HKU5 bat merbecoviruses engage bat and mink ACE2 as entry receptors

Abstract

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

Fig. 1. P. abramus bat and mink ACE2, but not DPP4, enable HKU5 pseudovirus entry.

a Phylogenetic analysis of the full-length amino acid sequences of coronavirus spikes from representative subgenera. Hosts and receptors are indicated. Asterisks (*) highlight bat coronaviruses used in this study. Animal illustrations were sourced from BioRender (https://BioRender.com/d86esp4). b HEK-293T cells were transiently transfected with the indicated animal DPP4 constructs, and infection of each spike pseudovirus was assessed. MERS-CoV pseudovirus uses diverse DPP4 orthologs, but other spike pseudoviruses do not use DPP4. c HEK-293T or BHK-21 cells were transiently transfected with the indicated animal ACE2 constructs. Pseudotyped virus infection with coronavirus spikes revealed that HKU5 uses P. abramus and N. vison ACE2 for entry. In contrast, SARS-CoV-2 uses diverse ACE2 orthologs. The mean of three technical replicates is plotted from one of two independent experiments. RLU relative light units, Sbc sarbecovirus, Merbc merbecovirus, Nobc nobecovirus, Emc embecovirus. Source data are provided as a Source Data file.

Fig. 2. HKU5 uses P. abramus bat and mink ACE2 as entry receptors.

HEK-293T cells expressing ACE2 (a) or DPP4 (b) orthologs from H. sapiens, P. abramus, P. pipistrellus, N. vison, or control vectors were infected with VSV pseudoviruses bearing SARS-CoV-2, HKU5, or MERS-CoV spikes. Entry was quantified via luciferase activity. c, d Cells expressing ACE2 orthologs were infected with eGFP-expressing pseudoviruses; GFP-positive cells were counted across 10 fields (100×). e MLV particles pseudotyped with P. abramus ACE2 and Gaussia luciferase infected HKU5 spike-expressing cells. Schematic illustration was generated using BioRender (https://BioRender.com/xige2n2). f Phylogenetic tree of full-length coronavirus spikes related to HKU5. g, h Entry efficiency of HKU5-related viruses in HEK-293T and BHK-21 cells expressing ACE2 orthologs was assessed via Renilla luciferase. i–l Vero81 cells stably expressing ACE2 constructs were infected with full-length HKU5 (FL-HKU5). i Brightfield and j fluorescence microscopy showed syncytia and nucleoprotein-positive cells in P. abramus ACE2-expressing cells. k Spike, ACE2, and nucleocapsid expression were assessed by Western blot at 24 hpi. l Plaque assays measured replication kinetics of FL-HKU5 in Vero81/ACE2 cells. RLU relative light units. Data represent three independent experiments unless otherwise stated. Data in (a, b, g, h) are pooled from two independent experiments with three biological replicates each. Images in (d) are representative of two experiments. Scale bars: 100 µm (d, i); 50 µm (j). Statistical analysis: two-tailed unpaired Student’s t-test and one-way repeated measures ANOVA. Data shown as mean ± s.e.m. ns, not significant; **p = 0.0069, ***p = 0.0022, ****p < 0.0001. Source data are provided as a Source Data file.

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.

Fig. 4. Cryo-EM structure of the HKU5 RBD in complex with P. abramus ACE2.

a Cryo-EM density (left) and refined model (right) for the HKU5^RBD in complex with P. abramus ACE2. The ACE2 is colored in green, and the RBD is colored in orange. b Footprints of the P. abramus ACE2 and HKU5^RBD complex are shown in open-book view. c Interactions at the interface. The complex is shown in the 45-degree rotated view compared to the orientation in (a). The interactions are divided into two patches. HKU5^RBD tyrosines provide more than 500 Ų binding surface for the P. abramus ACE2. Tyrosines in patch 1 form extensive interactions with a helical region on ACE2 that spans P324^ACE2, R328^ACE2, and D329^ACE2. Patch 2 interactions involve hydrogen bonding between HKU5^RBD tyrosines and A385^ACE2, glycan 387^ACE2, K352^ACE2, as well salt bridge between E510^RBD and K352^ACE2. The Y517^RBD is wedged between I92^ACE2 and I93^ACE2 and interacts with R26^ACE2 and D90^ACE2. d Comparison of binding modes of the RBDs of HKU5, SARS-CoV-2, NeoCoV and PDF-2180 CoV to ACE2. All structures (PDB ID: 6M0J, 7WPO, and 7WPZ) were superimposed onto the HKU5^RBD-bound to P. abramus ACE2. Footprints of the HKU5^RBD, SARS-CoV-2^RBD, PDF-2180^RBD, and NeoCoV^RBD are marked on the surface representation of P. abramus ACE2 and colored in the same color as their respective RBDs in the left panel. A zoomed-in view is shown to the lower right to compare the binding difference of HKU5^RBD and SARS-CoV-2^RBD relative to the ACE2 α1 helix. e, f Structural conservation map of the RBDs from the spike proteins of MERS-CoV, PDF-2180, and HKU5. Sequence conservation between HKU5, NeoCoV, PDF-2180, and MERS-CoV is mapped along the HKU5-P. abramus ACE2 binding footprint (outlined in black) on the HKU5 ^RBD (burnt orange) e. Pairwise sequence conservation between HKU5 and the RBD of NeoCoV (sand), PDF-2180 (pink), or MERS-CoV (salmon) is mapped to their respective binding footprints (outlined in black) f. Amino acid sequence with conservation colored blue and divergence red.

