SARS MERS S2

What is the S2 subunit in coronavirus spike proteins and how does it differ across SARS and MERS viruses?

The S2 subunit constitutes the membrane-anchored portion of coronavirus spike proteins that mediates virus-cell membrane fusion following receptor binding by the S1 subunit. In SARS-CoV, MERS-CoV, and SARS-CoV-2, the S2 domain contains critical functional elements including fusion peptides, heptad repeats (HR1 and HR2), a transmembrane domain, and cytoplasmic tail.

Structurally, the S2 subunit is significantly more conserved across coronaviruses than the S1 domain, with greater sequence homology between SARS-CoV and SARS-CoV-2 (approximately 90% identity) than with MERS-CoV. The S2 domain undergoes conformational rearrangements from a prefusion to a postfusion state during viral entry, a process that is mechanistically similar across these viruses though with specific differences in protease activation requirements .

For experimental characterization, researchers typically employ cryo-electron microscopy, X-ray crystallography, and molecular dynamics simulations to analyze structural features. The comparative analysis of S2 domains across coronaviruses reveals both conserved structural motifs essential for fusion function and virus-specific adaptations .

How do proteolytic activation patterns of S2 differ between SARS-CoV, MERS-CoV, and SARS-CoV-2?

Proteolytic activation represents a crucial difference in S2 function across coronaviruses. SARS-CoV-2 uniquely possesses a furin cleavage site at the S1/S2 boundary not present in SARS-CoV, allowing for pre-activation during biogenesis. This characteristic impacts:

• Viral entry pathways

• Cellular tropism

• Transmission efficiency

MERS-CoV also contains a furin-like protease site, whereas SARS-CoV relies primarily on target cell proteases. All three viruses require a second proteolytic event (S2') near the fusion peptide, typically mediated by TMPRSS2 or cathepsin L depending on the cell type and entry pathway .

Methodologically, researchers investigate these differences using:

• Pseudovirus systems with protease inhibitors

• Site-directed mutagenesis of cleavage sites

• Cell lines expressing different proteases

• Protease activation assays in various tissue contexts

The evolutionary retention of the furin site in SARS-CoV-2 throughout the pandemic, despite creating structural instability, suggests strong selective pressure for maintaining this feature for transmission advantage .

What evidence exists for cross-reactive antibodies targeting the S2 domain across different coronaviruses?

Cross-reactive antibodies targeting the S2 domain represent a significant finding in coronavirus immunology. Research demonstrates that COVID-19 convalescent sera frequently show binding activity against MERS-CoV S2, with approximately 57% (34 of 60) of convalescent samples exhibiting this cross-reactivity. Endpoint titers of these antibodies positively correlate with titers to SARS-CoV-2 S2, suggesting shared epitopes .

From memory B cells of COVID-19 convalescents, researchers have constructed monoclonal antibodies (mAbs) with remarkable cross-reactivity profiles:

Methodologically, these findings were established through:

• Systematic screening of convalescent sera against recombinant S2 domains

• Single B-cell sorting and antibody cloning

• Binding assays against multiple coronavirus spike proteins

• Pseudovirus neutralization assays

• Epitope mapping using peptide arrays and conformational analysis

This evidence supports the concept that S2-based immunogens might elicit broadly protective immune responses against multiple coronaviruses.

How do researchers identify and characterize conserved S2 epitopes with potential for broad coronavirus neutralization?

Identification and characterization of conserved S2 epitopes involves a multi-faceted approach combining structural biology, immunological screening, and functional analysis. A significant example is the identification of the S2 apex residues 980-1006 in the flexible hinge region, a conformational epitope present only in the prefusion core of β-coronaviruses .

Methodological approaches include:

• Structural Analysis: Cryo-EM and X-ray crystallography to define conformational states of S2, identifying regions that remain accessible in the prefusion state but are conserved across virus lineages.

• Immune Repertoire Mining: Isolation of memory B cells from convalescent donors followed by single-cell sequencing and antibody expression to identify naturally occurring cross-reactive antibodies.

• Epitope Mapping: Combination of hydrogen-deuterium exchange mass spectrometry, alanine scanning mutagenesis, and competition binding assays to precisely define epitope boundaries.

• Functional Characterization: Testing antibody effector functions beyond neutralization, such as antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC).

