Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage

  1. Nikita Shiliaev
  2. Tetyana Lukash
  3. Oksana Palchevska
  4. David K. Crossman
  5. Todd J. Green
  6. Michael R. Crowley
  7. Elena I. Frolova
  8. Ilya Frolov

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Abstract

The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes.

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  1. Version published to 10.1128/jvi.01357-21
  2. ScreenIT

    SciScore for 10.1101/2021.06.28.450274: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Biosafety: Virus rescue and analyses, which required infectious virus, were performed in BSL3 SEBLAB facility of the University of Alabama at Birmingham according to the protocols approved by the Institutional Biosafety Committee.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell cultures: The BHK-21 cells were kindly provided by Paul Olivo (Washington University, St. Louis, MO).
    BHK-21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    BHK-21, Vero E6, NIH 3T3 cells and their derivatives were maintained in alpha minimum essential medium supplemented with 10% fetal bovine serum (FBS) and vitamins.
    Vero E6, NIH 3T3
    suggested: None
    NIH
    suggested: None
    Huh7.5 and Calu-3 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% (FBS).
    Calu-3
    suggested: None
    This virus isolate was originally provided by Dr. Natalie Thornburg from the Centers for Disease Control and Prevention in Atlanta, GA (17), and amplified on Vero E6 cells at the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch at Galveston (UTMB).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Stable cell lines: Stable ACE2-expressing cell lines were generated by co-transfecting BHK-21, NIH 3T3, Vero E6 and Huh7.5 cells with PiggyBAC/ACE2 and integrase-encoding plasmids (System Biosciences, Inc.) using Lipofectamine 3000, according to the protocol recommended by the manufacturer (Invitrogen).
    Huh7.5
    suggested: RRID:CVCL_7927)
    The in vitro-synthesized RNAs (∼1 μg) were electroporated into BHK-21 or BHK/ACE2 cells containing VEErep/N/Pac under previously described conditions (22, 23).
    BHK/ACE2
    suggested: None
    Depending on the construct, viruses were harvested at 48 or 72 h post electroporation, and titers were determined by a plaque assay on Vero/ACE2 cells.
    Vero/ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid constructs: The cDNA of SARS-CoV-2 (NC_045512.2) genome was assembled in two fragments (5’ and 3’ fragments), which were cloned into low copy number plasmids pACNR1180 (19) and Proteus1 (20), and propagated in E.coli WM1100.
    pACNR1180
    suggested: None
    Software and Algorithms
    SentencesResources
    To identify mutational hot spots, the raw sequence fastq files were aligned to the reference genome using BWA version 0.7.17-r1188.
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Aligned reads were then sorted with Picard version 2.9.2 SortSam.
    Picard
    suggested: (Picard, RRID:SCR_006525)
    Naïve Variant Caller (Galaxy version 0.0.4) called the variants and VariantAnnotator (Galaxy version 1.3.2) counted the bases at each position.
    Galaxy
    suggested: (Galaxy, RRID:SCR_006281)
    Additional models of S trimers individually harboring I68R, N74K or S247R mutations were generated using COOT (27).
    COOT
    suggested: (Coot, RRID:SCR_014222)

    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:
    • 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.

    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 10.1101/2021.06.28.450274v1 on bioRxiv