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

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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. Here, 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 an animal model with previously prevalent variants. BA.2 S can fuse membranes slightly more efficiently than Omicron BA.1, but still less efficiently than other previous 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 of the Omicron subvariants.

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Fig. 1: Functional properties of Omicron BA.2 S.
Fig. 2: Purification of the Omicron BA.2 full-length S protein.
Fig. 3: Antigenic properties of the purified full-length BA.2 S protein.
Fig. 4: Cryo-EM structures of the full-length BA.2 S protein.
Fig. 5: Structural impact of the mutations in the BA.2 S.
Fig. 6: Antigenic surfaces of the RBD.

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Data availability

The atomic structure coordinates and EM maps are deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the following accession numbers: PDB ID 8D55 and EMDB ID EMD-27205 for the RBD-down conformation, PDB ID 8D56 and EMDB ID EMD-27206 for the one-RBD-up conformation, and PDB ID 8D5A and EMDB ID EMD-27207 for the RBD-intermediate conformation. All other related data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. The initial templates for model building include PDB IDs 7KRQ and 7KRR. Source data are provided with this paper.

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Acknowledgements

We thank the SBGrid team for computing support, S. Harrison and J. Abraham for computing resources, K. Arnett for support and advice on the BLI experiments, and S. Harrison for critical reading of the manuscript. EM data were collected at the Harvard Cryo-EM Center for Structural Biology at Harvard Medical School. We acknowledge support for COVID-19-related structural biology research at Harvard from the Nancy Lurie Marks Family Foundation and the Massachusetts Consortium on Pathogen Readiness (MassCPR). This work was supported by Fast Grants from Emergent Ventures (to B.C. and D.R.W.), COVID-19 Awards from MassCPR (to B.C., D.R.W. and M.S.S.), and NIH grants AI147884 (to B.C.), AI141002 (to B.C.), AI127193 (to B.C. and J. Chou), AI39538 (to D.R.W.), AI170715 (to D.R.W.), AI165072 (to D.R.W.) and AI169619 (to D.R.W). This work was also supported in part by the FDA Center for Biologics Evaluation and Research (CBER) intramural SARS-CoV-2 pandemic fund (to H.X.). The clinical isolate New York-PV09158/2020 (ATCC, NR-53516) and Omicron (B.1.1.529) BA.1 were obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), NIH: SARS-Related Coronavirus 2. The Delta (B.1.617.2) and Omicron BA.2 seed viruses were kindly provided by B. Zhou and C. Davis at CDC.

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Author notes

  1. These authors contributed equally: Jun Zhang, Weichun Tang, Hailong Gao.

Authors and Affiliations

  1. Division of Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Jun Zhang, Hailong Gao, Wei Shi, Hanqin Peng, Sophia Rits-Volloch, Tianshu Xiao & Bing Chen

  2. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Jun Zhang, Hailong Gao, Wei Shi, Tianshu Xiao & Bing Chen

  3. Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD, USA

    Weichun Tang, Matina Kosikova, Hyung Joon Kwon & Hang Xie

  4. Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Christy L. Lavine & Michael S. Seaman

  5. Institute for Protein Innovation, Harvard Institutes of Medicine, Boston, MA, USA

    Haisun Zhu & Krishna Anand

  6. Division of Allergy and Clinical Immunology, Department of Medicine, Brigham and Women’s Hospital; Ragon Institute of MGH, MIT and Harvard, Boston, MA, USA

    Pei Tong, Avneesh Gautam & Duane R. Wesemann

  7. Codex BioSolutions, Inc., Rockville, MD, USA

    Shaowei Wang & Jianming Lu

  8. The Harvard Cryo-EM Center for Structural Biology, Boston, MA, USA

    Megan L. Mayer

  9. Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, DC, USA

    Jianming Lu

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  1. Jun Zhang
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Contributions

B.C., T.X., J.Z. and H.G. conceived the project. H.G. expressed and purified the full-length S proteins with help from W.S. and H.P. T.X. designed and performed BLI and cell–cell fusion experiments, and was assisted by H.G. J.Z. prepared cryo grids and performed EM data collection with contributions from M.L.M., processed the cryo-EM data, and built and refined the atomic models. W.T., M.K., H.J.K. and H.X. carried out the animal study and in vitro virus replication kinetics using authentic viruses. C.L.L. and M.S.S. performed the neutralization assays using the HIV-based pseudoviruses. J.L. and S.W. created the BA.2 expression construct and performed the neutralization assays using the MLV-based pseudoviruses. H.Z. and K.A. performed the flow cytometry experiments. P.T., A.G. and D.R.W. produced anti-S monoclonal antibodies. S.R.-V. contributed to cell culture and protein production. All authors analyzed the data. B.C., T.X., J.Z., H.G. and H.X. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Tianshu Xiao, Hang Xie or Bing Chen.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Expression and cell-cell fusion of the S protein from Omicron BA.2.

