The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

  1. Izumi Kimura
  2. Daichi Yamasoba
  3. Hesham Nasser
  4. Jiri Zahradnik
  5. Yusuke Kosugi
  6. Jiaqi Wu
  7. Kayoko Nagata
  8. Keiya Uriu
  9. Yuri L. Tanaka
  10. Jumpei Ito
  11. Ryo Shimizu
  12. Toong Seng Tan
  13. Erika P. Butlertanaka
  14. Hiroyuki Asakura
  15. Kenji Sadamasu
  16. Kazuhisa Yoshimura
  17. Takamasa Ueno
  18. Akifumi Takaori-Kondo
  19. Gideon Schreiber
  20. Mako Toyoda
  21. Kotaro Shirakawa
  22. Takashi Irie
  23. Akatsuki Saito
  24. So Nakagawa
  25. Terumasa Ikeda
  26. Kei Sato

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Abstract

No abstract available

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  1. Version published to 10.1016/j.isci.2022.105720
  2. ScreenIT

    SciScore for 10.1101/2022.04.03.486864: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The protocols were approved by the Institutional Animal Care and Use Committee of National University Corporation Hokkaido University (approval ID: 20-0060).
    IRB: All protocols involving specimens from human subjects recruited at Kyoto University and Kuramochi Clinic Interpark were reviewed and approved by the Institutional Review Boards of Kyoto University (approval ID: G1309) and Kuramochi Clinic Interpark (approval ID: G2021-004).
    Consent: All human subjects provided written informed consent.
    Euthanasia Agents: Sera of infected hamsters were collected at 16 days postinfection (d.p.i.) using cardiac puncture under anesthesia with isoflurane and stored at −80°C until use.
    Sex as a biological variable, range: 29-56, 18% male) and sixteen vaccinees four weeks after their second mRNA-1273 (Moderna) vaccine (average age: 27, range: 20-47, 38% male)
    RandomizationWe then selected 12 genomes and randomly selected 100 genomes that met criteria 1 and 2.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For protein detection, the following antibodies were used: mouse anti-SARS-CoV-2 S monoclonal antibody (clone 1A9, GeneTex, Cat# GTX632604, 1:10,000)
    anti-SARS-CoV-2
    suggested: None
    mouse anti-HIV-1 p24 monoclonal antibody (183-H12-5C, obtained from the HIV Reagent Program, NIH, Cat# ARP-3537, 1:2,000), rabbit anti-beta actin (ACTB) monoclonal antibody (clone 13E5, Cell Signalling, Cat# 4970, 1:5,000)
    anti-HIV-1
    suggested: (NIH HIV Reagent Program Cat# 3537, RRID:AB_2832923)
    anti-beta actin
    suggested: None
    ACTB
    suggested: (Cell Signaling Technology Cat# 4970, RRID:AB_2223172)
    , mouse anti-tubulin (TUBA) monoclonal antibody (clone DM1A, Sigma-Aldrich, Cat# T9026, 1:10,000)
    anti-tubulin ( TUBA
    suggested: (Antibodies-Online Cat# ABIN387720, RRID:AB_10763362)
    , horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG antibody (Cell Signaling, Cat# 7076S, 1:2,000)
    anti-mouse IgG
    suggested: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
    HRP-conjugated donkey anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 711-035-152, 1:10,000) and HRP-conjugated donkey anti-mouse IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 715-035-150, 1:10,000).
    HRP-conjugated donkey anti-mouse IgG
    suggested: None
    To measure the surface expression level of S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-112048, 1:100)
    anti-SARS-CoV-2 S
    suggested: (Bio X Cell Cat# BE0357, RRID:AB_2894776)
    Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) was used as negative controls, and APC-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 111-136-144, 1:50) was used as a secondary antibody.
    anti-rabbit IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 111-136-144, RRID:AB_2337987)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T cells (a human embryonic kidney cell line; ATCC, CRL-3216)
    HEK293T
    suggested: None
    , HEK293 cells (a human embryonic kidney cell line; ATCC CRL-1573), and HOS-ACE2/TMPRSS2 cells, HOS cells stably expressing human ACE2 and TMPRSS2 (Ferreira et al., 2021; Ozono et al., 2021) were maintained in DMEM (high glucose) (Sigma-Aldrich, Cat# 6429-500ML) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS).
    HEK293
    suggested: None
    HOS-ACE2/TMPRSS2
    suggested: None
    HEK293-C34 cells, IFNAR1 KO HEK293 cells expressing human ACE2 and TMPRSS2 by doxycycline treatment (Torii et al., 2021), were maintained in Dulbecco’s modified Eagle’s medium (high glucose) (Sigma-Aldrich, Cat# R8758-500ML) containing 10% FBS, 10 μg/ml blasticidin (InvivoGen, Cat# ant-bl-1) and 1% PS.
    HEK293-C34
    suggested: None
    VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2; JCRB1819) (Matsuyama et al., 2020) were maintained in DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were transfected with pDSP1-7 (400ng).
    HEK293-ACE2
    suggested: None
    HEK293-ACE2/TMPRSS2
    suggested: None
    Briefly, 1 d before infection, VeroE6/TMPRSS2 cells (10,000 cells) were seeded into a 96-well plate.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Recombinant DNA
    SentencesResources
    The resulting PCR fragment was digested with KpnI and NotI and inserted into the corresponding site of the pCAGGS vector (Niwa et al., 1991).
    pCAGGS
    suggested: RRID:Addgene_127347)
    To prepare effector cells, HEK293 cells were cotransfected with the S expression plasmids (400 ng) and pDSP8-11 (400 ng) using TransIT-LT1 (Takara, Cat# MIR2300).
    pDSP8-11
    suggested: None
    To prepare target cells, HEK293 cells were cotransfected with pC-ACE2 (200 ng) and pDSP1-7 (400 ng)
    pC-ACE2
    suggested: None
