SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

  1. Michihito Sasaki
  2. Kentaro Uemura
  3. Akihiko Sato
  4. Shinsuke Toba
  5. Takao Sanaki
  6. Katsumi Maenaka
  7. William W. Hall
  8. Yasuko Orba
  9. Hirofumi Sawa

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Abstract

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

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  1. Version published to 10.1371/journal.ppat.1009233
  2. ScreenIT

    SciScore for 10.1101/2020.08.28.271163: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: Vero cells stably expressing TMPRSS2 (Vero-TMPRSS2) and 293T stably expressing ACE2 (293T-ACE2) were selected in the presence of blasticidin S or puromycin. Viruses: SARS-CoV-2 WK-521 strain was provided by Dr. Shimojima (National Institute of Infectious Diseases, Japan); the original stock of this virus (wild type, WT) was prepared by inoculation of Vero-TMPRSS2 cells with Mynox mycoplasma elimination reagent (Minerva Biolabs) [11].

    Table 2: Resources

    Antibodies
    SentencesResources
    The blots were incubated with the following primary antibodies: anti-SARS-CoV-2 N or anti-SARS-CoV-2 S (GTX632269, GTX632604, GeneTex).
    anti-SARS-CoV-2 N
    suggested: None
    anti-SARS-CoV-2 S ( GTX632269 , GTX632604
    suggested: None
    HRP-conjugated anti-β-actin antibody (PM053-7, MBL) was used to detect the loading control.
    anti-β-actin
    suggested: None
    After 24 h, cells were fixed with 3.7% buffered formaldehyde, permeabilized with ice-cold methanol, and incubated with anti-SARS-CoV-2 S antibody (GTX632604, GeneTex).
    anti-SARS-CoV-2 S
    suggested: None
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    Alexa Fluor Plus 488-conjugated anti-mouse IgG antibody (Invitrogen; Thermo Fisher Scientific) was used as the secondary antibody.
    Alexa Fluor Plus 488-conjugated anti-mouse IgG antibody
    suggested: None
    anti-mouse IgG
    suggested: (Thermo Fisher Scientific Cat# A-21042, RRID:AB_2535711)
    Experimental Models: Cell Lines
    SentencesResources
    Caco-2 (RIKEN BRC) cells were maintained in Eagle’s MEM supplemented with 10% FBS and non-essential amino acids.
    Caco-2
    suggested: None
    Vero E6 (ATCC) and 293T (JCRB cell bank) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS.
    Vero E6
    suggested: RRID:CVCL_XD71)
    For lentiviral vector preparation, 293T cells were co-transfected with the lentiviral vector plasmid and Lentiviral High Titer Packaging Mix (Takara Bio).
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Vero cells stably expressing TMPRSS2 (Vero-TMPRSS2) and 293T stably expressing ACE2 (293T-ACE2) were selected in the presence of blasticidin S or puromycin. Viruses: SARS-CoV-2 WK-521 strain was provided by Dr. Shimojima (National Institute of Infectious Diseases, Japan); the original stock of this virus (wild type, WT) was prepared by inoculation of Vero-TMPRSS2 cells with Mynox mycoplasma elimination reagent (Minerva Biolabs) [11].
    Vero
    suggested: None
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Deep sequencing of the S gene of passaged SARS-CoV-2 virions: The original stock of SARS-CoV-2 strain WK-521 was serially passaged in Vero, Vero-TMPRSS2, Calu-3, Caco-2, and 293T-ACE2 cells in complete culture medium or (for Vero) in serum free DMEM supplemented with 0.5 μg/ml trypsin (Gibco); three biological replicates were included for each of the cell lines.
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    Deep sequencing of the S gene of passaged SARS-CoV-2 virions: The original stock of SARS-CoV-2 strain WK-521 was serially passaged in Vero, Vero-TMPRSS2, Calu-3, Caco-2, and 293T-ACE2 cells in complete culture medium or (for Vero) in serum free DMEM supplemented with 0.5 μg/ml trypsin (Gibco); three biological replicates were included for each of the cell lines.
    Gibco)
    suggested: None

    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: 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  3. Version published to 10.1101/2020.08.28.271163 on bioRxiv