(a) Schematic representation of a full-length Omicron BA.2 spike (S) protein. The sequence is derived from an Omicron BA.2 subvariant (hCoV-19/Denmark/DCGC-327158/2022). Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; 630 loop, residues 620-640; S1/S2, the furin cleavage site at the S1/S2 boundary; S2’, S2’ cleavage site; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane segment; CT, cytoplasmic tail; and tree-like symbols for glycans. Positions of all mutations (from the amino-acid sequence of Wuhan-Hu-1) are indicated and those highlighted in red rectangles are also present in at least one of the previous VOCs. (b) Expression and processing of the full-length S constructs of various variants in HEK293 cells. S protein samples prepared from HEK293 cells transiently transfected with 10 μg of the full-length S expression plasmids were detected by anti-RBD polyclonal antibodies. Bands for the uncleaved S and S1 fragment are indicated. (c) HEK293T cells transfected with the untagged, full-length S protein expression plasmids were fused with ACE2-expressing cells. Cell-cell fusion led to reconstitution of α and ω fragments of β-galactosidase to form an active enzyme, and the fusion activity was then quantified by a chemiluminescent assay. No ACE2 and no S were negative controls. Error bars were generated with measurements on three biologically independent samples. All the experiments have been repeated at least twice with similar results.

Source data

Extended Data Fig. 2 Foci images of in vitro replication kinetics of the authentic viruses.

(a) and (b) Vero E6-TMPRSS2 cells were infected with the authentic G614 (B.1), Delta (B.1617.2), Omicron-BA.1 or BA.2 viruses at MOI of 0.005. Postinfection supernatants were titrated by a focus-forming assay. Foci as a cluster of cells expressing viral antigen were imaged and counted using AID vSpot Spectrum. (a) Extended virus replication kinetics in Vero E6-TMPRSS2 cells. Focus-forming units (FFU) per ml were determined and data are expressed as mean ± sem of n = 3 independent replicates/time point/virus. (b) images of foci (dark spots) titration shown in (a) with automatic counts in the lower corner of each well. (c) Vero E6 cells cells were infected with the authentic G614 (B.1), Delta (B.1617.2), Omicron-BA.1 or BA.2 viruses at MOI of 0.005. The data are summarized in Fig. 1d. (c) Vero E6-TMPRSS2 cells were infected with the authentic G614 (B.1), Delta (B.1617.2), Omicron-BA.1 or BA.2 viruses at MOI of 0.005. The data are summarized in Fig. 1e.

Source data

Extended Data Fig. 3 Infection of HEK293-ACE2 cells by MLV-based pseudotyped viruses.

Time course for single-cycle infection of HEK293-ACE2 cells by MLV-based pseudotyped viruses with various SARS-CoV-2 variant S constructs, as indicated, all containing a CT deletion. Infection was initiated by mixing viruses and target cells, and viruses were washed out at each time point as indicated. Delta AY.4.2 is a Delta subvariant. The relative light unit (RLU) of 8-hr infection of each pseudovirus was used as 100% infectivity for normalizing data at other time points. The relative infectivity (%) equals RLU(t)/RLU(8-hr)*100. The experiments were repeated at least three times with independent samples giving similar results. Error bars are standard deviations of 4 repeats of each data point (n = 3).

Source data

Extended Data Fig. 4 Tissue-specific viral burdens and hACE2 expression in K18-hACE2 mice at 1 and 3 days post infection (dpi).

Mice were intranasally inoculated with 100 TCID50/50 μl/mouse of G614 (B.1), Delta (B.1617.2), Omicron-BA.1 or BA.2. The viral RNA copies (a) or hACE2 expression (b) in various tissue homogenates were measured by RT-qPCR. Data are expressed as geometric means (bars) with geometric standard deviation (error bars). Individual results of infected mice (n = 4 mice/time point/group) and uninfected naïve mice (n = 5) are shown.

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Extended Data Fig. 5 Additional antigenic analysis of the purified full-length BA.2S protein.

(a) Antibody competition groups as described in ref. 36. Surface regions of the S trimer targeted by antibodies on S1 are highlighted by orange ellipses, including RBD-1, RBD-2, RBD-3, NTD-1 and NTD-2. The exact location of NTD-2 is uncertain and therefore marked with a dashed line. (b) Binding analysis of the prefusion S trimers from G614 and BA.2 with soluble monomeric ACE2 and selected monoclonal antibodies was performed by BLI. For ACE2 binding, purified ACE2 protein was immobilized to AR2G biosensors and dipped into the wells containing each purified S proteins at various concentrations. For antibody binding, various antibodies were immobilized to AHC biosensors and dipped into the wells containing each purified S protein at different concentrations. Binding kinetics were evaluated using a 1:1 Langmuir model except for antibody 12A2 targeting the RBD-2, which was analyzed by a bivalent binding model. The sensorgrams are in black and the fits in red. Binding constants highlighted by underlines were estimated by steady-state analysis as described in the Methods. RU, response unit. Binding constants are also summarized here and in Table 1. N.D., not determined. All experiments were repeated at least twice with essentially identical results. (c) Steady-state analysis by plotting steady-state responses against concentrations. KD values were derived from the fits. Error bars were generated by the steady-state analysis of Octet Data Analysis HT Version 12.0 (ForteBio), fitting each sensorgram to a single exponential function to extract the response at equilibrium (Req) as described in Methods.

