Recombinant Human respiratory syncytial virus A Small hydrophobic protein (SH)

What is the Respiratory Syncytial Virus Small Hydrophobic Protein?

The Small Hydrophobic (SH) protein is a type II transmembrane protein encoded by Respiratory Syncytial Virus (RSV), a member of the Paramyxoviridae family. The SH protein contains 64 amino acid residues in RSV subgroup A or 65 amino acid residues in RSV subgroup B . It is one of three viral-encoded proteins found in the RSV envelope, alongside the G (attachment) and F (fusion) glycoproteins . The SH protein is classified as a viroporin, which are small viral proteins that can form ion channels in host cell membranes. Initial studies of RSV SH protein suggested potential roles in viral fusion or membrane permeability alterations .

The function of SH remained unknown or poorly characterized for many years after its discovery, but recent research has significantly advanced our understanding of its structural and functional properties. Studies have demonstrated that while SH is not essential for virus replication in tissue culture, it plays an important role in viral pathogenesis as evidenced by the attenuation of SH-deleted viruses in animal models .

What is the structural organization of the RSV SH protein?

The RSV SH protein adopts a pentameric structure in the viral membrane, as demonstrated through comprehensive molecular dynamics simulations. Researchers conducted global searching molecular dynamic simulations using SH proteins from eight different viral strains, at different oligomeric states and with different lengths of the putative transmembrane domain . A total of 45 different simulations consistently identified a pentameric structure across all variants examined .

The model obtained reveals a channel-like homopentamer with a minimal transmembrane pore diameter of 1.46 Å . This structural arrangement is consistent with the proposed ion channel function of the protein. The transmembrane domain spans approximately from residues 14-41 (long TM segment) or 23-41 (short TM segment), with the pentameric conformation being more stable than other potential oligomeric states (trimers or tetramers) . The evidence for this pentameric structure includes lower Cα RMSD values when comparing structures across different variants and between simulations using different transmembrane segment lengths .

How does RSV SH protein function in viral pathogenesis?

The RSV SH protein appears to function primarily by inhibiting tumor necrosis factor alpha (TNF-α) signaling, based on studies with recombinant viruses. Researchers have demonstrated this function by generating and analyzing recombinant parainfluenza virus 5 (PIV5) lacking its own SH but containing RSV SH in its place (PIV5ΔSH-RSV SH), as well as RSV lacking its own SH (RSVΔSH) . These experiments revealed that RSV SH can functionally replace PIV5 SH in inhibiting TNF-α-induced apoptosis .

TNF-α is a pro-inflammatory cytokine that plays a key role in host defense against viral infections. By inhibiting TNF-α signaling, the SH protein likely helps RSV evade the host immune response, particularly inflammatory and apoptotic pathways that would otherwise limit viral replication and spread . This function explains why RSVΔSH viruses, while able to replicate efficiently in tissue culture, are attenuated in animal models where immune responses play a critical role in controlling infection .

What methodologies are most effective for expressing and purifying recombinant RSV SH protein for structural studies?

Recombinant expression and purification of RSV SH protein for structural studies requires specialized methodologies due to its hydrophobic nature and tendency to form oligomers. Based on successful approaches in the literature, an effective protocol would include:

• Expression system selection: E. coli expression systems with strong inducible promoters (T7) are commonly used, with the BL21(DE3) strain being particularly effective for membrane protein expression . For more complex studies requiring post-translational modifications, insect cell systems using baculovirus vectors may be preferable.

• Construct design: The SH protein should be expressed with affinity tags (e.g., His6 or GST) to facilitate purification. It's often beneficial to include a cleavable linker between the tag and the protein to allow tag removal after purification. For structural studies focusing specifically on the transmembrane domain, constructs containing residues 14-41 or 23-41 have been successfully employed .

• Membrane extraction: Effective solubilization requires detergents that maintain the native oligomeric state. Mild detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) are preferred for initial extraction, followed by purification in the presence of these detergents .

