Enhanced Stability of the SARS CoV-2 Spike Glycoprotein Following Modification of an Alanine Cavity in the Protein Core

Overview

Journal PLoS Pathog

Specialty Microbiology

Date 2023 May 18

PMID 37200378

Authors

Pantelis Poumbourios

Christine Langer

Irene Boo

Tasnim Zakir

Rob J Center

Anouschka Akerman

Vanessa Milogiannakis

Anupriya Aggarwal

Bronte A Johnstone

Jungmin Ha

Fasseli Coulibaly

Stuart G Turville

Heidi E Drummer

Affiliations

Soon will be listed here.

Abstract

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

References

Pallesen J, Wang N, Corbett K, Wrapp D, Kirchdoerfer R, Turner H . Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A. 2017; 114(35):E7348-E7357. PMC: 5584442. DOI: 10.1073/pnas.1707304114. View

Hoffmann M, Kleine-Weber H, Pohlmann S . A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell. 2020; 78(4):779-784.e5. PMC: 7194065. DOI: 10.1016/j.molcel.2020.04.022. View

Edwards T, Wyss S, Reeves J, Zolla-Pazner S, Hoxie J, Doms R . Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein. J Virol. 2002; 76(6):2683-91. PMC: 135967. DOI: 10.1128/jvi.76.6.2683-2691.2002. View

Niesen F, Berglund H, Vedadi M . The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007; 2(9):2212-21. DOI: 10.1038/nprot.2007.321. View

Bullough P, Hughson F, Skehel J, Wiley D . Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994; 371(6492):37-43. DOI: 10.1038/371037a0. View

Branche A, Rouphael N, Diemert D, Falsey A, Losada C, Baden L . SARS-CoV-2 Variant Vaccine Boosters Trial: Preliminary Analyses. medRxiv. 2022; . PMC: 9327623. DOI: 10.1101/2022.07.12.22277336. View

Jackson C, Farzan M, Chen B, Choe H . Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol. 2021; 23(1):3-20. PMC: 8491763. DOI: 10.1038/s41580-021-00418-x. View

Crawford K, Eguia R, Dingens A, Loes A, Malone K, Wolf C . Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses. 2020; 12(5). PMC: 7291041. DOI: 10.3390/v12050513. View

Chesebro B, Wehrly K, Nishio J, Perryman S . Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J Virol. 1992; 66(11):6547-54. PMC: 240149. DOI: 10.1128/JVI.66.11.6547-6554.1992. View

10.

Dejnirattisai W, Zhou D, Supasa P, Liu C, Mentzer A, Ginn H . Antibody evasion by the P.1 strain of SARS-CoV-2. Cell. 2021; 184(11):2939-2954.e9. PMC: 8008340. DOI: 10.1016/j.cell.2021.03.055. View

11.

Naldini L, Blomer U, Gage F, Trono D, Verma I . Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A. 1996; 93(21):11382-8. PMC: 38066. DOI: 10.1073/pnas.93.21.11382. View

12.

Ke Z, Oton J, Qu K, Cortese M, Zila V, McKeane L . Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature. 2020; 588(7838):498-502. PMC: 7116492. DOI: 10.1038/s41586-020-2665-2. View

13.

Wrapp D, Wang N, Corbett K, Goldsmith J, Hsieh C, Abiona O . Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367(6483):1260-1263. PMC: 7164637. DOI: 10.1126/science.abb2507. View

14.

McLellan J, Yang Y, Graham B, Kwong P . Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J Virol. 2011; 85(15):7788-96. PMC: 3147929. DOI: 10.1128/JVI.00555-11. View

15.

Tuekprakhon A, Nutalai R, Dijokaite-Guraliuc A, Zhou D, Ginn H, Selvaraj M . Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell. 2022; 185(14):2422-2433.e13. PMC: 9181312. DOI: 10.1016/j.cell.2022.06.005. View

16.

Shi R, Shan C, Duan X, Chen Z, Liu P, Song J . A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature. 2020; 584(7819):120-124. DOI: 10.1038/s41586-020-2381-y. View

17.

Wang Z, Muecksch F, Cho A, Gaebler C, Hoffmann H, Ramos V . Analysis of memory B cells identifies conserved neutralizing epitopes on the N-terminal domain of variant SARS-Cov-2 spike proteins. Immunity. 2022; 55(6):998-1012.e8. PMC: 8986478. DOI: 10.1016/j.immuni.2022.04.003. View

18.

Wilson I, Skehel J, Wiley D . Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981; 289(5796):366-73. DOI: 10.1038/289366a0. View

19.

Center R, Boo I, Phu L, McGregor J, Poumbourios P, Drummer H . Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine. J Biol Chem. 2020; 295(21):7179-7192. PMC: 7247312. DOI: 10.1074/jbc.RA120.013015. View

20.

Sliepen K, van Montfort T, Melchers M, Isik G, Sanders R . Immunosilencing a highly immunogenic protein trimerization domain. J Biol Chem. 2015; 290(12):7436-42. PMC: 4367253. DOI: 10.1074/jbc.M114.620534. View