The impact of S2 mutations on Omicron SARS-CoV-2 cell surface expression and fusogenicity

ABSTRACT

SARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1^614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

KEYWORDS:

• SARS-CoV-2
• Omicron
• spike
• cell–cell fusion
• spike cell surface expression
• “first-generation” Omicron sublineages
• “second-generation” Omicron sublineages

Acknowledgements

We thank Dr. Randy Albrecht for support with the BSL3 facility and procedures at the ISMMS as well as Richard Cadagan for excellent technical assistance. We also thank Dr. Viviana Simon and the Mount Sinai Pathogen Surveillance Program for collecting and sequencing clinical specimens used to generate the SARS-CoV-2 viral stocks from this study. We also want to thank Sadaf Aslam and Soner Yildiz for helping with the cloning techniques and cell–cell fusion quantification on the Celigo Image cytometer, respectively. Confocal microscopy images were taken at the Microscopy and Advanced Bioimaging CoRE at the Icahn School of Medicine at Mount Sinai. Schematic representation of the cell–cell fusion assay shown in Figure 2(A) was created using BioRender.

Author contributions

A.G.S and T.A conceived, designed, and supervised the study. T.A provided training to A.E. A.E performed all infection experiments including growth of SARS-CoV-2 viral stocks, viral infections in different cell lines, Western Blots and biotinilation experiments, generation of Omicron S plasmids, cell-cell fusion assay and immunofluorescence analysis with the help of T.A. M.L established GFP split cell lines, produced S expression plasmids, protein chimeras and single point mutants, performed cell-cell fusion assays and FACS analysis with the help of U.K. S.T. performed the phylogenetic analysis and estimation of evolutionary convergence of mutations close to the furin site and provided Figure 1. A.S.G-R, A.v.d.G., K.F and Z.K performed experiments for whole genome sequencing. H.v.B and D.S. provided methods and expertise. A.E and M.L analyzed data. A.E and T.A wrote the manuscript and prepared figures. All authors reviewed the manuscript.

Disclosure statement

The AGS laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck, outside of the reported work. AGS has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus and Pfizer, outside of the reported work. AGS has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott and Astrazeneca. AGS is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York. All other authors declare they have no competing interests.

Additional information

Data and materials availability

All data are available in the main text or the supplementary materials. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Adolfo García-Sastre ([email protected]).

Additional information

Funding

This work is partly supported by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract # 75N93021C00014) to A.G.-S and T.A; and Systems Biology Lens (SYBIL, # U19AI135972) to A.G.-S. M.L. was funded by the Belgian American Educational Foundation (BAEF). U.K. was funded by the Swiss National Science Foundation Postdoc. Mobility fellowship (P500PB_206818). This work was supported in part through the computational and data resources and staff expertise provided by Scientific Computing and Data at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences. Research reported in this publication was also supported by the Office of Research Infrastructure of the National Institutes of Health under award number S10OD026880 and S10OD030463.