• Purification strategy: Immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) has proven effective for obtaining pure, homogeneous preparations. The SEC step is particularly important for separating different oligomeric forms and ensuring pentameric assemblies are isolated .

• Quality assessment: Dynamic light scattering (DLS) and analytical ultracentrifugation can verify the homogeneity and oligomeric state of the purified protein. Circular dichroism (CD) spectroscopy should be used to confirm proper secondary structure formation.

This methodological approach has enabled successful structural characterization of the RSV SH protein, revealing its pentameric architecture and channel-like properties .

How do mutations in the RSV SH protein affect its structure-function relationship?

Mutations in the RSV SH protein can significantly impact its structure-function relationship, affecting both its pentameric assembly and its ability to inhibit TNF-α signaling. Comprehensive molecular dynamics simulations across eight different viral strains have revealed that despite sequence variations, the pentameric structure remains remarkably conserved, suggesting strong evolutionary pressure to maintain this oligomeric state .

Key regions that influence the structure-function relationship include:

Understanding these structure-function relationships is essential for rational drug design targeting the SH protein and for predicting how viral mutations might affect pathogenesis or drug susceptibility.

What experimental approaches can resolve contradictory data regarding RSV SH protein's role in membrane fusion?

Resolving contradictory data regarding RSV SH protein's role in membrane fusion requires a multi-faceted experimental approach that addresses both direct and indirect effects of the protein. Some studies have suggested SH may have a role in viral fusion, while others demonstrate that RSVΔSH viruses can form syncytia and replicate efficiently in vitro . These contradictions can be resolved through the following methodological approaches:

• Time-resolved fusion assays: Implementing fluorescence-based fusion assays that can detect both the rate and extent of membrane fusion. These assays should compare wild-type RSV with RSVΔSH using multiple cell types, as the effect may be cell-type dependent. Quantitative measures of fusion kinetics rather than endpoint measurements would reveal subtle effects that might be missed in binary assessments.

• Biophysical characterization of membrane properties: Using techniques such as fluorescence anisotropy, atomic force microscopy, and lateral diffusion measurements to determine if SH alters membrane fluidity, rigidity, or domain organization in ways that might indirectly influence fusion events without being essential for the basic fusion mechanism.

• Co-immunoprecipitation and proximity labeling studies: Investigating whether SH physically interacts with the F (fusion) and G (attachment) glycoproteins under various conditions. Techniques such as BioID or APEX2 proximity labeling could reveal transient or weak interactions that might be missed by traditional co-immunoprecipitation.

• Single-virus tracking: Utilizing high-resolution microscopy to track individual viruses during the entry process, comparing wild-type RSV with RSVΔSH to identify any differences in attachment, hemifusion, or pore expansion steps.

• Controlled expression systems: Developing cell lines with inducible expression of SH at different levels to determine if the effects on fusion are concentration-dependent, which might explain some contradictory observations if expression levels varied between studies.

How can the inhibition of TNF-α signaling by RSV SH protein be leveraged in experimental systems?

The RSV SH protein's ability to inhibit TNF-α signaling provides a valuable experimental tool for studying both viral pathogenesis and inflammatory signaling pathways. Researchers can leverage this property through several methodological approaches:

• Recombinant virus systems: Creating chimeric viruses where SH proteins from different paramyxoviruses are interchanged can help identify conserved mechanisms of TNF-α inhibition. Studies have already demonstrated that RSV SH (from strains A2 or B1) can functionally replace PIV5 SH in inhibiting TNF-α-induced apoptosis . This approach can be extended to other viruses to create an experimental platform for comparing the efficiency and mechanisms of different viral proteins in modulating TNF-α signaling.

• Cell-based reporter systems: Developing stable cell lines expressing luciferase or fluorescent reporters under the control of TNF-α responsive promoters (such as NF-κB responsive elements) allows for high-throughput screening of SH protein variants. This system can quantitatively assess how mutations in different regions of SH affect its TNF-α inhibitory function.

