key: cord-0990865-dblrxlt1
authors: Li, Yujun; Wang, Haimin; Tang, Xiaojuan; Ma, Danting; Du, Chengzhi; Wang, Yifei; Pan, Hong; Zou, Qing; Zheng, Jie; Xu, Liangde; Farzan, Michael; Zhong, Guocai
title: Potential host range of multiple SARS-like coronaviruses and an improved ACE2-Fc variant that is potent against both SARS-CoV-2 and SARS-CoV-1
date: 2020-04-11
journal: bioRxiv
DOI: 10.1101/2020.04.10.032342
sha: d984aa88e0341a41d75b65b83d192bbae6d3e644
doc_id: 990865
cord_uid: dblrxlt1

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a currently uncontrolled pandemic and the etiological agent of coronavirus disease 2019 (COVID-19). 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. Here we show that ACE2 orthologs of a wide range of domestic and wild animals support entry of SARS-CoV-2, as well as that of SARS-CoV-1, bat coronavirus RaTG13, and a coronavirus isolated from pangolins. Some of these species, including camels, cattle, horses, goats, sheep, pigs, cats, and rabbits may serve as potential intermediate hosts for new human transmission, and rabbits in particular may serve as a useful experimental model of COVID-19. We show that SARS-CoV-2 and SARS-CoV-1 entry could be potently blocked by recombinant IgG Fc-fusion proteins of viral spike protein receptor-binding domains (RBD-Fc) and soluble ACE2 (ACE2-Fc). Moreover, an ACE2-Fc variant, which carries a D30E mutation and has ACE2 truncated at its residue 740 but not 615, outperforms all the other ACE2-Fc variants on blocking entry of both viruses. Our data suggest that RBD-Fc and ACE2-Fc could be used to treat and prevent infection of SARS-CoV-2 and any new viral variants that emerge over the course of the pandemic.

Two subgroups of coronaviruses, including alphacoronaviruses (e.g. swine acute diarrhea syndrome 19 coronavirus, SADS-CoV) and betacoronaviruses (e.g. SARS coronavirus 1, SARS-CoV-1), infect 20 mammals and have broad host ranges spanning bats, rodents, domestic animals, and humans [1] [2] [3] [4] [5] [6] [7] . Bats 21 are considered to be the natural reservoir hosts for a number of pathogenic human coronaviruses, Receptor binding is a critical determinant for the host range of coronaviruses 6,12 . SARS-CoV-2 utilizes 39 ACE2, the SARS-CoV-1 receptor 13 , as an essential cellular receptor to infect cells 8,14,15 . Therefore, in 40 this study, we characterized seventeen ACE2 orthologs for their function to support entry of SARS- 

A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2, Pangolin-48 CoV, Bat-CoV RaTG13, and SARS-CoV-1 49 The RBD of SARS-CoV-2, like that of SARS-CoV-1, mainly relies on residues in its receptor binding 50 motif (RBM) to bind ACE2 (Figs. 1a and b) [17] [18] [19] . Through analyzing ACE2 residues that are within 5 51 Å from SARS-CoV-2 RBD atoms, we found that these residues are highly conserved across the 52 seventeen analyzed ACE2 orthologs, including human ACE2 and ACE2 orthologs of sixteen domestic Pangolin-CoV, and SARS-CoV-1 BJ01 (Extended Data Fig. 2a) . These RBD proteins were then 57 used to perform surface staining of 293T cells transfected with each of the seventeen ACE2 orthologs 58 or a vector plasmid control (Figs. 2a and b) . The flow cytometry data clearly show that, although 59 each of the four tested RBDs has a distinctive binding profile, they all can bind to a wide range of 60 ACE2 orthologs. Note that human ACE2 and ACE2 orthologs of all the domestic animals, except for 61 that of donkeys, guinea pigs, and chickens, support efficient binding to all the four tested RBDs. The 62 lack of any RBD binding to donkey ACE2 is likely due to a protein sequence defect, because 63 expression of the donkey ACE2 is detectable in 293T cells but a 22-residue fragment likely important 64 for ACE2 folding is missing in the Genbank sequence (Fig. 2c , and Extended Data Figs. 1 and 2b) . 65 These data suggest that ACE2 orthologs of domestic animals might be generally functional for 66 supporting cell entry of the four tested viruses. CoV-1, were respectively used to infect 293T cells expressing each of the seventeen ACE2 orthologs.

