Functional evaluation of the P681H mutation on the proteolytic activation the SARS-CoV-2 variant B.1.1.7 (Alpha) spike

  1. Bailey Lubinski
  2. Maureen H. V. Fernandes
  3. Laura Frazier
  4. Tiffany Tang
  5. Susan Daniel
  6. Diego G. Diel
  7. Javier A. Jaimes
  8. Gary R. Whittaker

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread.

Highlights

  • SARS-CoV-2 B.1.1.7 VOC has a P681H mutation in the spike that is predicted to enhance viral infection

  • P681H does not significantly impact furin cleavage, viral entry or cell-cell spread

  • Other mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates

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  1. Version published to 10.1101/2021.04.06.438731v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.04.06.438731: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (Cat: 40590-T62, Sinobiological) and an AlexaFluor 488 goat anti-rabbit antibody.
    anti-rabbit
    suggested: None
    Secondary antibody labeling was performed using AlexaFluorTM 488 goat anti-rabitt IgG antibody (Cat: A32731, Invitrogen Co.) at a 1/500 dilution for 45 minutes.
    anti-rabitt IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Pseudoparticle Generation: HEK293T cells were seeded at 3×10e5 cells/ml in a 6 well plate the day before transfection.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Pseudoparticle Infection Assay: Vero E6 and Vero TMPRSS2 cells were seeded at 3e5 cells/ml in a 24 well plate the day before infection.
    Vero E6
    suggested: None
    Vero TMPRSS2
    suggested: None
    Cell-Cell Fusion Assay: VeroE6 and Vero-TMPRSS2 cells were transfected with a plasmid harboring the spike gene of the SARS-CoV-2 isolate Wuhan-Hu 1, SARS-CoV-2 B.1.1.7 variant, the SARS-CoV-2 isolate Wuhan-Hu 1 with a P681H mutation, or a delta-spike pCDNA3.1+ plasmid, and evaluated through an immunofluorescence assay (IFA)
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)

    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.
    • 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.

    About SciScore

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  3. Version published to 10.1101/2021.04.06.438731v1 on bioRxiv