• Inducible expression systems: Creating tetracycline-inducible SH expression systems enables temporal control over SH protein levels, allowing researchers to study the kinetics of TNF-α signaling inhibition and recovery. This approach can reveal whether SH acts at early or late stages of the signaling cascade.

• Domain mapping through truncation variants: Expressing different portions of the SH protein can help identify the minimal domain required for TNF-α inhibition. This is particularly useful for separating the ion channel function from the TNF-α inhibitory function if these activities reside in different regions of the protein.

• Co-immunoprecipitation and protein interaction studies: Identifying cellular proteins that directly interact with SH through approaches like tandem affinity purification can reveal the molecular mechanisms underlying TNF-α inhibition. This information could potentially identify novel targets in the TNF-α pathway.

By leveraging these experimental approaches, researchers can gain insights into both RSV pathogenesis and the fundamental mechanisms of TNF-α signaling regulation, potentially leading to new antiviral or anti-inflammatory therapeutic strategies .

What are the best experimental models for studying the in vivo role of RSV SH protein in viral pathogenesis?

Selecting appropriate experimental models is crucial for understanding the in vivo role of RSV SH protein in viral pathogenesis. Several models have been employed with varying degrees of success in recapitulating human disease aspects:

Research has shown that RSVΔSH is attenuated in animals despite normal growth in vitro, indicating that SH plays an important role in viral pathogenesis that can only be properly studied in these in vivo or ex vivo models .

How can structural knowledge of the RSV SH protein be utilized for rational drug design?

The structural characterization of RSV SH protein as a pentameric ion channel provides a solid foundation for rational drug design approaches. The following methodologies can effectively leverage this structural knowledge:

• Structure-based virtual screening: Using the pentameric channel structure with its 1.46 Å pore diameter as a target for in silico screening of compound libraries . This approach should focus on:

  • Compounds that can physically block the narrow pore

  • Molecules that bind at the interfaces between monomers to disrupt assembly

  • Allosteric modulators that could alter channel gating

  The molecular dynamics simulations that identified the conserved pentameric structure across different viral strains provide multiple conformational states that can be used as targets for ensemble docking approaches .

• Peptide-based inhibitor design: Developing peptides that mimic portions of the SH transmembrane domain to disrupt pentamer formation. This approach can be guided by the identification of key residues involved in helix-helix interactions from the molecular dynamics simulations of the transmembrane segments (residues 14-41 or 23-41) .

• Small molecule ion channel blockers: Designing compounds similar to known viroporin inhibitors, such as amantadine derivatives, that have been successful against influenza M2 protein. The defined pore diameter of 1.46 Å provides critical constraints for the size and shape of potential channel blockers .

• Fragment-based drug discovery: Using NMR or X-ray crystallography to screen fragment libraries against the SH protein, identifying initial chemical matter that binds to critical regions of the pentamer. The resulting fragments can then be elaborated into more potent and selective inhibitors.

• Conformational stability modulators: Targeting the dynamics of the pentameric assembly rather than just the static structure. Molecular dynamics simulations have shown that the pentameric structure is conserved across variants but exhibits conformational fluctuations . Compounds that restrict these natural motions could inhibit channel function.

• Dual-target inhibitors: Designing molecules that can simultaneously inhibit the ion channel function and the TNF-α signaling inhibition function of SH. This would require mapping the domain responsible for TNF-α inhibition and identifying compounds that can engage both functional sites.

The successful application of these approaches relies on the detailed structural information provided by molecular dynamics simulations, which have identified a conserved pentameric architecture across different RSV strains . This conservation suggests that drugs targeting this structure would have broad efficacy against different viral variants.

What are the key considerations when designing experiments to study SH protein interactions with host cellular factors?