A VSV-G-pseudotyped reporter retrovirus whose entry is independent of ACE2 was used as a control 

CoV-1 88 Recombinant RBD and soluble ACE2 proteins have been shown to potently block SARS-CoV-1 89 entry 20,21 . To investigate whether similar approaches could also be applied to SARS-CoV-2, we first 90 produced IgG Fc fusion proteins of RBD (RBD-Fc) and soluble ACE2 (ACE2-Fc) variants (Fig. 4a) . 91 Specifically, the RBD variants include wildtype RBDs of SARS-CoV-1, Pangolin-CoV, and SARS- showed improved potency over the corresponding wildtypes (Fig. 4c) . We further tested three of the Fig. 4c) . Thus, the mechanism 107 of the D30E-mediated improvement is likely that the mutation enhances the salt-bridge interaction 108 between the residue 30 of the ACE2 and residue 417 of the SARS-CoV-2 RBD (Fig. 4f) , because this 109 interaction is only present in the ACE2 complex with the RBD of SARS-CoV-2 but not that of SARS- 

In this study, we found that ACE2 orthologs of a wide range of domestic animals and pangolins 113 efficiently support binding and entry of SARS-CoV-2, as well as that of SARS-CoV-1, a pangolin RBD residues in less than 5 Å from ACE2 atoms and ACE2 residues in less than 5 Å from RBD atoms are shown. b, The sequences of the SARS-CoV-2 WHU01, a pangolin coronavirus (Pangolin-CoV), a bat coronavirus (Bat-CoV RaTG13), and the SARS-CoV-1 BJ01 are aligned, with residues different from the corresponding ones in SARS-CoV-2 highlighted in blue. The stars indicate RBD residues in less than 5 Å from ACE2 atoms. The yellow lines indicate the RBM region, and the red lines or arrows indicate disulfide linkages. N-linked glycosylation motifs are indicated in green. c, Sequences of ACE2 orthologs from the 17 indicated species are aligned, with only residues in less than 5 Å from RBD atoms shown here and aligned full-length protein sequences shown in the Extended Data Figure 1 . The numbering is based on human ACE2 protein, and the residues different from the corresponding ones in human ACE2 are highlighted in blue. 

Receptor recognition and cross-species infections of SARS coronavirus

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Functional assessment of cell entry and receptor usage for SARS-CoV-188 2 and other lineage B betacoronaviruses

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a 190 Clinically Proven Protease Inhibitor

Are pangolins the intermediate host of the 2019 novel coronavirus (2019-nCoV) ? bioRxiv

Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2

Structure of SARS coronavirus spike receptor-binding domain 196 complexed with receptor

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

A 193-amino acid fragment of the SARS 200 coronavirus S protein efficiently binds angiotensin-converting enzyme 2

Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike 203 protein efficiently infect cells expressing angiotensin-converting enzyme 2

The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice. bioRxiv

Differences in innate immune response between man and mouse

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute 209 SARS-CoV infection

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of 211 human immune cells via a pH-and cysteine protease-independent FcgammaR pathway

Buffers were then exchanged to PBS and proteins were concentrated by 30 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (Millipore)

Flow cytometry for detecting interactions of RBD-Fc proteins with cell surface ACE2

Cells in each well were then transfected with 0.5 µL of lipofectamine 2000 (Life Technologies) in complex with 200 ng of plasmid encoding one of the seventeen ACE2 orthologs or a D30E mutant of the human ACE2. Culture medium was changed at 6 hours after transfection. Cells were then detached with 5 mM EDTA at 36 hours post transfection. Cells were then stained with 5 µg/mL RBD-Fc proteins at 37 o C for 10 min

After another three washes, cells were analyzed by Attune NxT flow cytometer (Thermo Fisher) and signals of 10,000 FSC/SSC gated cells were collected for each sample

Thirty-six hours after transfection, cells were lysed and 10 µg of total protein were used for western blot. ACE2-S-tag expression was detected by using 6.2, a mouse anti-S-tag monoclonoal antibody (Invitrogen, Cat. No. MA1-981), and an HRP-conjugated goat anti-mouse IgG Fc secondary antibody (Invitrogen, Cat. No. 31437). Spike-C9-tag expression was then detected by using 1D4, a mouse anti-C9-tag monoclonal antibody (Invitrogen, Cat

293T cells were seeded at 30% density in 150 mm dish at 12-15 hours before transfection. Cells were then transfected with 67.5 µg of polyethylenimine (PEI) Max 40,000 (Polysciences) in complex with 11.25 µg of plasmid encoding a coronavirus spike protein or VSV-G, 11.25 µg of plasmid encoding murine leukemia virus (MLV) Gag and Pol proteins, and 11.25 µg of a pQCXIPbased GFP or luciferase reporter plasmid. Eight hours after transfection, cell culture medium was refreshed and changed to growth

