Receptor utilization of angiotensin-converting enzyme 2 (ACE2) indicates a narrower host range of SARS-CoV-2 than that of SARS-CoV

Abstract

Coronavirus (CoV) pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, severe acute respiratory syndrome CoV (SARS-CoV) caused SARS pandemic in 2003 and SARS-CoV-2 caused the ongoing COVID-19 pandemic. Both viruses are most likely originated from bats. Thus, direct or indirect inter-species transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the inter-species transmission. Angiotensin-converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Especially, SARS-CoV-2 tended not to use murine or non-mammal ACE2s. Meanwhile, pangolin-CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. Nevertheless, the actual susceptibility of these animals to the coronaviruses should be further verified by in vivo studies. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acid residues that might determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin-CoV. Our studies enhance the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.

Keywords: SARS-CoV; SARS-CoV-2; angiotensin-converting enzyme 2 (ACE2); coronavirus; host range; inter-species transmission; receptor utilization.

FIGURE 1

Validation of pseudovirus preparation and ACE2 expression. (a) Schematic structure of the spike protein of SARSr‐CoVs (upper panel) and alignment of the amino acid sequences of the receptor‐binding motifs (RBMs) of SARS‐CoV, SARS‐CoV‐2 and pangolin‐CoV spike proteins (lower panel). (b) Western blot detection of spike proteins of SARS‐CoV‐BJ01, SARS‐CoV‐2 and pangolin‐CoV in the pseudovirus stock solutions using an antibody against the HA tag conjugated to the viral spike proteins. HIV‐1 p24, a protein of the carrier pseudovirus, was detected as the loading control. "Mock" indicates the cells without any treatment. "NC" indicates the cells packaging the negative‐control pseudovirus that does not carry any spike. (c) Detection of different ACE2 orthologs in HeLa cells after transfecting the corresponding plasmids using an antibody against the 6XHis tag conjugated to the ACE2 proteins. β‐actin was detected as the loading control. All the Western blots were repeated for three times and the results were subjected to densitometric measurement to quantify the intensity of the bands using ImageJ program. [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Entry efficiency of SARS‐CoV‐BJ01, SARS‐CoV‐2 and pangolin‐CoV pseudoviruses into ACE2‐expressing cells. HeLa cells expressing different ACE2 orthologs were infected by SARS‐CoV, SARS‐CoV‐2 or pangolin‐CoV pseudoviruses. At 48 hr post‐infection, pseudovirus entry efficiency was determined by measuring luciferase activity in cell lysates. The results were presented as the logarithm (base 10) of the mean relative luminescence units (RLUs) and the error bars indicated the logarithm (base 10) of the standard deviations of the RLUs (n = 9) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Phylogenetic analysis of the 20 ACE2 orthologs and the key amino acid residues for SARS‐CoV‐2 utilization. (a) The phylogenetic tree was constructed on the whole aa sequences of ACE2s using NJ method by MEGA7 with 1,000 bootstrap replicates (left panel) and the amino acids on the 9 critical sites predicted previously were listed (right panel). The colours in the figure do not contain any biological or technical meaning but just for easy reading. (b) The structure of the complex of SARS‐CoV‐2 spike and human ACE2 was adapted from Protein Data Bank (PDB ID: 6VW1). S477, T478 and Q498 of SARS‐CoV‐2 spike were labelled in green, blue and cyan, respectively. T20 and Q42 were labelled in red and yellow, respectively [Colour figure can be viewed at wileyonlinelibrary.com]