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Receptor binding and structural basis of raccoon dog ACE2 binding to SARS-CoV-2 prototype and its variants [1]

['Chunliang Luo', 'College Of Veterinary Medicine', 'Shanxi Agricultural University', 'Jinzhong', 'Cas Key Laboratory Of Pathogen Microbiology', 'Immunology', 'Institute Of Microbiology', 'Chinese Academy Of Sciences', 'Cas', 'Beijing']

Date: 2025-03 

Abstract Raccoon dog was proposed as a potential host of SARS-CoV-2, but no evidence support such a notion. In our study, we investigated the binding affinities of raccoon dog ACE2 (rdACE2) to the spike (S) protein receptor binding domain (RBD) of SARS-CoV-2 prototype (PT) and its variants. It revealed that the binding affinities of RBD from SARS-CoV-2 variants were generally lower than that of the PT RBD. Through structural and functional analyses, we found amino acids H34 and M82 play pivotal roles in maintaining the binding affinity of ACE2 to different SARS-CoV-2 sub-variants. These results suggest that raccoon dogs exhibit lower susceptibility to SARS-CoV-2 compared to those animal species with a high prevalence of SARS-CoV-2 transmission.

Author summary In our study, we investigated the potential role of raccoon dogs as hosts for SARS-CoV-2 by examining the binding affinities between the spike (S) protein receptor binding domain (RBD) of SARS-CoV-2 prototype (PT) and its variants with the ACE2 receptor from raccoon dogs (rdACE2). Our findings revealed that the binding affinities of the RBD from most of SARS-CoV-2 variants to rdACE2 were generally lower compared to the PT RBD. Detailed structural and functional analyses identified two key amino acids, H34 and M82, which are crucial in preserving the binding affinity of ACE2 to different SARS-CoV-2 sub-variants. These results indicate that raccoon dogs have a lower susceptibility to SARS-CoV-2 compared to other animal species known for a high prevalence of SARS-CoV-2 transmission. This study provides valuable insights into the host range of SARS-CoV-2 and highlights the importance of specific amino acid residues in ACE2 for virus-receptor interactions. Understanding these interactions is essential for assessing the zoonotic potential of SARS-CoV-2 and implementing effective measures to prevent interspecies transmission of the virus.

