Fig. 4: Mutational analysis of ACE2 interface residues and cross-species RBD interactions.

a Critical interface residues of PaPD involved in HKU5 RBD binding. b Effects of PaPD, PsPD, HsPD, and PaPD mutants on HKU5 RBD binding, evaluated by flow cytometry and tripartite split-fluorescence-based assays. The sfGFP fluorescence intensity was normalized to WT PaPD. Data represent mean ± SD from three independent experiments (n = 3). c HKU5 S pseudovirus (PSV) entry into Lenti-X 293T cells transiently expressing the indicated Pipistrellus abramus ACE2 (PaACE2) mutants. Data represent mean ± SD of four technical replicates from one representative experiment (three independent experiments performed in total). d Conservation analysis of Pipistrellus abramus (PaPD) interface residues in Homo sapiens (HsPD) and Pitta sordida (PsPD), highlighting conserved residues essential for cross-species interactions. e Entry of HKU5 S pseudovirus (PSV) into Lenti-X 293T cells stably transfected with three ACE2 orthologs, Pipistrellus abramus (P.abr), Pitta sordida (P.sor), and Homo sapiens (human). Data represent mean ± SD of four technical replicates from one representative experiment (three independent experiments performed in total). f HKU5 S-mediated cell–cell fusion using Caco-2 cells stably expressing the indicated ACE2 orthologs, Homo sapiens (HsACE2), Pipistrellus abramus (PaACE2) and Pitta sordida (PsACE2), with or without the addition of 10 μg/mL TPCK-treated trypsin. Untransfected Caco-2 cells under the same conditions served as the blank control. Representative images from three independent experiments showing similar results. g Cryo-EM map (Left) at a contour level of 4 σ and atomic model shown in cartoon (Right) of HKU5 RBD (marine blue) in complex with PsPD (purple). h Enlarged view of the interaction interface between HKU5 RBD and PsPD. i–k Detailed analysis of interface interactions between PsPD and HKU5 RBD, with polar interactions indicated by dashed black lines. Statistical significance was determined using two-sided unpaired Student’s t-tests. ns not significant (P ≥ 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.