Structural and functional characteristics of the sars-cov-2 omicron subvariant ba. 2 spike protein

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. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS SPIKE MUTATION D614G ALTERS SARS-COV-2 FITNESS Article 26

October 2020 SARS-COV-2 SPIKE PROTEIN: STRUCTURE, VIRAL ENTRY AND VARIANTS Article 06 May 2025 EXPLORING DISTINCT MODES OF INTER-SPIKE CROSS-LINKING FOR ENHANCED NEUTRALIZATION BY

SARS-COV-2 ANTIBODIES Article Open access 04 December 2024 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. REFERENCES * Hirotsu, Y. et al. SARS-CoV-2

Omicron sublineage BA.2 replaces BA.1.1: genomic surveillance in Japan from September 2021 to March 2022. _J. Infect._ 85, 174–211 (2022). CAS  PubMed  PubMed Central  Google Scholar  *

Lyngse, F. P. et al. Household transmission of SARS-CoV-2 Omicron variant of concern subvariants BA.1 and BA.2 in Denmark. _Nat. Commun._ 13, 5760 (2022). CAS  PubMed  PubMed Central  Google

Scholar  * Mefsin, Y. M. et al. Epidemiology of infections with SARS-CoV-2 Omicron BA.2 variant, Hong Kong, January–March 2022. _Emerg. Infect. Dis._ 28, 1856–1858 (2022). CAS  PubMed 

PubMed Central  Google Scholar  * Smith, D. J. et al. COVID-19 mortality and vaccine coverage — Hong Kong Special Administrative Region, China, January 6, 2022–March 21, 2022. _MMWR Morb.

Mortal. Wkly Rep._ 71, 545–548 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Yamasoba, D. et al. Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike. _Cell_ 185,

2103–2115.e19 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Wolter, N. et al. Clinical severity of omicron lineage BA.2 infection compared with BA.1 infection in South Africa.

_Lancet_ 400, 93–96 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Iketani, S. et al. Antibody evasion properties of SARS-CoV-2 Omicron sublineages. _Nature_ 604, 553–556 (2022). CAS

  PubMed  PubMed Central  Google Scholar  * Gruell, H. et al. SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns. _Cell Host Microbe._ 30, 1231–1241.e6 (2022). CAS 

PubMed  PubMed Central  Google Scholar  * Yu, J. et al. Neutralization of the SARS-CoV-2 Omicron BA.1 and BA.2 variants. _N. Engl. J. Med._ 386, 1579–1580 (2022). PubMed  Google Scholar  *

Mykytyn, A. Z. et al. Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct. _Sci Immunol._ 7, eabq4450 (2022). CAS  PubMed  Google Scholar  *

Cao, Y. et al. Omicron BA.2 specifically evades broad sarbecovirus neutralizing antibodies. Preprint at _bioRxiv_ https://doi.org/10.1101/2022.02.07.479349 (2022). * Zou, J. et al.

Cross-neutralization of Omicron BA.1 against BA.2 and BA.3 SARS-CoV-2. _Nat. Commun._ 13, 2956 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Chemaitelly, H. et al. Protection of

Omicron sub-lineage infection against reinfection with another Omicron sub-lineage. _Nat. Commun._ 13, 4675 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Stegger, M. et al.

Occurrence and significance of Omicron BA.1 infection followed by BA.2 reinfection. Preprint at _medRxiv_ https://doi.org/10.1101/2022.02.19.22271112 (2022). * Kirsebom, F. C. M. et al.

COVID-19 vaccine effectiveness against the omicron (BA.2) variant in England. _Lancet Infect. Dis._ 22, 931–933 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Bowen, J. E. et al.