Designing experiments to study RSV SH protein interactions with host cellular factors requires careful consideration of several methodological aspects:

• Protein expression systems: For studying SH-host protein interactions, the expression system must maintain the native conformation of SH while providing sufficient yields. Consider:

  • Mammalian expression systems (HEK293T or A549 cells) for maintaining relevant post-translational modifications

  • Inducible expression systems to control SH levels and minimize potential cytotoxicity

  • Epitope or fluorescent tags positioned to avoid interfering with the transmembrane domain or cytoplasmic tail

  The tag location is particularly important as SH is a small protein (64-65 amino acids), and improper tag placement could disrupt its pentameric assembly or interactions with host factors .

• Membrane environment preservation: As SH is a transmembrane protein that forms pentameric structures, preserving the membrane environment is crucial . Methodological approaches should include:

  • Mild detergents (DDM, LMNG) for extraction that maintain oligomeric state

  • Nanodisc or liposome reconstitution for functional studies

  • Membrane-based yeast two-hybrid systems for interaction screening

  • In situ proximity labeling (BioID, APEX2) to identify interactions in intact cells

• Controlling for indirect effects of ion channel activity: Since SH functions as an ion channel, some apparent interactions might be indirect consequences of altered cellular ion concentrations . Control experiments should include:

  • Channel-inactive mutants (identified through structure-guided mutagenesis)

  • Pharmacological ion channel blockers as controls

  • Comparison with other viral ion channels to distinguish RSV-specific interactions

• Focus on TNF-α pathway components: Given SH's established role in inhibiting TNF-α signaling, particular attention should be paid to interactions with this pathway . Experimental designs should include:

  • Directed screens against TNF receptor complex components

  • Co-immunoprecipitation studies under TNF-α stimulation and basal conditions

  • FRET/BRET-based interaction assays to detect potential transient interactions

  • Domain mapping to identify regions of SH responsible for TNF-α inhibition

• Temporal dynamics consideration: Interactions may be transient or occur only at specific stages of the viral life cycle. Time-course experiments and inducible systems can help capture these dynamic interactions.

Studies have shown that RSV SH can functionally replace PIV5 SH in inhibiting TNF-α signaling, suggesting conserved mechanisms of action . Therefore, comparative approaches examining interactions across different paramyxovirus SH proteins can help identify both conserved and virus-specific host factor interactions.

What are the methodological approaches for distinguishing between the ion channel function and TNF-α inhibitory properties of RSV SH protein?

Distinguishing between the ion channel function and TNF-α inhibitory properties of RSV SH protein requires sophisticated experimental approaches that can selectively isolate and measure each function. The following methodological strategies are effective:

• Structure-guided mutagenesis: Based on the pentameric channel structure revealed by molecular dynamics simulations , targeted mutations can be introduced to:

  • Disrupt pore formation by altering pore-lining residues

  • Modify channel selectivity without affecting assembly

  • Interfere with pentamer assembly through helix-helix interface mutations

  Each mutant should then be assessed for both ion channel activity and TNF-α inhibition to identify mutations that affect one function but not the other.

• Electrophysiological techniques: Direct measurement of ion channel activity using:

  • Patch clamp recordings of cells expressing SH protein

  • Planar lipid bilayer reconstitution of purified SH protein

  • Liposome-based ion flux assays using fluorescent indicators

  These methods provide quantitative data on channel conductance, ion selectivity, and gating properties that can be correlated with TNF-α inhibitory function measured in parallel.

• TNF-α signaling-specific assays: Quantitative assessment of TNF-α pathway inhibition through:

  • NF-κB reporter assays in the presence of SH variants

  • Measurement of TNF-α-induced apoptosis using flow cytometry

  • Quantification of downstream signaling events (e.g., IκB degradation, p65 nuclear translocation)

  • Protein interaction studies with TNF receptor complex components

• Domain separation approaches: Creating chimeric constructs that combine portions of RSV SH with other viroporins to map functional domains:

  • SH extracellular domain fused to heterologous transmembrane domains

  • SH transmembrane domain fused to heterologous cytoplasmic domains

  • Systematic truncation mutants to identify minimal functional units

• Pharmacological dissection: Using known ion channel blockers to inhibit the channel function while monitoring effects on TNF-α inhibition:

  • Small molecule viroporin inhibitors

  • Specific ions that block channel conductance

  • pH modifications that affect channel gating but not protein-protein interactions

Research has established that RSV SH, like PIV5 SH, can inhibit TNF-α signaling , while structural studies have confirmed its pentameric ion channel architecture . A methodical combination of the approaches outlined above would definitively establish whether these are independent functions or if the ion channel activity directly contributes to TNF-α signaling inhibition.

