Susceptibilities of Human ACE2 Genetic Variants in Coronavirus Infection

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1. Version published to 10.1128/jvi.01492-21

   Jan 12, 2022
2. 

   ScreenIT

   Jul 21, 2021

   SciScore for 10.1101/2021.07.18.452826: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: This animal experiment protocol was approved by the Animal Ethics Committee of the School of Basic Medical Sciences at Fudan University.
   Sex as a biological variable	SARS-CoV-2 infection of adenovirus transduced mice: Six to eight week-old male mice (BALB/c) were transduced intranasally with adenovirus expressing wild-type ACE2, the D355N variant or empty control (5×1010 viral particles per mouse).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were tested routinely and found to be free of mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were fixed with 4% paraformaldehyde in PBS, …

   SciScore for 10.1101/2021.07.18.452826: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: This animal experiment protocol was approved by the Animal Ethics Committee of the School of Basic Medical Sciences at Fudan University.
   Sex as a biological variable	SARS-CoV-2 infection of adenovirus transduced mice: Six to eight week-old male mice (BALB/c) were transduced intranasally with adenovirus expressing wild-type ACE2, the D355N variant or empty control (5×1010 viral particles per mouse).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were tested routinely and found to be free of mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were fixed with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with the rabbit polyclonal antibody against SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1 μg/ml) at 4 °C overnight.	SARS-CoV nucleocapsid protein ( Rockland , 200-401-A50 , 1 suggested: None
   After three washes, cells were incubated with the secondary goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Thermo #A11034, 2 μg/ml) for 2 h at room temperature, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI).	anti-rabbit suggested: (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217)
   To detect the expression of FLAG-tagged ACE2 delivered by adenovirus, the sections were incubated in blocking reagent and then with FLAG M2 antibody (1:100 dilution, Sigma-Aldrich #1804) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution, Invitrogen).	FLAG suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044) anti-mouse IgG suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
   For viral antigen detection, the sections were incubated with house-made mouse anti-SARS-CoV-2 nucleocapsid protein serum (1:5000) and HRP465 conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution, Invitrogen).	anti-SARS-CoV-2 nucleocapsid protein serum suggested: None
   Cells were stained with homemade mouse anti-SARS-CoV-2 N serum overnight at 4°C, incubated with the secondary goat anti-mouse HRP-conjugated antibody for 2 h at room temperature.	anti-SARS-CoV-2 suggested: None anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell cultures and SARS-CoV-2 virus: HEK293T cells (American Tissue Culture Collection, ATCC, Manassas, VA, CRL-3216), Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HeLa (ATCC #CCL-2) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, NY, USA) supplemented with 10% (vol/vol	Vero E6 suggested: None
   (Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Surface ACE2 binding assay: HeLa cells were transduced with lentiviruses expressing the ACE2 from different species for 48 h.	HeLa suggested: None
   The pShuttle-ACE2 plasmids were then electroporated 440 into BJ5183 AD-1 cells (Agilent), which were pretransformed with pAdEasy-1 to facilitate recombination with the pShuttle-CMV vector.	AD-1 suggested: None
   The transfected HEK293 cells were maintained until the cells exhibited complete cytopathic effect (CPE) and then harvested and freeze-thawed.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   SARS-CoV-2 infection of adenovirus transduced mice: Six to eight week-old male mice (BALB/c) were transduced intranasally with adenovirus expressing wild-type ACE2, the D355N variant or empty control (5×1010 viral particles per mouse).	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Plasmids: The cDNAs encoding ACE2 orthologs (Table S1) were synthesized by GenScript and cloned into pLVX-IRES-zsGreen1 vectors (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.	pLVX-IRES-zsGreen1 suggested: None
   (Addgene #12259) and psPAX2 (Addgene #12260) and the transfer vector with VigoFect DNA transfection reagent (Vigorous) into HEK293T cells.	psPAX2 suggested: RRID:Addgene_12260)
   Specifically, SARS-CoV-2 RBD (residues Thr333-Pro527), SARS-CoV RBD (residues Arg306–Leu515), or NL63-CoV RBD (residues Gln481-Ile616) with a N-terminal gp67 signal peptide for secretion and a C-terminal 6×His tag for purification was inserted into pFastBac-Dual vector (Invitrogen).	pFastBac-Dual suggested: RRID:Addgene_135584)
   Production of SARS-CoV-2 or SARS-CoV pseudotyped virus: Pseudoviruses were produced in HEK293T cells by co-transfecting the retroviral vector pTG-MLV-Fluc, pTG-MLV-Gag-pol, and pcDNA3.1 expressing the SARS-CoV spike, SARS-CoV-2 spike or VSV-G (pMD2.G (Addgene #12259)) using VigoFect (	pTG-MLV-Fluc suggested: None pTG-MLV-Gag-pol suggested: None pcDNA3.1 suggested: RRID:Addgene_79663) VSV-G suggested: RRID:Addgene_138479) pMD2 . G suggested: None
   Generation and production of recombinant adenovirus expressing ACE2 variants: cDNA of ACE2 variants with a FLAG tag at the C-terminus was cloned into pShuttle.	pShuttle suggested: RRID:Addgene_16402)
   The pShuttle-ACE2 plasmids were then electroporated 440 into BJ5183 AD-1 cells (Agilent), which were pretransformed with pAdEasy-1 to facilitate recombination with the pShuttle-CMV vector.	pShuttle-ACE2 suggested: None pAdEasy-1 suggested: RRID:Addgene_16400) pShuttle-CMV suggested: RRID:Addgene_16403)
   Software and Algorithms
   Sentences	Resources
   Images were processed using the ImageJ program (http://rsb.info.nih.gov/ij/).	ImageJ suggested: (ImageJ, RRID:SCR_003070)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   We note that our conclusions are based on experiments performed in cell culture and in an artificial animal model and therefore have certain limitations. It will be important to extend this work to a real-world study, which could bridge the gap between genotype, phenotype and epidemiology. In addition, we only considered the effect of ACE2 SNVs. As the serine protease TMPRSS2 can prime the viral spike for direct fusion with the plasma membrane39, 43, SNPs in TMPRSS2 might also impact SARS-CoV-2 cell entry and be useful target of future studies. Moreover, SNPs in the 5’UTR or other non-coding regions of ACE2 were not included in this study and may also be important in regulating ACE2 transcription or translation, leading to varied ACE2 protein levels in vivo that could affect virus entry and the severity of COVID-19. Due to these limitations, we urge caution not to over interpret the results of this study. Of note, SARS-CoV-2 has evolved rapidly in humans, and a variety of genomic changes, including mutations and deletions in the spike protein, have recently been identified44, 45. Given that genetic variants will continue to arise, it is likely that we will see more genomic changes in the spike protein that might affect the spike interaction with human ACE2. Thus, it will be important to continue testing the interactions of these mutated spike proteins with the ACE2 SNVs. In summary, our study suggest that ACE2 polymorphism could impact human susceptibility to SARS-CoV-2 infec...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.07.18.452826v1 on bioRxiv

   Jul 19, 2021