Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike

  1. Jun Zhang
  2. Weichun Tang
  3. Hailong Gao
  4. Christy L. Lavine
  5. Wei Shi
  6. Hanqin Peng
  7. Haisun Zhu
  8. Krishna Anand
  9. Matina Kosikova
  10. Hyung Joon Kwon
  11. Pei Tong
  12. Avneesh Gautam
  13. Sophia Rits-Volloch
  14. Shaowei Wang
  15. Megan L. Mayer
  16. Duane R. Wesemann
  17. Michael S. Seaman
  18. Jianming Lu
  19. Tianshu Xiao
  20. Hang Xie
  21. Bing Chen

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Abstract

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

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    SciScore for 10.1101/2022.04.28.489772: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: 2 variant at 100 TCID50/mouse under isoflurane anesthesia.
    IACUC: All procedures were performed according to the animal study protocols approved by the FDA White Oak Animal Program Animal Care and Use Committee.
    Sex as a biological variablenot detected.
    RandomizationIn the ABSL-3 lab, K18-hACE2 mice were randomly grouped and were inoculated intranasally with NY (G614), Delta, Omicron BA.1 or Omicron BA.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (58).
    anti-SARS-COV-2 S
    suggested: None
    Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.
    anti-Rabbit IgG
    suggested: None
    Control sensors with no ACE2 or antibody were also dipped in the S protein solutions and the running buffer as references.
    ACE2
    suggested: None
    For antibody staining, an Alexa Fluor 647 conjugated donkey anti-human IgG Fc F(ab’)2 fragment (Jackson ImmunoResearch, West Grove, PA) was used as secondary antibody at 5 μg/ml concentration.
    anti-human IgG
    suggested: None
    After washing, plates were probed with 1 μg/ml of inhouse developed rabbit polyclonal antibody specific for SARS-CoV-2 membrane/nucleocapsid (33) at 4°C overnight followed by peroxidase-conjugated goat anti-rabbit secondary antibody (SeraCare #5220-0336, 1:2000) for 2h at room temperature.
    anti-rabbit
    suggested: (SeraCare KPL Cat# 5220-0336, RRID:AB_2857917)
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, Expi293F cells transfected with monomeric ACE2 or dimeric ACE2 expression construct and the supernatant of the cell culture was collected.
    Expi293F
    suggested: RRID:CVCL_D615)
    Murine Leukemia Virus (MLV) particles (plasmids of the MLV components kindly provided by Dr. Gary Whittaker at Cornell University and Drs. Catherine Chen and Wei Zheng at National Center for Advancing Translational Sciences, National Institutes of Health), pseudotyped with various SARS-CoV-2 S protein constructs, were generated in HEK 293T cells, following a protocol described previously for SARS-CoV (59, 60).
    HEK 293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    To prepare for infection, 7.5×103 of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μl culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment.
    HEK 293
    suggested: None
    Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.
    293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Drs.
    293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Seed viruses were amplified in Vero E6 (ATCC CRL-1586) or Vero E6 with TMPRSS2 overexpression (BPS Bioscience #78081)
    Vero E6
    suggested: None
    In vitro virus replication and focus-forming assay: Vero-E6 cells were pre-seeded in 12-well tissue culture plates overnight and were infected with authentic viruses (G614, Delta, Omicron BA.1 or BA.2) at MOI of 0.01 in Gibco™ high glucose DMEM containing 3% FBS.
    Vero-E6
    suggested: None
    Briefly, 10-fold serially diluted postinfection were added at 100 μl/well to Vero E6-TMPRSS2 cells pre-seeded in 96-well tissue culture plates.
    Vero E6-TMPRSS2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse study: Hemizygous B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    In the ABSL-3 lab, K18-hACE2 mice were randomly grouped and were inoculated intranasally with NY (G614), Delta, Omicron BA.1 or Omicron BA.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    The S gene was fused with a C-terminal twin Strep tag (SGGGSAWSHPQFEKGGGSGGGSGGSSAWSHPQFEK) and cloned into a mammalian cell expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg,
    pCMV-IRES-puro
    suggested: None
    Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.
    pCMV DR8.2 , and luciferase reporter
    suggested: None
    pHR’
    suggested: None
    Serially diluted pCMV6-AC-ACE2-GFP plasmid or pCC1-CoV2-F7 plasmid expressing SARS-CoV-2 N (62) was used to construct a standard curve.
    pCMV6-AC-ACE2-GFP
    suggested: None
    pCC1-CoV2-F7
    suggested: None
    Software and Algorithms
    SentencesResources
    The KD was obtained by fitting Req value and its corresponding concentration to the model: “one site-specific” using GraphPad Prism 8.0.2 according to H.J. Motulsky, Prism 5 Statistics Guide, 2007, GraphPad Software Inc.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Automated data collection was carried out using SerialEM version 3.8.6 (63) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.83 Å) at an exposure rate of 13.761 electrons per pixel per second.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Local resolution was also determined using cryoSPARC.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Several rounds of manual building were performed in Coot.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Iteratively, refinement was performed in both Phenix (real space refinement) and ISOLDE (66), and the Phenix refinement strategy included minimization_global, local_grid_search, and adp, with rotamer, Ramachandran, and reference-model restraints, using 7KRQ and 7KRR as the reference models.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)

    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

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  2. Version published to 10.1101/2022.04.28.489772 on bioRxiv