Omicron spike function and neutralizing activity elicited by a comprehensive panel of vaccines. _Science._ 377, 890–894 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Dai, L. &

Gao, G. F. Viral targets for vaccines against COVID-19. _Nat. Rev. Immunol._ 21, 73–82 (2021). CAS  PubMed  Google Scholar  * Bosch, B. J., van der Zee, R., de Haan, C. A. M. & Rottier,

P. J. M. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. _J. Virol._ 77, 8801–8811 (2003). CAS  PubMed

  PubMed Central  Google Scholar  * Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. _Cell_ 181, 271–280.e8

(2020). CAS  PubMed  PubMed Central  Google Scholar  * Millet, J. K. & Whittaker, G. R. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated

activation of the spike protein. _Proc. Natl Acad. Sci. USA_ 111, 15214–15219 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Tortorici, M. A. & Veesler, D. Structural insights

into coronavirus entry. _Adv. Virus Res_ 105, 93–116 (2019). CAS  PubMed  PubMed Central  Google Scholar  * Jackson, C. B., Farzan, M., Chen, B. & Choe, H. Mechanisms of SARS-CoV-2 entry

into cells. _Nat. Rev. Mol. Cell Biol._ 23, 3–20 (2022). CAS  PubMed  Google Scholar  * Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. _Proc. Natl Acad. Sci. USA_ 117, 11727–11734

(2020). CAS  PubMed  PubMed Central  Google Scholar  * Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. _Science_ 367, 1260–1263 (2020). CAS  PubMed 

PubMed Central  Google Scholar  * Harvey, W. T. et al. SARS-CoV-2 variants, spike mutations and immune escape. _Nat. Rev. Microbiol._ 19, 409–424 (2021). CAS  PubMed  PubMed Central  Google

Scholar  * Hong, Q. et al. Molecular basis of receptor binding and antibody neutralization of Omicron. _Nature_ 604, 546–552 (2022). CAS  PubMed  Google Scholar  * Stalls, V. et al. Cryo-EM

structures of SARS-CoV-2 Omicron BA.2 spike. _Cell Rep._ 39, 111009 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Zhang, J. et al. Structural impact on SARS-CoV-2 spike protein by

D614G substitution. _Science_ 372, 525–530 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Cai, Y. et al. Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2

variants. _Science_ 373, 642–648 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Zhang, J. et al. Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

_Science_ 374, 1353–1360 (2021). CAS  PubMed  Google Scholar  * Zhang, J. et al. Structural and functional impact by SARS-CoV-2 Omicron spike mutations. _Cell Rep._ 39, 110729 (2022). CAS 

PubMed  PubMed Central  Google Scholar  * Cai, Y. et al. Distinct conformational states of SARS-CoV-2 spike protein. _Science_ 369, 1586–1592 (2020). CAS  PubMed  Google Scholar  * Ogando,

N. S. et al. SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology. _J. Gen. Virol._ 101, 925–940 (2020). CAS  PubMed  PubMed Central 

Google Scholar  * McCray, P. B.Jr. et al. Lethal infection of K18-_hACE2_ mice infected with severe acute respiratory syndrome coronavirus. _J. Virol._ 81, 813–821 (2007). CAS  PubMed 

Google Scholar  * Radvak, P. et al. SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains. _Nat. Commun._

12, 6559 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Tong, P. et al. Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike. _Cell_ 184, 4969–4980.e15 (2021). CAS

  PubMed  PubMed Central  Google Scholar  * Xiao, T. et al. A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent. _Nat. Struct. Mol. Biol._ 28, 202–209 (2021). CAS 

PubMed  PubMed Central  Google Scholar  * Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.

_Nat. Methods_ 14, 290–296 (2017). CAS  PubMed  Google Scholar  * Zhang, J., Xiao, T., Cai, Y. & Chen, B. Structure of SARS-CoV-2 spike protein. _Curr. Opin. Virol._ 50, 173–182 (2021).

CAS  PubMed  PubMed Central  Google Scholar  * Tian, F. et al. N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2. _eLife_ 10, e69091 (2021). CAS  PubMed

  PubMed Central  Google Scholar  * Mannar, D. et al. SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein-ACE2 complex. _Science._ 375, 760–764 (2022). CAS 

PubMed  PubMed Central  Google Scholar  * Yin, W. et al. Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody. _Science._ 375, 1048–1053 (2022). * McCallum, M. et

al. Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement. _Science_ 375, 864–868 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Cui, Z. et al. Structural and

functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron. _Cell._ 185, 860–871.e13 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Gobeil, S. M.-C. et al.

