Templated trimerization of the phage L decoration protein on capsidsWoodbury, Newcomer, Leroux
et alProtein Sci (2025) 34 (4), e70089
Abstract: The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec1-98) was used to demonstrate that the globular OB-fold domain folds independently of the C-terminal residues. However, Dec1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, thereby implying Dec trimer assembly in vivo is templated by the phage capsid. The thermodynamic hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.© 2025 The Protein Society.
SARS CoV-2 spike adopts distinct conformational ensembles in situGramm, Braet, Srinivasu
et albioRxiv (2025)
Abstract: Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.
SARS-CoV-2 serotyping based on spike antigenicity and its implications for host immune evasionRuan, Gao, Qu
et alEBioMedicine (2025) 114, 105634
Abstract: As SARS-CoV-2 continues to spread and evolve, new variants/sub-variants emerge, raising concerns about vaccine-induced immune escape. Here, we conducted a systematic analysis of the serology and immunogenicity of major circulating variants/sub-variants of SARS-CoV-2 since the outbreak.We expressed and purified trimeric S proteins from 21 SARS-CoV-2 variants, with SARS-CoV included as an outgroup. Mice were immunized, and the resulting antisera were tested for binding antibodies after the third dose injection, and for neutralizing antibodies (NAbs) after both the second and third doses. Using pseudovirus neutralization assays, we evaluated cross-neutralization among major circulating variants. By integrating serological classification, antigenic mapping, and 3D landscape analysis, we explored the antigenic relationships among different SARS-CoV-2 variants and their impact on serological responses.Based on the cross-neutralization activities of the sera from different S protein vaccinations and antigenicity analyses, we grouped the 21 lineages into six serotypes. Particularly, BA.2.86 and JN.1 had very weak cross-neutralization with all other SARS-CoV-2 sub-variants tested and were grouped into a separate serotype, Serotype VI.This systematic study contributes to a better understanding of the evolution of SARS-CoV-2 and its antigenic characteristics and provides valuable insights for vaccine development.This study was supported by the National Key R&D Program of China (2023YFC2307801, 2020YFA0509202 and 2021YFA1300803), the National Natural Science Foundation of China (82222040 and 82072289), CAS Project for Young Scientists in Basic Research (YSBR-083) and Beijing Nova Program of Science and Technology (20220484181).Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.
SARS-CoV-2 spike peptide analysis reveals a highly conserved region that elicits potentially pathogenic autoantibodies: implications to pan-coronavirus vaccine developmentDiaz, Mikulski, Leaman
et alFront Immunol (2025) 16, 1488388
Abstract: The SARS-CoV-2 pandemic, while subsiding, continues to plague the world as new variants emerge. Millions have died, and millions more battle with the debilitating symptoms of a clinical entity known as long Covid. The biggest challenge remains combating an ever-changing variant landscape that threatens immune evasion from vaccine and prior infection-generated immunity. In addition, the sequelae of symptoms associated with long Covid almost certainly point to multiple pathologies that range from direct damage to organs during infection to a potential role for infection-induced autoreactive antibodies in promoting autoimmune-like conditions in these patients. In this study, a peptide scan of the SARS-CoV-2 spike protein was done to detect novel, highly conserved linear epitopes that do not elicit autoantibodies. We identified eight predicted linear epitopes capable of eliciting anti-spike IgG antibodies. Immunizations alternating peptide conjugated to KLH with the full trimer yielded the highest antibody levels, but homologous immunization with some of the peptides also yielded high levels when an additional immunization step was added. Of all regions tested, the stem helix adjacent to the heptad repeat 2 (HR2) region also elicited high levels of autoreactive antibodies to known autoantigens in common systemic autoimmune disorders such as lupus and scleroderma and may contribute to the long Covid syndrome seen in some patients. Implications to vaccine design are discussed.Copyright © 2025 Diaz, Mikulski, Leaman, Gandarilla, Da Silva, Verkoczy, Zhang and Verkoczy.
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