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. 2020 Oct 27;94(22):e01283-20.
doi: 10.1128/JVI.01283-20. Print 2020 Oct 27.

SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

Yujun Li #  1 , Haimin Wang #  1 , Xiaojuan Tang #  1 , Shisong Fang #  2 , Danting Ma  1 , Chengzhi Du  1   3 , Yifei Wang  1 , Hong Pan  1   3 , Weitong Yao  1   3 , Renli Zhang  2 , Xuan Zou  2 , Jie Zheng  4 , Liangde Xu  5 , Michael Farzan  6 , Guocai Zhong  7   3
Affiliations

SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

Yujun Li et al. J Virol. .

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic has caused >20 million infections and >750,000 deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, has been found closely related to the bat coronavirus strain RaTG13 (Bat-CoV RaTG13) and a recently identified pangolin coronavirus (Pangolin-CoV-2020). Here, we first investigated the ability of SARS-CoV-2 and three related coronaviruses to utilize animal orthologs of angiotensin-converting enzyme 2 (ACE2) for cell entry. We found that ACE2 orthologs of a wide range of domestic and wild mammals, including camels, cattle, horses, goats, sheep, cats, rabbits, and pangolins, were able to support cell entry of SARS-CoV-2, suggesting that these species might be able to harbor and spread this virus. In addition, the pangolin and bat coronaviruses, Pangolin-CoV-2020 and Bat-CoV RaTG13, were also found able to utilize human ACE2 and a number of animal-ACE2 orthologs for cell entry, indicating risks of spillover of these viruses into humans in the future. We then developed potently anticoronavirus ACE2-Ig proteins that are broadly effective against the four distinct coronaviruses. In particular, through truncating ACE2 at its residue 740 but not 615, introducing a D30E mutation, and adopting an antibody-like tetrameric-ACE2 configuration, we generated an ACE2-Ig variant that neutralizes SARS-CoV-2 at picomolar range. These data demonstrate that the improved ACE2-Ig variants developed in this study could potentially be developed to protect from SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future.IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the currently uncontrolled coronavirus disease 2019 (COVID-19) pandemic. It is important to study the host range of SARS-CoV-2, because some domestic species might harbor the virus and transmit it back to humans. In addition, insight into the ability of SARS-CoV-2 and SARS-like viruses to utilize animal orthologs of the SARS-CoV-2 receptor ACE2 might provide structural insight into improving ACE2-based viral entry inhibitors. In this study, we found that ACE2 orthologs of a wide range of domestic and wild animals can support cell entry of SARS-CoV-2 and three related coronaviruses, providing insights into identifying animal hosts of these viruses. We also developed recombinant ACE2-Ig proteins that are able to potently block these viral infections, providing a promising approach to developing antiviral proteins broadly effective against these distinct coronaviruses.

Keywords: ACE2; ACE2-Ig; SARS-CoV-2; entry inhibitor; host range.

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Figures

FIG 1
FIG 1
SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J). RBD residues <5 Å from ACE2 atoms and ACE2 residues <5 Å from RBD atoms are shown. (B) The sequences of the SARS-CoV-2 WHU01, a pangolin coronavirus identified in Manis javanica (Pangolin-CoV-2020), a bat coronavirus identified in R. affinis (Bat-CoV RaTG13), and the SARS-CoV BJ01 are aligned, with residues different from the corresponding ones in SARS-CoV-2 highlighted in blue. The stars indicate RBD residues <5 Å from ACE2 atoms. The yellow lines indicate the RBM region. N-linked glycosylation motifs are indicated in green. (C) Sequences of ACE2 orthologs from the 16 indicated species are aligned, with only residues <5 Å from RBD atoms shown here. The numbering is based on human ACE2 protein, and the residues different from the corresponding ones in human ACE2 are highlighted in blue.
FIG 2
FIG 2
A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.
FIG 3
FIG 3
A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID50. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.
FIG 4
FIG 4
Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P < 0.001). The data shown are representative of two experiments independently performed by two different people with similar results, and data points represent the means ± the SD of four biological replicates.
FIG 5
FIG 5
The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.
FIG 6
FIG 6
A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC50 and IC90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.
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