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity. _Science_ 373, eabi6226 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Chi, X. et al. A

neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. _Science_ 369, 650–655 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Davies, N. G. et

al. Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. _Science_ 372, eabg3055 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Hart, W. S. et al.

Generation time of the alpha and delta SARS-CoV-2 variants: an epidemiological analysis. _Lancet Infect. Dis._ 22, 603–610 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Milne, G. et

al. Does infection with or vaccination against SARS-CoV-2 lead to lasting immunity? _Lancet Respir. Med_ 9, 1450–1466 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Levin, E. G. et

al. Waning immune humoral response to BNT162b2 Covid-19 vaccine over 6 months. _N. Engl. J. Med._ 385, e84 (2021). CAS  PubMed  Google Scholar  * Meng, B. et al. Altered TMPRSS2 usage by

SARS-CoV-2 Omicron impacts infectivity and fusogenicity. _Nature_ 603, 706–714 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Zeng, C. et al. Neutralization and stability of

SARS-CoV-2 Omicron variant. Preprint at _bioRxiv_ https://doi.org/10.1101/2021.12.16.472934 (2021). * Halfmann, P. J. et al. SARS-CoV-2 Omicron virus causes attenuated disease in mice and

hamsters. _Nature_ 603, 687–692 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Suzuki, R. et al. Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant. _Nature_

603, 700–705 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Shuai, H. et al. Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron. _Nature_ 603, 693–699 (2022).

CAS  PubMed  Google Scholar  * Sentis, C. et al. SARS-CoV-2 Omicron variant, lineage BA.1, is associated with lower viral load in nasopharyngeal samples compared to Delta variant. _Viruses_

14, 919 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Yinda, C. K. et al. K18-hACE2 mice develop respiratory disease resembling severe COVID-19. _PLoS Pathog._ 17, e1009195 (2021).

CAS  PubMed  PubMed Central  Google Scholar  * Romano, M., Ruggiero, A., Squeglia, F., Maga, G. & Berisio, R. A structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis,

proofreading and final capping. _Cells_ 9, 1267 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Hansen, J. et al. Studies in humanized mice and convalescent humans yield a SARS-CoV-2

antibody cocktail. _Science_ 369, 1010–1014 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Pinto, D. et al. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV

antibody. _Nature_ 583, 290–295 (2020). CAS  PubMed  Google Scholar  * Tortorici, M. A. et al. Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms.

_Science_ 370, 950–957 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Starr, T. N. et al. SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. _Nature_ 597,

97–102 (2021). CAS  PubMed  PubMed Central  Google Scholar  * Huo, J. et al. Neutralization of SARS-CoV-2 by destruction of the prefusion spike. _Cell Host Microbe_ 28, 445–454.e6 (2020).

CAS  PubMed  PubMed Central  Google Scholar  * Chen, J. et al. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein. _Science_ 349, 191–195 (2015).

CAS  PubMed  PubMed Central  Google Scholar  * Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. _Nat. Methods_ 9, 671–675 (2012). CAS 

PubMed  PubMed Central  Google Scholar  * Chen, C. Z. et al. Identifying SARS-CoV-2 entry inhibitors through drug repurposing screens of SARS-S and MERS-S pseudotyped particles. _ACS

Pharmacol. Transl. Sci._ 3, 1165–1175 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Millet, J. K. & Whittaker, G. R. Murine leukemia virus (MLV)-based coronavirus

spike-pseudotyped particle production and infection. _Bio Protoc._ 6, e2035 (2016). PubMed  Google Scholar  * Case, J. B., Bailey, A. L., Kim, A. S., Chen, R. E. & Diamond, M. S. Growth,

detection, quantification, and inactivation of SARS-CoV-2. _Virology_ 548, 39–48 (2020). CAS  PubMed  Google Scholar  * Xie, X. et al. An infectious cDNA clone of SARS-CoV-2. _Cell Host

Microbe_ 27, 841–848.e3 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. _J.