The RAY53 antibody exemplifies this approach, binding a conserved hinge epitope present in both MERS-CoV and SARS-CoV-2 spikes and mediating ADCP and ADCC against SARS-CoV-2 spike in vitro. Importantly, mutations that ablate antibody binding to this epitope compromise pseudovirus infectivity, indicating the functional importance of this region .

Researchers must carefully evaluate how spike protein dynamics affect epitope accessibility. For example, conformational changes associated with spike opening, including those in Omicron BA.1, can occlude S2 core epitopes and potentially evade antibodies targeting this region .

What strategies are being pursued to develop S2-focused vaccines with broad coronavirus protection?

Development of S2-focused vaccines represents a promising strategy for creating broadly protective coronavirus immunogens. Research efforts center on stabilizing the S2 domain in its prefusion conformation to maintain critical epitopes that might otherwise be lost during conformational changes. Several methodological approaches are being pursued:

• Computational Design and Molecular Dynamics: Weighted ensemble (WE) molecular dynamics simulations are employed to characterize the opening mechanism of the S2 trimer and identify potential stabilizing mutations. This approach has successfully identified cavity-filling tryptophan substitutions (V991W and T998W) that alter the conformational landscape of S2 and stabilize the closed prefusion conformation .

• Structure-Guided Protein Engineering: Introduction of disulfide bonds, proline substitutions, and cavity-filling mutations to prevent premature conformational changes. The HexaPro-SS-2W construct exemplifies this approach, incorporating both the original HexaPro stabilizing mutations and additional tryptophan substitutions .

• Thermostability Enhancement: Selection of constructs with improved expression yields and higher melting temperatures, which correlate with greater stability of the prefusion conformation. Engineering efforts have produced S2 constructs with significantly improved thermostability profiles:

• Conformational Epitope Preservation: Development of constructs that maintain broadly neutralizing epitopes while eliminating non-neutralizing or potentially harmful epitopes. This approach requires careful mapping of S2 regions that elicit protective versus non-protective responses .

Animal studies with stabilized S2 antigens such as HexaPro-SS-2W have demonstrated neutralizing activity against recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from SARS-CoV-2 Wuhan-1 and Omicron BA.1, suggesting potential for broad protection .

What are the experimental challenges in evaluating S2-based vaccine candidates compared to S1/RBD-focused approaches?

Evaluation of S2-based vaccine candidates presents distinct experimental challenges compared to S1/RBD-focused approaches:

• Neutralization Assay Limitations: Standard neutralization assays may underestimate the protective potential of S2-targeting antibodies if they predominantly act through Fc-mediated effector functions rather than direct neutralization. Researchers must employ:

  • Antibody-dependent cellular phagocytosis (ADCP) assays

  • Antibody-dependent cellular cytotoxicity (ADCC) assays

  • Complement-dependent cytotoxicity (CDC) assays

  • In vivo challenge studies to fully assess protection mechanisms

• Conformational Complexity: The S2 domain undergoes significant conformational changes during fusion, making it challenging to ensure that vaccine-induced antibodies target the relevant prefusion epitopes. This necessitates:

  • Sophisticated structural verification using cryo-EM

  • Conformational probes to confirm epitope presentation

  • Thermal stability assessments to verify maintenance of desired conformation

• Lower Immunogenicity: S2 epitopes may be less immunodominant than RBD epitopes, particularly in natural infection. Overcoming this requires:

  • Adjuvant optimization studies

  • Prime-boost strategies with heterologous constructs

  • Multimerization approaches to enhance immunogenicity

• Correlates of Protection: The field lacks established correlates of protection for S2-targeted immunity, unlike RBD-binding antibody titers which correlate well with protection for current vaccines. Researchers must:

  • Design longitudinal studies correlating S2 antibody responses with protection

  • Develop standardized assays specific for S2-directed immunity

  • Establish appropriate animal models that reflect human S2-directed immune responses

Despite these challenges, S2-based approaches offer significant advantages in addressing viral evolution. While the RBD is highly variable across variants (with the NTD changing even faster), the S2 subunit remains relatively conserved, potentially offering longer-lasting protection against emerging variants and even different coronavirus species .

How do conformational dynamics of the S2 subunit influence antibody targeting and vaccine design strategies?

The conformational dynamics of the S2 subunit critically impact both antibody recognition and vaccine design strategy. S2 undergoes dramatic rearrangements during the fusion process, transitioning from a metastable prefusion state to a highly stable postfusion state, with numerous intermediate conformations.

