARS-CoV-2 has been revealed to infect mammals and other species, which potentially promotes the virus adaptation to broader species and the emergence of new variants. The host range of different SARS-CoV-2 variants are mainly determined by the affinity of the receptor-binding domain (RBD) of the spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Thus, this study aims to elucidate the detailed mechanisms of such dynamic adaptation of indicated SARS-CoV-2 variants. Methods: In this study, flow cytometry and surface plasmon resonance (SPR) assays were used to assess the binding affinity between RBDs and avian ACE2. Then, infection assays with MLV-based SARS-CoV-2 spike pseudovirus or authentic viruses were performed to verify the avian ACE2 mediated viral entry. Finally, mutagenesis studies were conducted to identify key amino acids of avian ACE2 orthologs and RBDs. Results: Our previous findings revealed that wild-type SARS-CoV-2 RBD does not bind chicken ACE2. Here, we found that ACE2 orthologs from chicken and mallard were capable to support binding to RBDs of the Alpha, Beta, and Gamma variants, which further enabled the viral entry. On the contrary, the RBD of BA.1 failed to bind avian ACE2. Whereas, a triple-residue reversal mutant (S446G, S496G, H505Y) restored ACE2 binding and enabled efficient viral entry. Additionally, several key residues within RBD were characterized as the determinant of its affinity to avian ACE2. Discussion: Our findings reveal that higher mutation rates in emerging variants might lead to future cross-species receptor usage or even spillover. Understanding such cross-species transmission mechanisms provides new insights to the virological features and potential host range of emerging SARS-CoV-2 variants.