    Target HEK293 cells in selected wells were cotransfected with pC-TMPRSS2 (40 ng) in addition to the plasmids above.
    pC-TMPRSS2
    suggested: None
    HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were transfected with pDSP1-7 (400ng).
    pDSP1-7
    suggested: None
    An enhanced yeast display platform for SARS-CoV-2 S RBD [wild-type (B.1.1), residues 336-528] yeast surface expression was established using Saccharomyces cerevisiae EBY100 strain and pJYDC1 plasmid (Addgene, Cat# 162458) as previously described (Dejnirattisai et al., 2022; Kimura et al., 2022; Motozono et al., 2021; Yamasoba et al., 2022; Zahradnik et al., 2021).
    pJYDC1
    suggested: RRID:Addgene_162458)
    Software and Algorithms
    SentencesResources
    Sequencing reads were trimmed using fastp v0.21.0 (Chen et al., 2018) and subsequently mapped to the viral genome sequences of a lineage A isolate (strain WK-521; GISAID ID: EPI_ISL_408667) (Matsuyama et al., 2020) using BWA-MEM v0.7.17 (Li and Durbin, 2009).
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    Variant calling, filtering, and annotation were performed using SAMtools v1.9 (Li et al., 2009) and snpEff v5.0e (Cingolani et al., 2012).
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    snpEff
    suggested: (SnpEff, RRID:SCR_005191)
    The 48 Omicron genomes with two outgroup genomes EPI_ISL_402125 (strain Wuhan-Hu-1, B lineage) and EPI_ISL_406862 (B.1 lineage; one of the earliest sequences carrying the S D614G mutation) were aligned using FFT-NS-1 in MAFFT suite v7.407 (Katoh and Standley, 2013).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    BEAST v1.10.4 (Suchard et al., 2018) was used to construct a timetree under an exponential growth coalescent model using a strict molecular clock.
    BEAST
    suggested: (BEAST, RRID:SCR_010228)
    FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to show the tree.
    FigTree
    suggested: (FigTree, RRID:SCR_008515)
    To further determine the population history of the Omicron genomes, we generated a Bayesian skyline plot using the same model (2 × 108 chain length for MCMC) and summarized the results using Tracer v1.7.1 (Rambaut et al., 2018)
    Tracer
    suggested: (Tracer, RRID:SCR_019121)
    mouse anti-HIV-1 p24 monoclonal antibody (183-H12-5C, obtained from the HIV Reagent Program, NIH, Cat# ARP-3537, 1:2,000), rabbit anti-beta actin (ACTB) monoclonal antibody (clone 13E5, Cell Signalling, Cat# 4970, 1:5,000)
    HIV Reagent Program
    suggested: None
    (LI-COR Biosciences) or Fiji software v2.2.0 (ImageJ).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Surface expression level of S protein was measured using FACS Canto II (BD Biosciences) and the data were analyzed using FlowJo software v10.7.1 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Nucleotide sequences were determined by a DNA sequencing service (Fasmac), and the sequence data were analyzed by Sequencher v5.1 software (Gene Codes Corporation).
    Sequencher
    suggested: (Sequencher, RRID:SCR_001528)
    Information on the unexpected mutations detected is summarized in Table S4, and the raw data are deposited in Gene Expression Omnibus (accession number: GSE196649 and xxxx).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    The stained cells were washed with tap water and dried, and the size of plaques was measured using Adobe Photoshop 2021 v22.4.1 (Adobe)
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    The assay of each serum was performed in triplicate, and the 50% neutralization titer (NT50) was calculated using Prism 9 (GraphPad Software)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Protein structure: All protein structural analyses were performed using the PyMOL molecular graphics system v2.5.0 (Schrödinger).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    The background binding subtracted fluorescent signal was fitted to a standard noncooperative Hill equation by nonlinear least-squares regression using Python v3.7 (https://www.python.org) as previously described (Zahradnik et al., 2021).
    Python
    suggested: (IPython, RRID:SCR_001658)
    https://www.python.org
    suggested: (CVXOPT - Python Software for Convex Optimization, RRID:SCR_002918)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Limitations of the study: Here, we showed the importance of the S375F mutation to the virological properties of Omicron; however, the following issues remain unclear. First, although we showed that the S375F mutation determines the virological features of Omicron S, it remains unclear which mutations in Omicron S determine the pronounced immune resistance of Omicron S. Second, in addition to the Omicron BA.1 variant that we focused on this study, another recently emerged Omicron lineage, BA.2, also bears the S375F mutation. However, we have recently shown that the fusogenicity of BA.2 S is significantly higher than that of BA.1 S (Yamasoba et al., 2022). Together with the results shown in this study, these observations suggest that BA.2 S acquired certain compensatory mutation(s) that increased fusion efficacy. Further investigations will be needed to unveil the full evolutionary history of the Omicron lineage. Furthermore, the question of why the acquisition of the S375F mutation caused explosive spread despite reducing infectivity in tissue culture, S cleavage efficacy and fusogenicity also needs to be elucidated in detail by further studies.

    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

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  3. Version published to 10.1101/2022.04.03.486864 on bioRxiv