Citation: Luo C, Li L, Gu Y, Zhang H, Xu Z, Sun J, et al. (2024) Receptor binding and structural basis of raccoon dog ACE2 binding to SARS-CoV-2 prototype and its variants. PLoS Pathog 20(12): e1012713. https://doi.org/10.1371/journal.ppat.1012713 Editor: Leo L. M. Poon, University of Hong Kong, HONG KONG Received: June 27, 2024; Accepted: October 31, 2024; Published: December 5, 2024 Copyright: © 2024 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The cryo-EM maps and the atomic structure coordinates have been deposited in the RCSB Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under accession codes 8YF2 / EMD-39208 (Cryo-EM structure of SARS-CoV-2 prototype RBD in complex with rdACE2 (local refinement)), EMD-39209 (Cryo-EM structure of SARS-CoV-2 prototype spike protein in complex with rdACE2), 8YFT / EMD-39229 (Cryo-EM structure of SARS-CoV-2 alpha variant spike protein in complex with rdACE2 (local refinement)), EMD-39224 (Cryo-EM map of SARS-CoV-2 alpha variant spike protein in complex with rdACE2). Funding: This work was supported by the National Key R&D Program of China (2022YFF1203203). KL was supported by Young Elite Scientists Sponsorship Program by CAST (2021QNRC001), fellowships from the China Postdoctoral Science Foundation (2022T150688) and the Postdoctoral Science Foundation of China (2021M700161). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction To date, seven coronaviruses have been identified capable of infecting humans: hCoV-NL63, hCoV-229E, hCoV-OC43, hCoV-HKU1, SARS-CoV-1, MERS-CoV and SARS-CoV-2 [1–8]. Additionally, Hu-PDCoV and CCoV-HuPn-2018 have been found to cause sporadic infections in children [9, 10]. Almost all these coronaviruses originate from animals [11]. SARS-CoV-2 has been continuously evolving into new variants since its outbreak in the end of 2019[12]. The World Health Organization (WHO) has identified five variants of concern (VOCs): Alpha, Beta, Gamma, Delta and Omicron. Moreover, Omicron is undergoing further evolution into multiple sub-variants with distinct characteristics [13–16]. In the early stages of the pandemic, our group discovered the virus entry receptor and assessed the receptor binding of SARS-CoV-2. We raised concerns about its broad interspecies binding spectrum and found that the Omicron variant further expanded the host range in subsequent studies [17–20]. Consequently, numerous animals have been reported to be naturally infected by SARS-CoV-2, totaling 45 species including pets, livestock, captive animals, and wildlife (https://www.fao.org/animal-health/situation-updates/sars-cov-2-in-animals/en). Receptor binding plays a critical role in SARS-CoV-2 infection. The receptor binding domain (RBD) of the spike (S) protein binds to its host receptor, angiotensin-converting enzyme 2 (ACE2), facilitating virus entry [20]. The structures of SARS-CoV-2 and its variants binding to ACE2 receptors from humans, intermediate horseshoe bats, big-eared horseshoe bats, cats, dogs, horses, rabbits, white-tailed deer, mink, fox, hippopotami, minke whale and sea lion have been elucidated [17,20–30]. Additionally, the binding affinities of ACE2 receptors from intermediate horseshoe bats, white-tailed deer, fox, rabbits and hippopotami to various SARS-CoV-2 variants were assessed [22,23,26,27,29]. However, the origin of SARS-CoV-2 is yet under debate, and further investigation is needed to explore potential hosts. During the SARS-CoV-1 pandemic, the virus was detected in raccoon dogs, suggesting their role as one of the hosts for SARS-CoV-1 [31]. With the emergence of the SARS-CoV-2 pandemic, raccoon dogs have become important suspected hosts [32]. Animal infection experiments have demonstrated that raccoon dogs typically display mild clinical signs, with viral replication and tissue lesions predominantly found in the nasal conchae [32]. After surveillance for SARS-CoV-2 was reported, some scientists suspected raccoon dog is the natural host of SARS-CoV-2[33,34]. However, the environmental samples cannot sufficiently prove the evidence for animal infection. A study examining coronaviruses in wild animals during the early stages of the COVID-19 outbreak, including 15 raccoon dogs, did not detect SARS-CoV-2 [35]. Another research team collected blood, tissue and swab samples from 229 raccoons and 11 raccoon dogs in Germany between 2021 and 2022 [36]. Despite thorough analysis using both molecular biological and serological methods, no evidence of SARS-CoV-2 was found in these samples. Until now, there has been no evidence to suggest that raccoon dogs can naturally become infected with SARS-CoV-2. Recently, the structure of chimeric SARS-CoV-2 RBD (containing the core structure from SARS-CoV-1 and receptor-binding motif (RBM) from SARS-CoV-2) with a chimeric raccoon dog ACE2 (rdACE2) (containing the core structure from human ACE2 (hACE2) and virus-binding motif (VBM) from rdACE2) was determined [37]. However, it’s worth noting that this construct is an artificial creation and may not fully reflect real interactions. In structural biology, it’s considered a best practice to rely on the original amino acid sequences, as any mutations, whether located on the binding motif or not, can potentially impact functionality. For instance, the R346T substitution, located far from the RBM of SARS-CoV-2 RBD, was found to enhance ACE2 binding only when interacting with residue R493 but not Q493, which occurred through a mechanism involving long-range conformational changes [38,39]. Herein, we evaluated the binding affinities of rdACE2 with RBDs of SARS-CoV-2 PT and its variants. To elucidate the molecular basis of rdACE2 binding to SARS-CoV-2 PT and Alpha RBDs, respectively, we obtained cryo-EM rdACE2/S complex structures.

