Fig. 3: The receptor-binding domain of the HKU5 spike protein is required for species-specific ACE2 binding.

Flow cytometry analysis of SARS-CoV-2^RBD-Fc (a), MERS-CoV^RBD-Fc (b), PDF-2180^RBD-Fc (c), and HKU5^RBD-Fc-fusion protein (d) binding to HEK-293T cells expressing the indicated receptors. The mean fluorescence intensity (MFI) was calculated. Data are plotted from two independent experiments with two biological replicates per experiment. The same mock samples were used in (a–d), as the experiments were processed together. HKU5^RBD-Fc directly binds the P. abramus ACE2. e BLI analysis reveals binding kinetics of HKU5^RBD with P. abramus ACE2. The reported K_D values correspond to avidities due to the use of dimeric ACE2 constructs. Analysis was conducted using curve-fitting kinetic global fitting (1:1 binding model). f–h Neutralization assays using SARS-CoV-2 vaccine sera, MERS-CoV-S2P mouse vaccine sera, and a monoclonal antibody against MERS-CoV (MERS-CoV-27) in BHK-21 cells revealed that HKU5 is resistant to SARS-CoV-2- and MERS-CoVelicited antibodies. Each point represents the mean neutralization value of two biologically independent infection replicates. Nonlinear regression (4-parameter logistic model) was performed using Python, with curve fitting implemented via scipy.optimize.curve_fit, and graphs were generated using GraphPad Prism v10.4.2. For panel f, the SARS-CoV-2 IC₅₀ is presented from a single biological experiment for clarity in presentation. ^#Not reliable: The confidence intervals could not be reliably estimated in some cases due to poor curve fit, likely resulting from flat or noisy responses. a–d Statistical analyses were performed using two-tailed unpaired Student’s t-tests and one-way ANOVA. Data are represented as mean ± s.e.m. ns not significant, *p = 0.0365, ***p = 0.0003, ****p < 0.0001. Source data are provided as a Source Data file.