Fig. 1: Construction of the alphacoronavirus spike protein library and screening of the receptor libraries.

a, Schematic of the selection process used to create the spike protein library. Full-length amino acid sequences of alphacoronaviruses were recovered from public repositories (1) and aligned (2) to infer the alphacoronavirus genus phylogeny (3), from which cophenetic distances between all taxa were estimated. In total, 40 representatives spike protein sequences were selected using an optimal greedy-based algorithm (4). b, Screening of known human coronavirus receptors using the alphacoronavirus spike protein library pseudotypes. As expected, entry was observed for HCoV-229E inf1 and HCoV-NL63 with APN and ACE2, respectively. Entry of other viruses in the library was not supported by previously described human coronavirus receptors. c, Wider receptor usage by the alphacoronavirus library. To assess whether APN or ACE2 proteins from nonhuman species could be used as receptors for spike proteins in the alphacoronavirus library, we examined receptors from different mammalian species. Positive results were reproduced at least three times using independent biological replicates. All experiments were performed in technical triplicate with pseudotype (PV) entry indicated as log₁₀ relative light units (RLU). The average of the repeats, minus background, is shown.