Advanced simulation studies using weighted ensemble (WE) molecular dynamics have revealed key insights about these dynamics:

• Asymmetric Opening Pathway: The S2 trimer apex transitions from closed to open conformations through an asymmetric protomer-protomer separation rather than a symmetric pathway. This mechanistic understanding has directly informed the design of stabilizing mutations .

• Key Interfacial Interactions: Contact map analysis of interfacial helices (CHs, UHs, and HR1) identifies specific residue-residue interactions critical during the opening pathway. Disruption of these interactions can significantly alter S2 stability and function .

• Targeted Stabilization: Cavity-filling substitutions with tryptophan at positions V991 and T998 have been shown to provide kinetic stabilization by slowing S2 trimer opening. Free energy perturbation (FEP) calculations confirm increased stability of the closed prefusion conformation in engineered variants .

These dynamic considerations have profound implications for antibody targeting and vaccine design:

• Epitope Accessibility: Many functionally important epitopes in S2 are transiently exposed or conformationally dependent. For example, the hinge epitope recognized by antibody RAY53 is only present in the prefusion state and becomes occluded during spike opening dynamics, including changes observed in Omicron BA.1 .

• Conformational Masking: Changes in spike opening dynamics, including those driven by mutations distant from key epitopes, can effectively evade pre-existing antibodies targeting the S2 core through conformational masking .

• Stabilization Strategies: Vaccine designs must balance the need to stabilize specific conformations while preserving the native-like presentation of key epitopes. Excess stabilization could potentially alter important epitopes or create non-native structures .

For experimental evaluation of these dynamics, researchers employ:

• Hydrogen-deuterium exchange mass spectrometry to map conformational flexibility

• Single-molecule FRET to observe real-time conformational changes

• Negative-stain electron microscopy to visualize conformational distributions

• Antibody binding kinetics to probe accessibility of different epitopes across conformational states

What is the role of the S2 subunit in coronavirus tissue tropism and how does this influence pathogenesis?

The S2 subunit plays a complex role in coronavirus tissue tropism and pathogenesis, extending beyond its primary fusion function. While receptor binding via S1 is the primary determinant of tissue tropism, S2 contributes through several mechanisms:

• Protease Activation Requirements: Different coronaviruses have evolved distinct protease activation sequences in S2 that align with protease availability in target tissues. SARS-CoV-2's acquisition of a furin cleavage site allows pre-activation during virion production, expanding potential target tissues beyond those expressing TMPRSS2 or cathepsins .

• Fusion Kinetics: The intrinsic fusion kinetics encoded within S2 structures influence cell type susceptibility. Comparative studies of SARS-CoV-2 and SARS-CoV S2 domains reveal differential replication abilities in lung and intestinal tissues despite using the same receptor .

• Post-Fusion Cytopathic Effects: The formation of the S2 postfusion structure during viral entry may contribute to membrane damage and cytopathic effects in infected cells, with virus-specific differences potentially contributing to pathogenicity profiles.

Cellular susceptibility data demonstrates important differences across coronaviruses:

Methodologically, researchers investigate S2's contribution to tropism through:

• Pseudotyped virus entry assays with chimeric spike proteins

• Site-directed mutagenesis of S2 protease cleavage sites

• Organoid infection models representing different tissues

• Comparative viral kinetics in primary cell cultures from different organs

The broader tissue tropism of MERS-CoV correlates with its clinical manifestations, including more frequent extrapulmonary organ dysfunction and greater need for renal replacement therapy compared to SARS and COVID-19 . Understanding how S2 features contribute to these differences may help predict the pathogenic potential of emerging coronaviruses.

What are the optimal experimental systems for studying S2-mediated fusion and evaluating antibody neutralization mechanisms?

