Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation

  1. Hsu-Yu Chen
  2. Chun Huang
  3. Lu Tian
  4. Xiaoli Huang
  5. Chennan Zhang
  6. George N. Llewellyn
  7. Geoffrey L. Rogers
  8. Kevin Andresen
  9. Maurice R. G. O’Gorman
  10. Ya-Wen Chen
  11. Paula M. Cannon

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Abstract

Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors.

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  1. Version published to 10.1128/jvi.00966-21
  2. ScreenIT

    SciScore for 10.1101/2021.06.21.449352: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Lung bud organoid differentiation and transduction: Lung bud organoids were generated from human pluripotent stem cells (hPSCs) and validated as previously described (77). hPSC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of IMDM/Ham’s F-12 (3:1) (Life Technologies, Carlsbad, CA) supplemented with the following: 1 × N2 (Life Technologies), 0.5 × B27 (Life Technologies), 50 μg/ml ascorbic acid, 1 × Glutamax (Gibco), 0.4 μM monothioglycerol, 0.05% BSA, 10 μM Y27632, 0.5 ng/ml human BMP4 (R&D Systems), 2.5 ng/ml human FGF2 (R&D Systems, Minneapolis, MN), and 100 ng/ml human Activin (R&D Systems), in a 5% CO2/5% O2 atmosphere at 37 °C for 72-76 h.

    Table 2: Resources

    Antibodies
    SentencesResources
    Spike S1 antibody at 1:1000 (Prosci, Cat.# 9083); the S2 subunit was detected using anti-SARS-CoV/SARS-CoV-2 (COVID-19)
    anti-SARS-CoV/SARS-CoV-2
    suggested: None
    spike antibody clone [1A9] at 1:1000 (GeneTex, Cat.# GTX632604); HIV-1 p24 was detected using a polyclonal anti-HIV-1 SF2 p24 rabbit antiserum at 1:6000 (obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: ARP-4250, contributed by DAIDS/NIAID; produced by BioMolecular Technologies).
    anti-HIV-1 SF2
    suggested: None
    VSV M protein was detected using anti-VSV M antibody clone [23H12] (KeraFast, Boston, MA, Cat.#
    anti-VSV M
    suggested: None
    HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies were used as secondary antibodies (Santa Cruz Biotechnology, Dallas, TX).
    anti-rabbit
    suggested: None
    One million cells were re-suspend in 100 μl PBS and either immediately incubated with 0.25 μg anti-ACE2 antibody (R&D systems, Cat.# AF933) or first incubated at 37 °C for 6 hours with shaking to allow recovery of cell surface proteins after trypsinization.
    anti-ACE2
    suggested: None
    Alexa Fluor 647 conjugated donkey anti-goat antibody (1:200 dilution, Thermo Fisher Scientific, Waltham, MA, Cat.# A21447) was used as a secondary antibody and ACE2 expression was determined by flow cytometry (Guava easyCyte).
    anti-goat
    suggested: (Molecular Probes Cat# A-21447, RRID:AB_141844)
    APC-conjugated goat anti-mouse and ducky anti-rabbit antibodies were used as secondary antibodies (1:100 dilution, Invitrogen), and expression was detected by flow cytometry (Guava easyCyte).
    anti-mouse
    suggested: None
    Convalescent sera was confirmed to be positive for IgG class antibodies against SARS-CoV-2 Spike using anti-SARS-CoV-2 ELISA (IgG) (EUROIMMUN, Lübeck, Germany) (78).
    anti-SARS-CoV-2 ELISA (IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: 293T, HeLa, HeLa-ACE2, Vero, VeroE6 and Huh7.5 cells were maintained in Dulbecco’s modified Eagle medium (DMEM), and Calu-3 cells were maintained in Eagle’s Minimum Essential Medium (EMEM).
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Huh7.5
    suggested: RRID:CVCL_7927)
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    HeLa-ACE2 cells were transduced with Spike pseudotyped LV by seeding 1×104 cells per well in 96-well plates and adding 50μl of unconcentrated vector stocks the next day.
    HeLa-ACE2
    suggested: None
    Spike protein cell surface expression: VSV pseudovector-producing 293T cells were harvested to examine cell surface expression of the Spike protein.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    Plasmids: Full-length (S) and 18 amino acid cytoplasmic tail truncated (SΔ18) Spike proteins for the Wuhan-Hu-1 isolate of SARS-CoV-2 (GenBank: MN908947.3) were provided by Dr. James Voss (The Scripps Research Institute) in a plasmid pcDNA3.3 backbone.
    pcDNA3.3
    suggested: RRID:Addgene_26820)
    Lentiviral vector production, concentration and transduction: Lentiviral vectors were generated by transfection of 10 cm plates of 293T cells at 75% confluency with 2 μg of Spike expression plasmid, 10 μg of packaging plasmid pCMVdeltaR8.2 (Addgene Cat.# 12263) and 10 μg of a GFP-expressing vector genome plasmid FUGW (Addgene Cat.# 14883).
    pCMVdeltaR8.2
    suggested: None
    A suitable dose of Spike pseudotyped VSV-Luc vectors was used in the neutralization assays to produce approximately 105 relative light unit (RLU) of luciferase activity on HeLa-ACE2 cells in the absence of serum.
    VSV-Luc
    suggested: None
    Software and Algorithms
    SentencesResources
    spike antibody clone [1A9] at 1:1000 (GeneTex, Cat.# GTX632604); HIV-1 p24 was detected using a polyclonal anti-HIV-1 SF2 p24 rabbit antiserum at 1:6000 (obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: ARP-4250, contributed by DAIDS/NIAID; produced by BioMolecular Technologies).
    HIV Reagent Program
    suggested: None
    BioMolecular
    suggested: (Nemours Biomolecular Core Facility, RRID:SCR_018265)
    Densitometry was measured using ImageJ software (http://rsb.info.nih.gov/ij/).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

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

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  3. Version published to 10.1101/2021.06.21.449352 on bioRxiv