Struct. Biol._ 152, 36–51 (2005). PubMed  Google Scholar  * Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. _J. Struct. Biol._ 180,

519–530 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. _Acta Crystallogr. D

Biol. Crystallogr._ 66, 213–221 (2010). CAS  PubMed  PubMed Central  Google Scholar  * Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution

electron-density maps. _Acta Crystallogr. D Struct. Biol._ 74, 519–530 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Pettersen, E. F. et al. UCSF ChimeraX: structure visualization

for researchers, educators, and developers. _Protein Sci._ 30, 70–82 (2021). CAS  PubMed  Google Scholar  * Morin, A. et al. Collaboration gets the most out of software. _eLife_ 2, e01456

(2013). PubMed  PubMed Central  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * These authors contributed equally: Jun Zhang, Weichun Tang, Hailong Gao. AUTHORS AND

AFFILIATIONS * 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 *

Department of Pediatrics, Harvard Medical School, Boston, MA, USA Jun Zhang, Hailong Gao, Wei Shi, Tianshu Xiao & Bing Chen * 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 * Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, USA Christy L. Lavine & Michael S.

Seaman * Institute for Protein Innovation, Harvard Institutes of Medicine, Boston, MA, USA Haisun Zhu & Krishna Anand * 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 * Codex BioSolutions, Inc., Rockville, MD,

USA Shaowei Wang & Jianming Lu * The Harvard Cryo-EM Center for Structural Biology, Boston, MA, USA Megan L. Mayer * Department of Biochemistry and Molecular and Cellular Biology,

Georgetown University, Washington, DC, USA Jianming Lu Authors * Jun Zhang View author publications You can also search for this author inPubMed Google Scholar * Weichun Tang View author

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Lavine View author publications You can also search for this author inPubMed Google Scholar * Wei Shi View author publications You can also search for this author inPubMed Google Scholar *

Hanqin Peng View author publications You can also search for this author inPubMed Google Scholar * Haisun Zhu View author publications You can also search for this author inPubMed Google

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publications You can also search for this author inPubMed Google Scholar * Duane R. Wesemann View author publications You can also search for this author inPubMed Google Scholar * Michael S.

Seaman View author publications You can also search for this author inPubMed Google Scholar * Jianming Lu View author publications You can also search for this author inPubMed Google

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Google Scholar * Bing Chen View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW

PEER REVIEW INFORMATION _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. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional

claims in published maps and institutional affiliations. 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. Source

data 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. Source data 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. Source data 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. Source data 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. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–4. REPORTING SUMMARY SOURCE DATA SOURCE DATA

FIG. 1 Source data for plots in Fig. 1a–f. SOURCE DATA FIG. 2 Source data for gel-filtration trace in Fig. 2b. SOURCE DATA FIG. 2 Unprocessed gel in Fig. 2b. SOURCE DATA FIG. 3 Source data

for sensorgrams and fits in Fig. 3. SOURCE DATA EXTENDED DATA FIG. 1 Unprocessed western blot in Extended Data Fig. 1b. SOURCE DATA EXTENDED DATA FIG. 1 Source data for plot in Extended Data

Fig. 1c. SOURCE DATA EXTENDED DATA FIG. 2 Source data for plot in Extended Data Fig. 2a. SOURCE DATA EXTENDED DATA FIG. 3 Source data for plot in Extended Data Fig. 3. SOURCE DATA EXTENDED

DATA FIG. 4 Source data for plots in Extended Data Fig. 4. SOURCE DATA EXTENDED DATA FIG. 5 Source data for sensorgrams and fits in Extended Data Fig. 5b and plot in Extended Data Fig. 5c.

SOURCE DATA EXTENDED DATA FIG. 6 Source data for plot in Extended Data Fig. 6. SOURCE DATA EXTENDED DATA FIG. 8 Unprocessed western blot in Extended Data Fig. 8c. RIGHTS AND PERMISSIONS

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CITE THIS ARTICLE 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 Download citation * Received: 17 May 2022 * Accepted: 17 May 2023 * Published: 10 July 2023 * Issue Date: July 2023 * DOI:

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