Studying S2-mediated fusion and antibody neutralization requires specialized experimental systems that capture the complex, multi-step nature of coronavirus entry. Researchers employ several complementary approaches:

• Pseudovirus Systems:

  • VSV-based pseudotyping offers a BSL-2 alternative to live coronavirus

  • Lentiviral pseudotypes enable quantitative luminescence/fluorescence readouts

  • Spike protein variants can be rapidly generated and compared

  • Limitations include potential differences from authentic virus and absence of other viral proteins

• Cell-Cell Fusion Assays:

  • Split reporter systems (luciferase or fluorescent proteins) allow quantification of fusion events

  • Real-time monitoring of fusion kinetics possible with some designs

  • Direct visualization of syncytia formation provides morphological data

  • Effectively isolates the fusion step from other entry processes

• Structural Analysis Platforms:

  • Single-particle cryo-EM captures prefusion, intermediate, and postfusion conformations

  • Hydrogen-deuterium exchange mass spectrometry maps conformational changes and antibody binding sites

  • X-ray crystallography provides atomic resolution of stable domains and antibody-epitope complexes

  • Molecular dynamics simulations predict conformational transitions and epitope accessibility

• Authentic Virus Systems:

  • Plaque reduction neutralization tests (PRNT) with live virus remain the gold standard

  • Focus reduction neutralization tests (FRNT) offer faster readouts

  • BSL-3 containment needed for SARS-CoV-2 and MERS-CoV

  • Recombinant viruses with reporter genes facilitate high-throughput screening

For antibody mechanism evaluation, additional methodologies are essential:

• Pre/Post-Attachment Assays: Determine whether antibodies block initial attachment or subsequent fusion steps

• Protease Inhibitor Studies: Evaluate whether antibodies interfere with proteolytic activation

• Fc Function Assays: Measure ADCC, ADCP, and complement activation by S2-targeting antibodies

• Single-Molecule Techniques: Assess how antibodies affect S protein conformational dynamics

For in vivo protection studies:

• Transgenic Mice: ACE2-humanized mice for SARS-CoV/SARS-CoV-2; DPP4-humanized mice for MERS-CoV

• Syrian Hamsters: Natural susceptibility to SARS-CoV-2 with similar pathology to humans

• Ferrets: Useful for upper respiratory tract infection and transmission studies

• Non-Human Primates: Most physiologically relevant but limited by ethical and practical constraints

How can computational approaches accelerate the design and optimization of S2-based antiviral strategies?

Computational approaches have become increasingly central to S2-based antiviral development, offering powerful tools for rational design and optimization. Several methodologies have demonstrated particular value:

• Advanced Sampling Techniques: Weighted ensemble molecular dynamics simulations have proven crucial for characterizing low-probability conformational transitions in the S2 trimer that would be inaccessible to standard simulations. This approach has directly enabled the design of stabilized S2 immunogens by:

  • Revealing asymmetric opening pathways of the S2 trimer

  • Identifying key residue-residue interactions during conformational changes

  • Predicting stabilizing mutations at the cavity interfaces (V991W and T998W)

  • Confirming kinetic stabilization of engineered variants through reduced opening rates

• Free Energy Perturbation (FEP) Calculations: These calculations quantify the energetic impact of mutations on protein stability, allowing researchers to:

  • Rank candidate mutations by their predicted stabilizing effect

  • Calculate ΔΔG mutation-folding values to predict conformational preferences

  • Prioritize constructs for experimental validation

  • Optimize combinations of mutations for additive or synergistic effects

• Epitope Prediction and Analysis:

  • Conservation analysis across coronavirus species identifies constrained regions less likely to tolerate escape mutations

  • B-cell epitope prediction algorithms identify regions likely to be immunogenic

  • Antibody-epitope docking predicts binding modes and contact residues

  • Sequence co-evolution analysis detects coordinated mutation patterns that might indicate functional constraints

• Molecular Dynamics-Based Drug Discovery:

  • Virtual screening of small molecule libraries against S2 fusion machinery

  • Identification of druggable pockets that appear transiently during conformational changes

  • Fragment-based approaches to design fusion inhibitors targeting conserved S2 regions

  • Pharmacophore modeling based on known neutralizing antibody binding sites

These computational approaches offer significant advantages for S2-based strategies, including:

• Efficiency: Screening thousands of potential mutations or compounds in silico before experimental testing

• Mechanistic Insight: Understanding the physical basis of stabilization or inhibition rather than empirical discovery

• Novel Hypothesis Generation: Identifying non-obvious targets or approaches that might be missed by conventional methods

• Integration with Experimental Data: Using structural and binding data to refine and validate computational models

The HexaPro-SS-2W construct exemplifies the success of this approach, where computational prediction led to significantly improved expression yields and thermostability, enabling high-resolution cryo-EM characterization and demonstrating neutralization potential against multiple SARS-CoV-2 variants .