Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2

  1. Samuel J. Dicken
  2. Matthew J. Murray
  3. Lucy G. Thorne
  4. Ann-Kathrin Reuschl
  5. Calum Forrest
  6. Maaroothen Ganeshalingham
  7. Luke Muir
  8. Mphatso D. Kalemera
  9. Machaela Palor
  10. Laura E. McCoy
  11. Clare Jolly
  12. Greg J. Towers
  13. Matthew B. Reeves
  14. Joe Grove

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Abstract

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARS-CoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.22.436468: (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
    Antibodies: The following antibodies were used in this study: mouse anti-spike S2 (1A9, Gene Tex), mouse anti-p55/24 (ARP366, Centre for AIDS Reagents), goat anti-ACE2 (AF933, R& D Systems), rabbit anti-ACE2 (EPR4435, abcam), rabbit anti-TMPRSS2 (EPR3861, abcam), mouse anti--actin (ab49900, abcam)
    anti-spike S2
    suggested: (Imported from the IEDB Cat# S2, RRID:AB_2833224)
    anti-p55/24
    suggested: None
    anti-ACE2
    suggested: None
    anti-TMPRSS2
    suggested: (LSBio (LifeSpan Cat# LS-C105697, RRID:AB_2240746)
    anti--actin
    suggested: (Cell Signaling Technology Cat# 8844, RRID:AB_10998933)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HeLa ACE2 (a kind gift from Dr. James Voss, SCRIPPS (
    HeLa ACE2
    suggested: None
    51)), HEK 293T cells and Calu-3 cells were maintained at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% foetal calf serum (FCS), 1% non-essential amino acids and 1% penicillin/streptomycin. Plasmids: Codon optimised open reading frames encoding spike proteins were synthesised (GeneArt, Thermo Fisher) and cloned into pCDNA3.1 and/or pD603 (ATUM) expression plasmids.
    HEK 293T
    suggested: None
    Calu-3
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were acquired using an LSR Fortessa II (BD Biosciences) and analysed using FlowJo version 10.5.3 (FlowJo LLC, Becton Dickinson).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    MUSCLE) method (67).
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    Statistics: All statistical analysis (T-test, one-way ANOVA with Dunnet’s correction for multiple comparisons, curve fitting and F-test) were performed in GraphPad Prism.
    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.

    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.

  2. Version published to 10.1101/2021.03.22.436468v1 on bioRxiv