SARS-CoV-2 Delta spike protein enhances the viral fusogenicity and inflammatory cytokine production

  1. Zhujun Ao
  2. Maggie Jing Ouyang
  3. Titus Abiola Olukitibi
  4. Xiaojian Yao

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Abstract

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  1. Version published to 10.1016/j.isci.2022.104759
  2. ScreenIT

    SciScore for 10.1101/2021.11.23.469765: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The rabbit polyclonal antibody against SARS-CoV-2 SP/RBD (Cat# 40592-T62) or human SARS-CoV-2 S-NTD antibody (E-AB-V1030) was obtained from Sino Biological or Elabscience.
    SP/RBD
    suggested: None
    Mouse monoclonal antibody (1A9) against SARS-CoV-2 SP-S2 (Cat# ab273433) was obtained from Abcam.
    SARS-CoV-2
    suggested: (Abcam Cat# ab273433, RRID:AB_2891068)
    SP-S2
    suggested: None
    Anti-HIVp24 monoclonal antibody was described previously (Ao et al., 2007; Qiu et al., 2011)
    Anti-HIVp24
    suggested: None
    Anti-human ACE2 antibody (sc-390851) was obtained from Santa Cruz Biotechnology Inc.
    Anti-human ACE2 antibody
    suggested: None
    Anti-human ACE2
    suggested: None
    SPΔCwt/mutant expression in the different transduced A549 cells was evaluated by WB using an anti-RBD antibody.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549-expressing human ACE2 (A549ACE2) cells were generated by transducing A549 cells with the ACE2-expressing lentiviral vector (pLenti-C-mGFP-ACE2) (Origene, Cat# PS100093) and then selected with puromycin according to the manufacturer’s procedure.
    A549ACE2
    suggested: None
    Virus production and infection experiments: SARS-CoV-2 SPΔC pseudotyped viruses (CoV-2-SPΔC-PVs, CoV-2-SPΔCG614-PVs and CoV-2-SPΔCDelta-PVs) or pseudotyped virus-like particles (VLPs) were produced by transfecting HEK293T cells with pCAGGS-SPΔCWT, pCAGGS-SPΔCG614 or pCAGGS-SPΔCDelta and pCMVΔ8.2 with or without a Gluc-expressing HIV vector ΔRI/E/Gluc (Ao et al., 2021a).
    HEK293T
    suggested: None
    To measure the infection ability of SARS-CoV-2 SPΔC pseudotyped VPs, equal amounts of each SPΔC-PVs stock (as adjusted by p24 levels) were used to infect A549ACE2, Calu-3 cells, human MDMs or MDDCs.
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Generation of different SPΔC-expressing A549 stable cell lines: Production of lentiviral vectors expressing different SPΔC: 293T cells were cotransfected with pEF1-SPΔCwt, pEF1-SPΔCG614 or pEF1-SPΔCDelta with packaging plasmid Δ8.2 and VSV-G expressing plasmid.
    293T
    suggested: None
    Then, each produced lentiviral particle was used to transduce A549 cells, and the transduced cells were selected with puromycin for one week.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    For syncytium formation of the stable cell line, A549-SPΔCWT or A549-SPΔCDelta cells were detached with 0.05% trypsin and mixed with A549 or A549ACE2 cells.
    A549-SPΔCDelta
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid constructs: The SARS-CoV-2 SP protein-expressing plasmids (pCAGGS-nCoVSPΔC and pCAGGS-nCoVSPΔCG614) were described previously (Ao et al., 2021a).
    pCAGGS-nCoVSPΔC
    suggested: None
    pCAGGS-nCoVSPΔCG614
    suggested: None
    The gene encoding SPΔCDelta or SPΔCDelta-PD was synthesized (Genescript) and cloned into the pCAGGS plasmid, and each mutation was confirmed by sequencing.
    pCAGGS
    suggested: RRID:Addgene_127347)
    pEF1-SPΔCwt, pEF1-SPΔCG614 or pEF1-SPΔCDelta was constructed by inserting the cDNA encoding SPΔCwt, SPΔCG614 or SPΔCDelta through the BamHI and NheI sites into the pEF1-pcs-puro vector (Ao et al., 2008).
    pEF1-SPΔCG614
    suggested: None
    pEF1-pcs-puro
    suggested: None
    A549-expressing human ACE2 (A549ACE2) cells were generated by transducing A549 cells with the ACE2-expressing lentiviral vector (pLenti-C-mGFP-ACE2) (Origene, Cat# PS100093) and then selected with puromycin according to the manufacturer’s procedure.
    pLenti-C-mGFP-ACE2
    suggested: None
    Virus production and infection experiments: SARS-CoV-2 SPΔC pseudotyped viruses (CoV-2-SPΔC-PVs, CoV-2-SPΔCG614-PVs and CoV-2-SPΔCDelta-PVs) or pseudotyped virus-like particles (VLPs) were produced by transfecting HEK293T cells with pCAGGS-SPΔCWT, pCAGGS-SPΔCG614 or pCAGGS-SPΔCDelta and pCMVΔ8.2 with or without a Gluc-expressing HIV vector ΔRI/E/Gluc (Ao et al., 2021a).
    pCAGGS-SPΔCWT
    suggested: None
    pCAGGS-SPΔCG614
    suggested: None
    pCAGGS-SPΔCDelta
    suggested: None
    pCMVΔ8.2
    suggested: None
    Generation of different SPΔC-expressing A549 stable cell lines: Production of lentiviral vectors expressing different SPΔC: 293T cells were cotransfected with pEF1-SPΔCwt, pEF1-SPΔCG614 or pEF1-SPΔCDelta with packaging plasmid Δ8.2 and VSV-G expressing plasmid.
    pEF1-SPΔCwt
    suggested: None
    pEF1-SPΔCDelta
    suggested: None
    VSV-G
    suggested: RRID:Addgene_138479)
    Immunofluorescence assay: 293T cells transfected with various SARS-CoV-2 SPΔC-expressing plasmids were grown on glass coverslips (12 mm2) in a 24-well plate.
    SPΔC-expressing
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistics: Statistical analysis of cytokine levels, including the results of GLuc assay, Luciferase assay, and various cytokine/chemokines assay, were performed using the unpaired t-test (considered significant at P≥0.05) by GraphPad Prism 9 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 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.

    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.

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.11.23.469765v1 on bioRxiv