Discussion The evolution of SARS-CoV-2 is constrained by the trade-off between enhancing immune evasion and maintaining an optimal binding affinity to its host receptor [38,42]. Previous studies have shown that the affinities of SARS-CoV-2 and its variants to hACE2 are limited within a certain range [38]. Additionally, it has been observed that SARS-CoV-2 can naturally infect white-tailed deer, mink, hamster and hippopotamuses. Of particular concern is the broad spread of SARS-CoV-2 among white-tailed deer, hamster and mink, leading to the emergence of new variants. Similar to hACE2, the binding of SARS-CoV-2 and most of its variants to ACE2 receptors from white-tailed deer, mink and hamsters is also limited within a certain range (within two orders of magnitude in nanomolar) [23,25,27]. In this study, we investigated the binding affinities of RBDs from various SARS-CoV-2 variants to rdACE2 receptor. We found that these affinities were generally lower compared to the PT RBD, falling into the range of three digital numbers. Notably, animal infection assays revealed that raccoon dogs typically exhibit mild clinical signs when infected with the PT virus. The decreased binding affinities observed in SARS-CoV-2 variants may indicate a reduced ability to infect raccoon dogs effectively. From our structural analysis, we discovered that the rdACE2 mutants Y34H and T82M significantly enhance the binding affinity to the PT, Alpha, and BA.2 RBDs. More notably, the K417N mutation in the PT RBD substantially diminishes its binding to rdACE2. However, when the rdACE2 undergoes mutations from Y34 to H34 or from T82 to M82, these two mutants exhibit comparable binding affinities to the PT RBD K417N as seen in the original rdACE2/PT RBD interaction. This finding indicates that these two residues play a pivotal role in maintaining the binding affinity of ACE2 to different SARS-CoV-2 sub-variants. Although structures determined by cryo-EM and those determined by crystallography may differ in their interaction details [40], the substantial differences in ACE2 conformation could be related to the protein constructs used. Additionally, the N103 glycosylation was not observed to insert into the gap between α2 and α4 in previously solved structures [20,28,39,40,43], which may be attributed to conformational effects caused by the chimeric constructs. Some scientists raised the possibility that raccoon dogs could serve as natural hosts for SARS-CoV-2 [33, 34]. However, this finding alone falls short of conclusively establishing raccoon dogs as natural hosts. To date, while 45 species have been documented as naturally infected with SARS-CoV-2, there has been no confirmation of raccoon dogs being naturally infected with or carrying the virus (https://www.fao.org/animal-health/situation-updates/sars-cov-2-in-animals/en). Furthermore, investigations during the early stages of the COVID-19 epidemic failed to detect SARS-CoV-2 in raccoon dogs [35]. Thus, there is currently no direct evidence supporting that raccoon dogs are the natural hosts of SARS-CoV-2. In summary, our data suggests that the binding affinities of rdACE2 to most of the SARS-CoV-2 sub-variants were reduced compared to PT. Additionally, structural analysis identified several key residues crucial for receptor binding. Our result indicated that the susceptibility of raccoon dog is lower than that of some animals with widespread transmission of SARS-CoV-2, such as white-tailed deer and hamster.

