Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2

Abstract

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

Keywords: SARS-CoV-2; animal ACE2; animal hosts; entry; furin cleavage; receptor.

FIG 1

Schematic diagram of domain structures and critical ACE2-binding residues of the spike (S) protein of SARS-CoV-2. The S protein is cleaved into S1 and S2 subunits during biogenesis at the polybasic furin cleavage site (RRAR↓), which is not present in SARS-CoV and other animal SARS-CoV-2-like CoVs. The S1 subunit is required for binding to ACE2 receptor, while the S2 subunit containing a fusion peptide mediates membrane fusion. In SARS-CoV-2, the S1 subunit contains an N-terminal domain and an independently folded domain known as the RBD, which harbors a region called the receptor binding motif (RBM), that is primarily in contact with receptor. The most critical hACE2-binding residues in the RBM of several SARS-CoV-2-related CoVs are highlighted in yellow and inferred from the crystal structure of RBD/hACE2 complex (16). The only difference in the RBMs between PCoV-GD and SARS-CoV-2 is Q498H (underlined). The GenBank numbers for these CoVs are as follows: SARS-CoV-2 isolate Wuhan-Hu-1, MN908947; SARS-CoV isolate Tor2, NC_004718.3; bat ZC45, MG772933.1; bat RaTG13, MN996532.1; PCoV-GX isolate P4L, MT040333.1; PCoV-GD isolate MP789, MT084071.1. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; FP, fusion peptide; TM, transmembrane domain; CT, cytoplasmic tail; PCoV-GX, pangolin CoV isolate GX-PL4; PCoV-GD, pangolin CoV isolate MP789.

FIG 2

Human ACE2 served as a receptor for SARS-CoV-2. (A) ACE2 supported HIV-Luc-based pseudotyped virus entry. 293T cells were transfected with empty vector pcDNA3.1, APN (receptor for HCoV-229E), DDP4 (receptor for MERS-CoV), ACE1, or ACE2. At 48 h posttransfection, the cells were infected by SARS-CoV-2 S protein pseudotyped virus (SARS-CoV-2pp). At 48 h postinfection, luciferase activity was measured. Ab, antibody. (B) Human ACE2 antibody inhibited virus entry in a dose-dependent manner. 293T cells were transfected with ACE2. At 48 h posttransfection, the cells were preincubated with the indicated concentration of hACE2 antibody or control antibody (anti-IDE) for 1 h and then infected by pseudotyped virus particles of SARS-CoV-2, influenza virus A (IAVpp) or human coronavirus (HCoV) OC43 (HCoV-OC43pp) in the presence of the indicated concentration of hACE2 antibody or control antibody (anti-IDE) for another 3 h, and then the virus and antibodies were removed. At 48 h postinfection, luciferase activity was measured and normalized to the level of the control antibody for SARS-CoV-2pp. Error bars represent the standard deviations of the means from four biological repeats. (C) Syncytium formation assay. 293T cells transfected with a plasmid expressing the S protein of SARS-CoV-2 or SARS-CoV were mixed at a 1:1 ratio with cells transfected with a plasmid expressing ACE1 or ACE2. Twenty-four hours later, syncytium formation was recorded.

FIG 3

Multiple ACE2 orthologs served as receptors for SARS-CoV-2. (A) Transient expression of ACE2 orthologs in 293T cells. The cell lysates were detected by Western blot assay using an anti-C9 monoclonal antibody. (B) HIV-Luc-based pseudotyped virus entry. 293T cells were transfected with ACE2 orthologs. At 48 h posttransfection, the cells were infected by pseudotyped virus of wild-type SARS-CoV-2 or a mutant lacking furin (ΔFurin). At 48 h postinfection, luciferase activity was measured and normalized to that of human ACE2. Error bars represent the standard deviations of the means from four biological repeats. (C) IP assay. The upper panel shows the input of ACE2 protein with a C9 tag and S1 and RBD with an IgG Fc tag (S1-Ig or RBD-Ig). The lower panel shows the ACE2 pulled down by an S1-Ig or RBD-Ig fusion protein. (D) SARS-CoV spike-mediated entry. 293T cells were transfected with ACE2 orthologs. At 48 h posttransfection, the cells were infected by the pseudotyped virus of SARS-CoV. At 48 h postinfection, luciferase activity was measured and normalized to that of human ACE2. Error bars represent the standard deviations of the means from four biological repeats.

FIG 4

Phylogenetic clustering of ACE2s correlates with their receptor activities. At top is a phylogenetic tree of 14 ACE2s. The tree was constructed based on nucleotide sequences using the neighbor-joining method implemented in the program MEGA X. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree was rooted by the ACE2 of platypus (Ornithorhynchus anatinus). The taxonomic orders into which these animals are classified are shown on the right-hand side of the tree. A heat bar summarizing the relative levels of pseudotyped virus entry supported by different animal ACE2s is shown below the tree.

FIG 5

Critical RBD-binding residues in ACE2 orthologs. The top panel shows the 23 RBD-binding residues at the contact interface between hACE2 and the RBD of SARS-CoV-2. Human ACE2 (PDB accession no. 6VW1) in the bound conformation was extracted from the SARS-CoV-2 RBD/ACE2 complex and used as a template for homology modeling (16). Critical RBD-binding residues in ACE2 orthologs are shown in the bottom panel. Residue substitutions highlighted in red and orange are those unique to both mouse and rat ACE2s and to both bat species, respectively. Other residue substitutions are highlighted in yellow. Rs bat, Rhinolophus sinicus; Tb bat, Tadarida brasiliensis.

FIG 6

Structural models of key residue substitutions in ACE2 of mouse, rat, and bats. Human ACE2 (PDB accession no. 6VW1) in the bound conformation was extracted from the SARS-CoV-2 RBD/ACE2 complex and used as a template for homology modeling (16). ACE2 homology models were generated using the one-to-one threading algorithm of Phyre2 (63). The models were then aligned and compared to that of the intact SARS-CoV-2 RBD/ACE2 complex in PyMOL. Rs bat, Rhinolophus sinicus; Tb bat, Tadarida brasiliensis.