Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Binding to ACE2 Receptors from Human, Pets, Farm Animals, and Putative Intermediate Hosts

Abstract

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a pandemic. Here, we used X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptors allows the definition of residues important for binding. From the 20 amino acids in ACE2 that contact S, up to 7 can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or acquisition of N-glycosylation sites located near the S interface. Of note, pigs and dogs, which are not infected or are not effectively infected and have only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with that from bat coronavirus strain RaTG13 (Bat-CoV-RaTG13) and pangolin coronavirus (Pangolin-CoV) strain hCoV-19/pangolin/Guangdong/1/2019 revealed that the latter contains only one substitution, whereas Bat-CoV-RaTG13 exhibits five. However, ACE2 of pangolin exhibits seven changes relative to human ACE2, and a similar number of substitutions is present in ACE2 of bats, raccoon dogs, and civets, suggesting that SARS-CoV-2 may not be especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-CoV-2.IMPORTANCE SARS-CoV-2 is threatening people worldwide, and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear, and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes, indicating that the species barrier might be low. Exceptions are dogs and especially pigs, which revealed relatively low ACE2 expression levels in the respiratory tract. Monitoring of animals is necessary to prevent the generation of a new coronavirus reservoir. Finally, our analysis also showed that SARS-CoV-2 may not be specifically adapted to any of its putative intermediate hosts.

Keywords: SARS-CoV-2; angiotensin-converting enzyme 2; livestock; severe acute respiratory syndrome coronavirus 2.

FIG 1

(A) Interaction surface of SARS-CoV-2 S (green) and human ACE2 (blue). Amino acids involved in contact are shown in magenta for S or as orange (amino acids forming hydrogen bonds) and cyan (hydrophobic interaction) sticks for ACE2. D30 forms a salt bridge with K417 in S of SARS-CoV-2. The figure was created with PyMol from PDB file 6MOJ. (B) Interaction surface between SARS-CoV S (green) and human ACE2 (blue). Amino acids making contact are shown in magenta (S) or orange (ACE2) sticks. The three amino acids in ACE2 that are not involved in binding of S from SARS-CoV-2 are shown as cyan sticks. The side chain of D30 that forms a salt bridge with S of SARS-CoV-2 is pointing away from the interacting surface. Inset, detail of the interaction between R426 that forms a salt bridge with E329 and a hydrogen bond with Q325. The figure was created with PyMol from PDB file 2AJF.

FIG 2

(A) Amino acid changes of human ACE2 and pig ACE2. Amino acid changes in ACE2 from pigs in comparison with human ACE2 are highlighted in red. N90 is not an interaction site, but the change N90T destroys the N-glycosylation site present in human ACE2. (B) Amino acid changes in human ACE2 and civet ACE2. Amino acid changes in ACE2 from civet in comparison with human ACE2 are highlighted in red. (C) Amino acid changes in human and mouse ACE2. Amino acid changes in human ACE2 in comparison with mouse ACE2 are highlighted in red. P84 in human ACE2 does not interact with amino acids in S of SARS-CoV-2 but might affect the secondary structure. N90T and N322H destroy the N-glycosylation site in human ACE2.

FIG 3

(A) Amino acid changes between human ACE2 and dog ACE2. (B) Amino acid changes between human ACE2 and ferret ACE2. (C) Amino acid changes between human ACE2 and chicken ACE2.

FIG 4

The relative expression of ACE2 in pig (A) and dog (B) tissues was determined by qRT-PCR. (A) The relative expression of ACE2 in pig was determined in heart, liver, spleen, lung, kidney, duodenum, trachea, turbinate bone, and stomach. (B) The relative expression of ACE2 in dog was determined in heart, liver, spleen, lung, kidney, trachea, and turbinate bone. The experiments were repeated three times.

FIG 5

Amino acids in ACE2 important for binding to S of SARS-CoV-2. See the text for details.

FIG 6

(A) Amino acid changes in the RBD of S from SARS-CoV-2 and viruses from bats (white sticks) and pangolins (red stick). Hydrogen bonds between S from SARS-CoV-2 and the corresponding amino acids in ACE2, which are likely to be weakened or destroyed by the changes, are shown as dotted lines. (B) Amino acid changes between human ACE2 and pangolin ACE2.

FIG 7

Distribution of glycosylation sites and RGD motifs in S from SARS-CoV-2 and bat and pangolin CoV. (A) Schematic representation of SARS-CoV-2 S1 protein. The sequences of SARS-CoV-2 and bat and pangolin CoV were aligned using MegAlign. The RGD motif is highlighted in red. The RBM starting position is highlighted in blue. (B) Cryo-EM structure of S from SARS-CoV-2 (15). N-glycosylation sites unique for SARS-CoV-2 and bat and/or pangolin coronavirus are represented by red or orange spheres. The RGD amino acid sequences present in S of SARS-CoV-2 and Pangolin-CoV are represented by a cyan sphere. In a monomer in the open conformation, it is located near the ACE2 binding sites on top of the molecule. The location of the RGD motif in a monomer in a closed conformation (gray) is also indicated. The locations of the furin cleavage site present only in S of SARS-CoV-2 and of the S2´ cleavage site are also represented by red spheres.