Fig. 5. Molecular determinants of HKU5-ACE2-mediated entry.

a Schematic of the ACE2 gene highlighting substituted residues and domains. Positions in gray had minimal effect on HKU5 entry, while substitutions that reduced entry are shown in red. b Wild-type P. abramus ACE2, individual, and combinatorial ACE2 mutants were transiently transfected into BHK-21 cells and then infected with VSV-HKU5^spike-RLuc or VSV-SARS-CoV^spike-RLuc. c We generated two patches of substitutions (1, 2) on the HKU5^RBD and tested each for infectivity on BHK-21 cells stably expressing P. abramus ACE2 (Patch 1: Y507A/Y544Q/Y557S^RBD; patch 2: E510K/Y512S/Y517S/Y521E/Y523A^RBD. Patches 1 and 2 of the HKU5^RBD were necessary for P. abramus ACE2 utilization, while individual substitutions Y507A^RBD and E510K^RBD were sufficient to block infection. d We generated four patches of substitutions (3, 4, 5, 6) on the HKU5^RBD (orange) and tested each for infectivity on BHK-21 cells stably expressing P. abramus ACE2 (green). Patch 3: S457P/D459S/Y463D/A471P^RBD; patch 4: T509N/E510K/Y512S/T514L/S515L/A516F/Y517D/G518D/K519R/Y521E^RBD; patch 5: 542-548→EDGDYYRKQLSPLEG^RBD; patch 6: T553A/T555S/Y557S/I558T/Y559V^RBD). HKU5 patches 4, 5, and 6 were required for P. abramus ACE2 use, with individual substitutions K519R^RBD and Q545P^RBD sufficient to block infection. Dots represent the mean from each of three independent experiments, each done in technical triplicate. Statistical analyses were performed using two-tailed unpaired Student’s t-tests and one-way ANOVA. Data are mean ± s.e.m. b *D90N-I92T-I93V: p = 0.0426, D90N: p = 0.0317, **V30D: p = 0.0089, **I93V: p = 0.0094, **R328E: p = 0.0020, ****p < 0.0001. c *p = 0.0445, ***p = 0005, ****p < 0.0001. d *Y463D: p = 0.0294, *T514L: p = 0.0473, *S515L: p = 0.0176, *A516F: p = 0.0158, **Patch 1: p = 0.0060, **K543D: p = 0.0047, ****p < 0.0001. e HKU5^RBD patches 3, 4, 5, and 6. Residue positions for each set of substitutions are shown in stick and dot representations. K519^RBD and Q545^RBD, which showed infection-blocking effects when mutated, are labeled along with other residues for positional reference. Source data are provided as a Source Data file.

Fig. 6. HKU5 interacts with mink ACE2 in a manner distinct from that of bat ACE2.

a Phylogenetic tree of mustelid species for which ACE2 constructs were tested. Shown are the nine amino acid positions that differ between N. vison (susceptible to HKU5) and M. putorius (resistant to HKU5). The figure was created using PhyloPic, an open-access database licensed under Creative Commons. b Wild-type mustelid ACE2 constructs were transiently transfected into BHK-21 cells, and VSV-HKU5-^spike-RLuc and VSV-SARS-CoV-^spike-RLuc were used to assess entry. N. vison (American mink) and M. erminea (stoat) ACE2, but not the ACE2 of other mustelids, were sufficient for HKU5 entry. SARS-CoV-2 could use all tested mustelid ACE2s. c Wild-type and mutant N. vison ACE2 and M. putorius ACE2s were transiently transfected into BHK-21 cells, and entry was assessed using VSV-HKU5-^spike-RLuc and VSV-SARS-CoV-^spike-RLuc. N. vison ACE2 residues at A387^ACE2 and R548^ACE2 (highlighted in red) are necessary for HKU5 use of N. vison ACE2. No individual substitutions were sufficient for HKU5 use of M. putorius ACE2. Data are means from three independent experiments, each done in triplicate. d Key amino acids are mapped onto the P. abramus ACE2 structure. Positions 387^ACE2 and 548^ACE2 are necessary for HKU5 to utilize mink ACE2. Statistical analyses were performed using two-tailed unpaired Student’s t-tests and one-way ANOVA. Data are mean ± s.e.m. b *M. erminea-HKU5: p = 0.0412, **N. vison ACE2-HKU5: p = 0.0032, *A. collaris ACE2-SARS-CoV-2: p = 0.0109, *M. nigripes-ACE2: p = 0.0172, **M. meles ACE2-SARS-CoV-2: p = 0.0067, **L. canadensis ACE2-SARS-CoV-2: p = 0.0089, **G. gulo ACE2-SARS-CoV-2: p = 0.0052, **L. lutra ACE2-SARS-CoV-2: p = 0.0016, **M. moschata ACE2-SARS-CoV-2: p = 0.0065, **M. putorius ACE2-SARS-CoV-2: p = 0.0013, **M. lutreola ACE2-SARS-CoV-2: p = 0.0042, ***p = 0.0002. c N. vison ACE2-HKU5: **p = 0.0024, ****p < 0.0001; M. putorius ACE2-HKU5: *p = 0.0207, **p = 0.0061 ***p = 0.0009; N. vison ACE2-SARS-CoV-2: *K309E, p = 0.0245, *K313T, p = 0.0284; *H354R, p = 0.0202; ***p = 0.0002; M. putorius ACE2-SARS-CoV-2: ****p < 0.0001. Source data are provided as a Source Data file.