Cell culture supernatants were collected at 36-48 hours post transfection, spun down at 3000×g for 10 min, and filtered through 0.45 µm filter units to remove cell debris. Coronavirus spikepseudotyped viruses were then concentrated 10 times at 2000×g using 100 kDa cut-off Amicon Ultra-15 Centrifugal Filter Units (Millipore)

Pseudovirus infection of 293T cells expressing ACE2 orthologs. 293T cells were seeded at 20% density in poly-lysine pre-coated 48-well plates 12-15 hours before transfection. Cells in each well were then transfected with 0.5 µL of lipofectamine 2000 (Life Technologies) in complex with 200 ng of a vector control plasmid or a plasmid encoding one of the seventeen ACE2 orthologs

Additional 18 hours later, cells in each well were infected with 50 µL of SARS-CoV-2 wildtype pseudovirus (10×concentrated), 20 µL of SARS-CoV-2 ΔFurin pseudovirus (10×concentrated), 50 µL of Bat-CoV RaTG13

Culture medium was refreshed at 2 hours post pseudovirus infection and the medium was refreshed every 12 hours. For the luciferase reporter virus-infected cells, cell culture supernatants were collected and subjected to a Gaussia luciferase assay at 48 hours post infection. The GFP reporter virusinfected cells were stained with Hoechst 33342 (Invitrogen) and subjected to fluorescent microscopy (IX73 microscope

To measure Gaussia luciferase expression, 20 µL of cell culture supernatant of each sample and 100 µL of assay buffer containing 4 µM coelenterazine native (Biosynth Chemistry & Biology) were added to one well of a 96-well black opaque assay plate (Corning), and measured with the Tristar5 multifunctional microplate reader

SARS-CoV-2 or SARS-CoV-1 spike protein-pseudotyped luciferase reporter virus was diluted in DMEM (2% FBS, heat-inactivated) containing titrated amounts of RBD-Fc or ACE2-Fc variant proteins. Virus-inhibitor mixtures were then added to cells for 2 h at 37 o C. Cells were then washed with serum-free medium and incubated in 250 µL of DMEM (2% FBS) at 37 o C. Cell culture supernatants were collected for the Gaussia luciferase assay at 48 h post infection. Statistical analysis. Data expressed as mean values ± s.d. or s.e.m. Statistical analyses were performed using two-sided two-sample Student's t-test using GraphPad Prism 6.0 software when applicable. Differences were considered significant at P < 0.01. Data availability. Our research resources, including methods, plasmids, and protocols, are available upon reasonable request to qualified academic investigators for noncommercial research purposes

Are pangolins the intermediate host of the 2019 novel coronavirus (2019-nCoV) ? bioRxiv

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Extended Data Fig. 1 | Protein sequence alignment of the seventeen ACE2 orthologs evaluated in this study. Detailed information about the species and accession numbers of the ACE2 orthologs are presented in the Extended Data Table 1 . Fig. 1 (continued) | Protein sequence alignment of the seventeen ACE2 orthologs evaluated in this study. Detailed information about the species and accession numbers of the ACE2 orthologs are presented in the Extended Data Table 1 . Figure 2 . a, SDS-PAGE image of the purified recombinant RBD-Fc fusion proteins used in the cell-surface ACE2 staining experiments shown in Figure 2a . b, SARS-CoV-2 RBD in complex with human ACE2. The residues absent in donkey ACE2, as shown in Extended Data Figure 1 , are indicated as magenta. c, Gating strategy for the flow cytometry data shown in Figures 2a and b . The left panel shows the preliminary FSC/SSC gate of the starting cell population. The right panel shows the gates that defines "negative" (red) and "positive" (purple) staining cell populations, respectively. Fig. 3 | Optimization of the coronavirus pseudotype systems. a, Fluorescent microscopic images showing that SARS-CoV-2 wildtype spike-based pseudovirus system is not very robust and should be optimized. b, Original reporter expression data used for generating the heatmaps shown in Figure 3c . Data shown are representative of three experiments independently performed by three different people with similar results, and data points represent mean ± s.d. of three biological replicates. Fig. 4 | Key reagents (a, b) used in the experiments described in Figure 4 and flow cytometry data (c) supporting the D30E improvement of neutralization potency shown in Figure  4c .