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Extended Data Fig. 6 Antigenic properties of the cell-surface BA.2S protein assessed by flow cytometry.

Antibody binding to the full-length S proteins of the G614 and Omicron variants, as well as the uncleaved wildtype spike and an S2 construct expressed on the cell surfaces analyzed by flow cytometry. BA.2_notag, the unmodified, full-length S protein from the BA.2 subvariant. BA.2_streptag, the intact BA.2S protein fused with a C-terminal twin Strep tag. The antibodies and their targets are indicated. A designed ACE2-based fusion inhibitor ACE2615-foldon-T27W was used for detecting receptor binding37. No major difference in the trimeric ACE2 binding to non-tagged BA.2 and G614 spikes was observed in this assay, probably because the BA.2 trimer is less stable than G614 in the presence of ACE2 under the extensive washing conditions for flow cytometry. MFI, mean fluorescent intensity. Error bars represent standard errors of mean from measurements using three independently transfected cell samples. The flow cytometry assays were repeated three times with essentially identical results.

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Extended Data Fig. 7 Cryo-EM structures of the full-length BA.2S protein.

(a) Three structures of the BA.2S trimer, representing the closed prefusion conformation, one-RBD-intermediate conformation and one-RBD-up conformations, were modeled based on corresponding cryo-EM density maps at 2.7 Å, 3.5 Å and 2.9 Å resolution, respectively. Three protomers (a, b, c) are colored in red, blue and green, respectively. RBD locations are indicated. Particle percentage for each class in the data processing is also indicated, but it may not accurately reflect the conformation distribution of the S trimer in solution. (b) Superposition of the three conformations of the BA.2 S trimer aligned by the invariant S2 with only one protomer for each shown for clarity. Three RBDs representing the closed prefusion, one-RBD-intermediate and one-RBD-up conformations are colored in cyan, yellow and orange, respectively.

Extended Data Fig. 8 Additional ordered residues near the furin site in the BA.2 structure.

(a) Superposition of the structure of the BA.2S trimer in ribbon representation and various colors with the structures of the BA.1 S in gray and G614 S in yellow aligned by S2, showing the region near the furin cleavage site. (b) Density near the furin cleavage site in the BA.2 map. (c) Processing of the revertant BA.2 mutations near the furin cleavage site. S protein samples prepared from HEK293 cells transiently transfected with 10 μg of the full-length S expression plasmids of G614, Delta, Omicron BA.2, BA.2-K679N, BA.2-H681P and BA.2-K679N/H681P were detected by anti-RBD polyclonal antibodies. Bands for the uncleaved S and S1 fragment are indicated. The experiment was repeated three times independently with similar results.

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Extended Data Fig. 9 Local regions of mutations in BA.2S.

(a) A close-up view of the unique mutations in the BA.2 RBD. Superposition of the BA.2 RBD structure in ribbon representation and cyan with the structures of the RBDs of G614 S in yellow and BA.1 in gray. The mutated residues and the N-linked glycans at Asn343 are in stick model. NAG, N-acetylglucosamine. (b) Density in the BA.2 map near the short helix formed by residues 365-371 and the model fitting. (c) Possibly ordered 70-80 loop. The N-terminal segment of the BA.2S is shortened by the three-residue deletion (L24del-P25del-P26del) and also constrained by the disulfide bond between Cys15 and Cys136. There is reasonable density in which the 70-80 loop, disordered in many previous S trimer structures, could be modeled (shown in red). Such a structured loop can create a knot in this region, however, which will need a higher resolution map to confirm.

Extended Data Table 1 Neutralization of the SARS-CoV-2 Omicron subvariants
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Supplementary information

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Source data for plots in Fig. 1a–f.

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Source data for gel-filtration trace in Fig. 2b.

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Unprocessed gel in Fig. 2b.

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Source data for sensorgrams and fits in Fig. 3.

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Unprocessed western blot in Extended Data Fig. 1b.

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Source data for sensorgrams and fits in Extended Data Fig. 5b and plot in Extended Data Fig. 5c.

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Source data for plot in Extended Data Fig. 6.

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Unprocessed western blot in Extended Data Fig. 8c.

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Zhang, J., Tang, W., Gao, H. et al. Structural and functional characteristics of the SARS-CoV-2 Omicron subvariant BA.2 spike protein. Nat Struct Mol Biol 30, 980–990 (2023). https://doi.org/10.1038/s41594-023-01023-6

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