Spike protein cleavage-activation mediated by the SARS-CoV-2 P681R mutation: a case-study from its first appearance in variant of interest (VOI) A.23.1 identified in Uganda

  1. Bailey Lubinski
  2. Laura Frazier
  3. My Phan
  4. Daniel Bugumbe
  5. Jessie L Cunningham
  6. Tiffany Tang
  7. Susan Daniel
  8. Matthew Cotten
  9. Javier A. Jaimes
  10. Gary Whittaker

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The African continent like all other parts of the world with high infection/low vaccination rates can, and will, be a source of novel SARS-CoV-2 variants. The A.23 viral lineage, characterized by three spike mutations F157L, V367F and Q613H, was first identified in COVID-19 cases from a Ugandan prison in July 2020, and then was identified in the general population with additional spike mutations (R102I, L141F, E484K and P681R) to comprise lineage A.23.1 by September 2020, with this virus being designated a variant of interest (VOI) in Africa and with subsequent spread to 26 other countries. The P681R spike substitution of the A.23.1 VOI is of note as it increases the number of basic residues in the sub-optimal SARS-CoV-2 spike protein furin cleavage site; as such, this substitution may affect viral replication, transmissibility or pathogenic properties. The same P681R substitution has also appeared in B.1.617 variants, including B.1.617.2 (Delta). Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from A.23.1 and B.1.617.2 and observed significantly increased cleavability with furin, compared to sequences derived from the original Wuhan-Hu1 S1/S2. We performed functional infectivity assays using pseudotyped MLV particles harboring SARS-CoV-2 spike proteins and observed an increase in transduction for A.23.1-pseudotyped particles compared to Wuhan-Hu-1 in Vero-TMPRSS2 and Calu-3 cells (with a presumed early entry pathway), although lowered infection in Vero E6 cells (with a presumed late entry pathway). However, these changes in infectivity were not reproduced in the original Wuhan-Hu-1 spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, which may affect viral infection and transmissibility, this substitution alone is not sufficient and needs to occur on the background of other spike protein changes to enable its full functional consequences.

Article activity feed

  1. Version published to 10.1101/2021.06.30.450632v6 on bioRxiv
  2. Version published to 10.1101/2021.06.30.450632v5 on bioRxiv
  3. Version published to 10.1101/2021.06.30.450632v4 on bioRxiv
  4. Version published to 10.1101/2021.06.30.450632v3 on bioRxiv
  5. Version published to 10.1101/2021.06.30.450632v2 on bioRxiv
  6. ScreenIT

    SciScore for 10.1101/2021.06.30.450632: (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
    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
    A mouse monoclonal antibody against MLV p30 (ab130757, Abcam) and an AlexaFluor 488 goat anti-mouse antibody (Invitrogen) were used as a loading control.
    A mouse monoclonal antibody against MLV p30 (ab130757, Abcam) and an AlexaFluor 488 goat anti-mouse antibody (Invitrogen)
    suggested: None
    antibody against MLV p30 (ab130757, Abcam)
    suggested: None
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells were seeded at 2.5×10^5 cells/ml in a 6-well plate the day before transfection.
    HEK293T
    suggested: DSMZ Cat# ACC-873, RRID:CVCL_A5EF)
    Vero E6 and Vero-TMPRSS2 cells were seeded at 4.5×10^5 cells/ml in a 24-well plate the day before infection.
    Vero E6
    suggested: None
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Calu-3 cells were seeded at 10×10^5 cells/ml in a 24-well plate three days before infection.
    Calu-3
    suggested: None
    Recombinant DNA
    SentencesResources
    Synthesis and cloning of the A.23.1 spike protein: The sequence for the A.23.1 spike gene from isolate SARS-CoV-2 A.23.1 hCoV-19/Uganda/UG185/2020 (EPI_ISL_955136) was obtained from GISAID, and codon-optimized, synthesized and cloned into a pcDNA 3.1+ vector for expression (GenScript).
    pcDNA 3.1+
    suggested: None
    Mutagenesis was carried out on a pcDNA-SARS-CoV-22 Wuhan-Hu 1 S plasmid using the Agilent QuickChange Lightning Mutagenesis kit (The original plasmid was generously provided by David Veesler, University of Washington USA).
    pcDNA-SARS-CoV-22
    suggested: None
    The mutated pcDNA-SARS-CoV-2 Wuhan-Hu1 P681R S plasmid was used to transform XL-10 gold ultracompetent cells, which were grown up in small culture, and then plasmid was extracted using the Qiagen QIAprep Spin Miniprep Kit.
    pcDNA-SARS-CoV-2 Wuhan-Hu1 P681R S
    suggested: None
    Cells were transfected with 800 ng of pCMV-MLV-gagpol, 600 ng of pTG-luc, and 600 ng of a plasmid containing the viral envelope protein of choice.
    pCMV-MLV-gagpol
    suggested: None
    pTG-luc
    suggested: None
    Viral envelope plasmids included pcDNA-SARS-CoV-2 Wuhan-Hu1 S as the WT, pcDNA-SARS-CoV-2 Wuhan-Hu1 P681R S, and pcDNA-SARS-CoV-2 A.23.1 S. pCAGGS-VSV G was used as a positive control and pCAGGS was used for a Δ-envelope negative control.
    pcDNA-SARS-CoV-2 Wuhan-Hu1
    suggested: None
    pcDNA-SARS-CoV-2
    suggested: None
    pcDNA-SARS-CoV-2 A.23.1
    suggested: None
    pCAGGS-VSV
    suggested: None
    pCAGGS
    suggested: RRID:Addgene_18926)

    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.

    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.

  7. Version published to 10.1101/2021.06.30.450632v1 on bioRxiv