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. 2020 Sep 29;94(20):e00902-20.
doi: 10.1128/JVI.00902-20. Print 2020 Sep 29.

Evolutionary Arms Race between Virus and Host Drives Genetic Diversity in Bat Severe Acute Respiratory Syndrome-Related Coronavirus Spike Genes

Hua Guo #  1   2 , Bing-Jie Hu #  1 , Xing-Lou Yang  1 , Lei-Ping Zeng  1 , Bei Li  1 , Songying Ouyang  3 , Zheng-Li Shi  4
Affiliations

Evolutionary Arms Race between Virus and Host Drives Genetic Diversity in Bat Severe Acute Respiratory Syndrome-Related Coronavirus Spike Genes

Hua Guo et al. J Virol. .

Abstract

The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiencies in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS-CoV and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with that of their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that some key residues were under positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics.IMPORTANCE Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoVs in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS-CoV and SARSr-CoV infection but with different binding affinities to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity for spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.

Keywords: ACE2; Chinese horseshoe bat; SARS-related coronavirus; genetic diversity; receptor; spike gene.

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Figures

FIG 1
FIG 1
Phylogenetic tree of R. sinicus ACE2. (A) The maximum-likelihood tree (left panel) was produced using MEGA6 software, based on the alignment of ACE2 amino acid sequences of R. sinicus with the Jones-Taylor-Thornton (JTT+G+I) model and a bootstrap value (%) of 1,000 replicates (72). The eight key residues which are involved in interacting with the SARS-CoV spike are indicated in the panel on the right. The eight residues of human ACE2 are indicated at the bottom. The numbers at the top are amino acid positions in ACE2. (B) Frequencies of ACE2 alleles among the R. sinicus population. The number of R. sinicus ACE2 sequences is shown in the center of the pie chart. Different colored sectors with percentages in the pie chart indicate allele frequencies of ACE2 in the R. sinicus population used in this study. ACE2 sequences of Rs-411, Rs-832, Rs-3357, and Rs-ACT66275 have been published previously and were downloaded from GenBank. (C) Analysis of different R. sinicus ACE2 usage of SARSr-CoVs determined by immunofluorescence assay. Determination of bat SARSr-CoV infectivity in HeLa cells with and without the expression of ACE2 from R. sinicus or human (hACE2) at a multiplicity of infection (MOI) of 1. The original immunofluorescence assay images are not shown. The cross indicates that the allele is not susceptible to bat SARSr-CoV.
FIG 2
FIG 2
Infectivity of SARSr-CoV in HeLa cells expressing R. sinicus ACE2. (A to D) Determination of bat SARSr-CoV infectivity in HeLa cells with and without the expression of ACE2 from R. sinicus or human (hACE2) at an MOI of 0.01. The growth curves were determined by real-time PCR. Numbers in red square indicate samples that are not susceptible to bat SARSr-CoV. The values reported at each time point were averaged from two independent experiments. (E) The infectivity of the SARS-CoV-BJ01 pseudotyped was used for the assay at MOIs of 1 and 0.1 due to biosafety regulation in China. Error bars represent the standard error of the mean (SEM) from two independent transfections; each assay was performed in triplicate. (F) The expression of ACE2 in HeLa cells in SARS-CoV BJ01 pseudotyped infection assay. ACE2 expression was detected with mouse anti-S tag monoclonal antibody followed by horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody. β-Actin was detected with mouse anti-β-action monoclonal antibody by HRP-labeled goat anti-mouse IgG antibody.
FIG 3
FIG 3
Binding affinity assay between different RBDs and ACE2s by biolayer interferometry. (A to D) Binding assay of human ACE2 or bat ACE2 to RsWIV1-RBD. (E to H) Binding assay of human ACE2 or bat ACE2 to RsSHC014-RBD. (I to L) Binding assay of human ACE2 or bat ACE2 to SARS-CoV BJ01-RBD. Binding assay of human DPP4 to WIV1 RBD was used as the negative control (data not shown). The parameters of the equilibrium dissociation constant (KD) value (M) are shown on the upper right side of the picture. Different RBD proteins were immobilized on the sensors and tested for binding with graded concentrations of human ACE2 and R. sinicus ACE2s. The y axis shows the real-time binding response. Values reported represent the global fit to the data. The coefficient of determination (R2) for these interactions was close to 1.0. The RsSHC014-RBD/R. sinicus ACE2-3357 and BJ01-RBD/R. sinicus ACE2-3357 did not show obviously binding activity. For details of the kinetic analysis between different RBD and ACE2 proteins, see Table 1.
FIG 4
FIG 4
Structure modeling at the interface between SARSr-CoV spike and human or bat ACE2. Detailed view of the interaction between RBD and ACE2. Several important residues in RBD and ACE2 that are involved in the interactions are shown. ACE2 residues are in green and RBD residues are in magenta and yellow. (A) Structure complex of the SARS-CoV hTor2 RBD and human ACE2 (PDB code 2AJF). (B, C) Structural details of the interfaces between human ACE2 (M82, Y41) and SARS-CoV RBD. (D) Predicted structure complex of R. sinicus ACE2 3357 with RsWIV1 RBD. (E) Predicted structure complex of R. sinicus ACE2 1434 with RsSHC014 RBD. (F) Structural details of the interfaces between R. sinicus ACE2 1434 (T31, E35) and RsSHC014 RBD. The two predicted models (D, E) were built based on the structure of hTor2 RBD with hACE2 (yellow) and civet-optimized RBD with hACE2 (magenta) (PDB codes 2AJF and 3SCJ).
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