Materials and methods Gene synthesis The PD of the rdACE2 protein (UniProt, B4XEP4) and its mutants with the original signal peptide at the amino-terminal, and a 6×His tag connected to the carboxy-terminal (C-terminal), was designed to generate the deoxyribonucleic acid (DNA) sequence by codon-optimization. Then the DNA sequence was synthesized before being fused to the expression vector pcDNA3.1 [43]. For the S proteins of the SARS-CoV-2 PT and the Alpha variant, the residues 1–1208 were reserved while the transmembrane regions of the C-terminals (residues 1209–1273) were replaced by a trimerization tag (YIPEAPRDGQAYVRKDGEWVLLSTFL) followed by a strep tag (SAWSHPQFEK) and a 6×His tag. There were a "GSG" linker in front of the trimerization tag and a single “G” added the end of this tag. There were no linkers between other tags. The sequence was internally modified with the “6P” mutations reported to improve stability [44]. The codon-optimized DNA sequences were synthesized before being fused to the expression vector pCAGGS. The RBD (corresponding to residues 319–541 of the S protein) of each variant was fused with a signal peptide from interleukin-10 (IL-10) at the amino-terminal, and a 6×His tag at the carboxy-terminal. The codon-optimized DNA sequences were synthesized before being fused to the expression vector pCAGGS, as previously reported [26]. All the genes and subcloned-plasmids mentioned above were produced by GenScript (www.genscript.com). Protein expression and purification HEK293F cells (Gibco, Cat# 11625–019) were cultured at 37°C, 5% CO 2 in SMM 293-TII expression medium (Sino Biological, Cat# M293TII) to express soluble proteins. When the cell density reached 2×106 cells/ml, each plasmid and its three times mass of polyethylenimine (PEI) were separately diluted within phosphate buffered saline (PBS) and mixed for 20 minutes before being added to 293F cells, 100 μg plasmid was transfected into 100 ml cells. Five days later, the supernatants were centrifuged and filtered to remove the cell debris, then the His-tagged proteins were captured by the His columns (Cytiva, Cat# 17524802). All purification steps were carried out in a PBS-based buffer. Specifically, the irrelevant host proteins were washed off with the PBS solution containing 20 mM imidazole, while the target proteins were washed off and collected with the PBS solution containing 300 mM imidazole. The purified proteins were further purified by size exclusion chromatography using different types of molecular sieves, with the HiLoad 16/600 Superdex 200 pg (Cytiva, Cat# 28989335) for S protein and the Superose 6 Increase 10/300 GL (Cytiva, Cat# 29091596) for RBD and ACE2 proteins. The protein samples corresponding to the UV absorption peaks were collected and identified by SDS-PAGE (sodium dodecyl sulfate -polyacrylamide gel electrophoresis). SPR assays All affinity assays were conducted in PBST (0.005% Tween-20 in PBS) and measured by a BIAcore 8K instrument (GE Healthcare) at 25°C. Before the measurements, the rdACE2 protein was immobilized on the CM5 sensor chip (Cytiva, Cat# 29149603) with amine coupling reaction, the target immobilization level 5000 response units (RU), and the actual immobilization levels were 1300–6700 RU. In the preliminary experiment, each RBD variant was diluted to concentrations ranging from 200 nM to 12.5 nM, and a single-cycle experiment was performed. To ensure that the objective values fall within the fitting range, the concentration ranges of the RBDs were adjusted as shown in S3–S7 Tables. The K D values from SPR experiments were obtained using BIAcore 8K evaluation software (GE Healthcare), with a 1:1 Langmuir binding model. Values of single-cycle analysis are the mean ± standard deviation (SD) of three independent experiments. Pseudovirus infection assay BHK-21 cells were transfected with the pEGFP-N1-rdACE2s plasmids. After 24 h, the EGFP-positive cells were sorted, reseeded in 96-well plates at 2 × 104 cells/well, and cultured for another 24 h. The pseudovirus particles of SARS-CoV-2 PT and its variants were diluted to the same amount, according to the RT-PCR results. Then, 100 μL of each pseudovirus was added to the sorted eGFP-positive cells. At 15 h post-transfection, imaging and analysis of fluorescent cells were performed using a CQ1 confocal image cytometer (Yokogawa, Japan). Each group contained three replicates. Cryo-sample preparation Before cryo-EM sampling, all proteins were dialyzed from PBS to buffer A (20mM Tris, 150 mM NaCl, pH 8.0), and adjusted to 2 mg/ml. Then 5 μl rdACE2 was added into the equal volume of spike proteins, respectively. The mixtures were placed on ice for 1.5 h. At the same time, the 300 mash gold grids with 2 μm/2 μm C-flat films (Quantifoil) were glow-discharged in 15 mA electric current for 30 s using an easiGlow machine (PELCO). After being applied with 4μl protein solution, each grid was frozen in liquid ethane using a Vitrobot device (ThermoFisher Scientific), in which 4°C temperature, 100% humidity, 7 s blot time and −5 blot force was settled for all the samples. The grids were accepted into plastic boxes and stored in liquid nitrogen before use. Cryo-EM data collection The grids were packed and loaded into a 300 kV Titan Krios transmission electron microscope equipped with Gatan K3 camera and matched with the EPU software (ThermoFisher Scientific). All the movies were collected at ×105,000 magnification with the pixel size of 0.69 Å over a defocus range of −1.0 μm to −2.0 μm in super-resolution counting mode with a total dose of 60 e-/Å2. Each movie contains 32 frames. Cryo-EM data processing All the movies were processed on the cryoSPARC platform [45]. Generally, by Motion Correction [46] and CTF (contrast transfer function) estimation [47], each micrograph was generated from a movie, accordingly. The diameter ranges from 80 to 500 Å was set to picked out and extracted the particles, after 5–9 rounds of interactive 2D classification, serval particle stacks were selected for Ab-initial Reconstruction to generated 4~8 volumes, followed by the Heterogeneous Refinement [48,49]. And the best volumes were chosen for Non-uniform Refinement, CTF Refinement and DeepEMhancer sharpening [50]. The resolution was determined by the FCS cut-off at 0.143 [51]. To conduct the local refinements, partial maps of ACE2/RBD complexes were extracted from the overall maps utilizing the volume eraser tool in ChimeraX [52]. Subsequently, these partial maps were imported into Cryosparc to generate masks with a dilution radius of "6", a soft padding width of "3", and an appropriate threshold that minimally includes homologous structures. Each local map was produced through two iterations of localized refinements. For the data set of PT RBD/rdACE2 complex, 17,940 micrographs were generated form the same number of movies, and 2,620,451 particles were extracted from the micrographs. After 2D-classfication, 1,452,120 particles were chosen for Ab-initial reconstruction, in which 309,215 particles were sorted out for the refinement of the final volume, and the resolution was calculated to be 2.52 Å. The local refinement was performed to generate the RBD/ACE2-focused map with the resolution of 2.64 Å (S2 Fig). For the data set of Alpha RBD/rdACE2 complex, 6,307 micrographs were generated form the same number of movies, and 1,299,569 particles were extracted from the micrographs. After 2D-classfication, 396,183 particles were chosen for 3D reconstruction, in which 306,176 particles were sorted out for the refinement of the final volume, and the resolution was calculated to be 2.86 Å. The local refinement was performed to generate the RBD/ACE2-focused map with the resolution of 3.16 Å (S3 Fig). Structural model building and refinement The PT RBD/ACE2 structure labeled 6LZG was used as the initial models. After molecular replacement, the structures were fitted to the local refined maps using ChimeraX and COOT [53], the main chains and side chains were adjusted manually. Then the maps together with the structures were processed by real-space refinement from PHINEX [54]. The structural figures were presented by PyMOL (https://pymol.org/). Detailed information for the cryo-EM data set is available in S1 Table.

Acknowledgments We thank Y.Q. Mi, H. Feng, and X.X. Bai at the Cryo-EM Center, Shanxi Academy of Advanced Research and Innovation for their technical support on the Cryo-EM. We thank Y. Chen, Z. Yang, B. Zhou (Institute of Biophysics, Chinese Academy of Sciences), and Z. Fan (Institute of Microbiology, Chinese Academy of Sciences) for technical help with SPR experiments.

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