Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
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Abstract
Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.
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SciScore for 10.1101/2020.09.20.297242: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cells were tested routinely and found to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4 °C overnight. SARS-CoV nucleocapsid proteinsuggested: (Creative …SciScore for 10.1101/2020.09.20.297242: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cells were tested routinely and found to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4 °C overnight. SARS-CoV nucleocapsid proteinsuggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)anti-FLAGsuggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)After three washes, cells were incubated with a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A32732, 2 μg/ml) and goat anti-mouse antibody conjugated with Alexa Fluor 647 (Thermo #A21235, 2 μg/ml) for 2 h at room temperature, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI). anti-rabbitsuggested: (Thermo Fisher Scientific Cat# A32732, RRID:AB_2633281)anti-mousesuggested: (Molecular Probes Cat# A-21235, RRID:AB_2535804)Experimental Models: Cell Lines Sentences Resources Cell culture and SARS-CoV-2 virus: HEK293T (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and A549 (ATCC) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol Vero E6suggested: NoneA549suggested: None(Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Although human ACE2 transgenic mice can be infected with SARS-CoV-2, the non-physiological expression and tissue distribution of human ACE2 in such models represent a major limitation for conducting pathogenesis studies and accurately modeling the disease features observed in severe cases of COVID-1916,17. Thus, animal models which recapitulate human disease are urgently required. Genetic modifications to minimally humanize the hepatitis C virus (HCV) receptors CD81 and OCLN in mice by knocking in critical domains rendered the mice susceptible to HCV entry40-42. Based on our findings here, a similar knock-in strategy to generate an ACE2 humanized mouse is worth considering to aid in the development of novel mouse models capable of supporting SARS-CoV-2 infection as well as antiviral therapies. Altogether, our study sheds more light on the species-specificity of interactions between SARS-CoV-2 and its cellular receptor ACE2 and uncovers evidence for a molecular arms race between SARS-CoV-2 and animal species. Here we identified the genetic basis defining the host range of SARS-CoV-2, which could potentially inform the development of novel animal models for SARS-CoV-2 research.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.09.20.297242: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Cells were tested routinely and found to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4 °C overnight. SARS-CoV nucleocapsid …SciScore for 10.1101/2020.09.20.297242: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Cells were tested routinely and found to be free of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4 °C overnight. SARS-CoV nucleocapsid proteinsuggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)anti-FLAGsuggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)After three washes, cells were incubated with a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A32732, 2 μg/ml) and goat anti-mouse antibody conjugated with Alexa Fluor (Thermo #A21235, 2 μg/ml) for 2 h at room temperature, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI). anti-rabbitsuggested: (Thermo Fisher Scientific Cat# A32732, RRID:AB_2633281)anti-mousesuggested: (Molecular Probes Cat# A-21235, RRID:AB_2535804)Experimental Models: Cell Lines Sentences Resources The SARS-CoV-2 strain nCoV-SH01 (GenBank accession no. MT121215) was isolated from a COVID-19 patient and propagated in Vero E6 cells for use. Vero E6suggested: RRID:CVCL_XD71)(Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells. HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)A549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. A549suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
Although human ACE2 transgenic mice can be infected with SARS-CoV-2, the non-physiological expression and tissue distribution of human ACE2 in such models represent a major limitation for conducting pathogenesis studies and accurately modeling the disease features observed in severe cases of COVID-1916,17. Thus, animal models which recapitulate human disease are urgently required. Genetic modifications to minimally humanize the hepatitis C virus (HCV) receptors CD81 and OCLN in mice by knocking in critical domains rendered the mice susceptible to HCV entry40-42. Based on our findings here, a similar knock-in strategy to generate an ACE2 humanized mouse is worth considering to aid in the development of novel mouse models capable of supporting SARS-CoV-2 infection as well as antiviral therapies. Altogether, our study sheds more light on the species-specificity of interactions between SARS-CoV-2 and its cellular receptor ACE2 and uncovers evidence for a molecular arms race between SARS-CoV-2 and animal species. Here we identified the genetic basis defining the host range of SARS-CoV-2, which could potentially inform the development of novel animal models for SARS-CoV-2 research.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
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