ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

  1. Daniel J. Rawle
  2. Thuy T. Le
  3. Troy Dumenil
  4. Kexin Yan
  5. Bing Tang
  6. Cameron Bishop
  7. Andreas Suhrbier

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Abstract

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR -/- and IL-28RA -/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARY

SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo , with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.09.430547: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Mouse work was approved by the QIMR Berghofer Medical Research Institute animal ethics committee (P3600, A2003-607).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFemale mice were 8 weeks to 1 year old (age matched between groups) at the start of the experiment.
    Cell Line AuthenticationContamination: Cells were routinely checked for mycoplasma (MycoAlert Mycoplasma Detection Kit MycoAlert, Lonza) and FCS was assayed for endotoxin contamination before purchase (Johnson et al., 2005).

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    USA), HEK293T, AE17, and NIH-3T3 cells were cultured in medium comprising RPMI1640 (Gibco) supplemented with 10% fetal calf serum (FCS), penicillin (100 □IU/ml)/streptomycin (100□μg/ml) (Gibo/Life Technologies) and L-glutamine (2 mM) (Life Technologies).
    NIH-3T3
    suggested: None
    The virus was determined to be mycoplasma free using co-culture with a non-permissive cell line (i.e. HeLa) and Hoechst staining as described (La Linn et al., 1995).
    HeLa
    suggested: None
    CCID50 assays: Vero E6 cells were plated into 96 well flat bottom plates at 2×104 cells per well in 100 μl of medium.
    Vero E6
    suggested: None
    Lentivirus production, titration and cell line transduction: ACE2 lentivirus was produced by co-transfection of HEK293T cells with the pCDH-ACE2 plasmid, VSV-G and Gag-Pol using Lipofectamine 2000 Reagent (Thermo Fisher Scientific) or Xfect Transfection Reagent (Takara Bio) as per manufacturer instructions.
    HEK293T
    suggested: None
    Neutralization assay: Mouse serum was heat inactivated at 56°C for 30 min and incubated with 100 CCID50 SARS-CoV-2 for 2 hr at 37°C before adding 105 Vero cells/well in a 96 well plate to 200 μl.
    Vero
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Cell lines and SARS-CoV-2 culture: Vero E6 (C1008, ECACC, Wiltshire, England; Sigma Aldridge, St. Louis, MO,
    SARS-CoV-2 culture: Vero E6
    suggested: None
    Mice intranasal lentivirus transduction and SARS-CoV-2 infection: C57BL/6J, IFNAR-/- (Swann et al., 2007) (originally provided by P. Hertzog, Monash University, Melbourne, VIC, Australia) and IL-28RA-/- (Ank et al., 2008; Galani et al., 2017) mice were bred in-house at QIMRB.
    C57BL/6J
    suggested: None
    IL-28RA-/-
    suggested: RRID:IMSR_TIGM:IST11948G1)
    K18-hACE2 mice (McCray et al., 2007) were purchased from The Jackson Laboratory and bred in-house at QIMRB with C57BL/6J mice.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Cell lines and SARS-CoV-2 culture: Vero E6 (C1008, ECACC, Wiltshire, England; Sigma Aldridge, St. Louis, MO,
    England; Sigma Aldridge
    suggested: None
    The quality of raw sequencing reads was assessed using FastQC (Simons, 2010) (v0.11.8) and trimmed using Cutadapt (Martin, 2011) (v2.3) to remove adapter sequences and low-quality bases.
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Trimmed reads were aligned using STAR (Dobin et al., 2013) (v2.7.1a) to a combined reference that included the mouse GRCm38 primary assembly and the GENCODE M23 gene model (Harrow et al., 2012), SARS-CoV-2 isolate Wuhan-Hu-1 (NC_045512.2; 29903 bp) and the human ACE2 mouse codon optimized sequence (2418 bp).
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Analyses of K18-hACE2 and Ad5-hACE2 RNA-seq data: RNA-seq datasets generated from the Winkler et al. study (Winkler et al., 2020), and Sun et al. study (Sun et al., 2020a) were obtained from the Gene Expression Omnibus (GSE154104 and GSE150847 respectively) and trimmed using Cutadapt (v2.3).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Cutadapt
    suggested: (cutadapt, RRID:SCR_011841)
    Mouse gene expression was estimated using RSEM (v1.3.0)
    RSEM
    suggested: (RSEM, RRID:SCR_013027)
    Reads aligned to SARS-CoV-2 and hACE2 were counted using SAMtools (v1.9)
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Differential gene expression in the mouse was analyzed using EdgeR (3.22.3) and modelled using the likelihood ratio test, glmLRT().
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    Diseases and Functions and canonical pathways enriched in differentially expressed genes in direct and indirect interactions were investigated using Ingenuity Pathway Analysis (IPA) (QIAGEN).
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    Network Analysis: Protein interaction networks of differentially expressed gene lists were visualized in Cytoscape (v3.7.2)
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Enrichment for biological processes, molecular functions, KEGG pathways and other gene ontology categories in DEG lists was elucidated using the STRING database (Szklarczyk et al., 2019) and GO enrichment analysis (Mi et al., 2019)
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    STRING
    suggested: (STRING, RRID:SCR_005223)
    GSEA (Subramanian et al., 2005) was performed on a desktop application (GSEA v4.0.3) (http://www.broadinstitute.org/gsea/) using the “GSEAPreranked” module.
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    http://www.broadinstitute.org/gsea/
    suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 25. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from scite Reference Check: We found one citation with an erratum. We recommend checking the erratum to confirm that it does not impact the accuracy of your citation.

    DOIStatusTitle
    10.1371/journal.ppat.1003106Has correctionRecombinant HIV Envelope Proteins Fail to Engage Germline Ve…

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