What analytical techniques can be used to characterize the oligomeric state of RSV SH protein in different membrane environments?

Characterizing the oligomeric state of RSV SH protein in different membrane environments requires a combination of biophysical, biochemical, and imaging techniques. The following analytical approaches provide complementary information about SH oligomerization:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): This technique separates protein complexes based on size and directly measures their molecular weight, independent of shape. For membrane proteins like SH:

  • Protein must be extracted with appropriate detergents that maintain oligomeric state

  • Different detergents can be compared to assess environment-dependent oligomerization

  • Absolute molecular weight determination can distinguish between different oligomeric states (pentamer vs. tetramer or trimer)

  This approach has been valuable in confirming the pentameric assembly suggested by molecular dynamics simulations .

• Analytical ultracentrifugation (AUC): Sedimentation velocity and sedimentation equilibrium experiments provide information about both size and shape of protein complexes:

  • Can be performed in detergent micelles or lipid nanodiscs

  • Directly measures the sedimentation coefficient and diffusion coefficient

  • Can detect multiple oligomeric species in a mixture and their relative proportions

• Chemical cross-linking coupled with mass spectrometry (XL-MS): This approach captures transient interactions and can map the interfaces between monomers in the pentamer:

  • Membrane-permeable crosslinkers can be used in native cellular environments

  • MS analysis identifies specific residues involved in monomer-monomer contacts

  • Different crosslinker spacer lengths can provide distance constraints

• Förster resonance energy transfer (FRET): By labeling SH monomers with donor and acceptor fluorophores, FRET can detect oligomerization in intact membranes:

  • Can be performed in living cells to assess oligomerization in native environment

  • Acceptor photobleaching or fluorescence lifetime measurements provide quantitative data

  • Different fluorophore positions can map the geometry of the oligomer

• Single-molecule techniques: Methods such as single-molecule photobleaching or single-molecule tracking can directly visualize individual oligomers:

  • Stepwise photobleaching counts the number of monomers in a complex

  • Can distinguish between pentamers and other oligomeric states

  • Works in both artificial membranes and cellular membranes

• Native mass spectrometry: Recent advances allow membrane proteins to be analyzed directly:

  • Requires careful optimization of detergent removal during ionization

  • Can determine exact stoichiometry of complexes

  • Can detect bound lipids that might stabilize the oligomer

• Electron microscopy (EM): Negative stain EM or cryo-EM can directly visualize oligomers:

  • Provides structural information about the arrangement of monomers

  • Can be applied to protein in detergent, nanodiscs, or liposomes

  • Single-particle analysis can generate 3D models to complement molecular dynamics simulations

Molecular dynamics simulations across different RSV SH variants have consistently identified a pentameric structure with a Cα RMSD of <0.99 Å between different variants . These computational predictions should be validated using the experimental techniques described above, particularly in different membrane environments that might influence the stability of the pentameric assembly.

How might emerging structural biology techniques enhance our understanding of RSV SH protein dynamics?

Emerging structural biology techniques offer unprecedented opportunities to enhance our understanding of RSV SH protein dynamics, potentially revealing aspects of function that have remained elusive with traditional approaches. These cutting-edge methodologies can address key questions about SH protein behavior in physiologically relevant contexts:

• Time-resolved cryo-electron microscopy (cryo-EM): This technique captures structural snapshots of proteins at different functional states:

  • Millisecond time resolution can capture ion channel opening and closing events

  • Multiple conformational states can be classified through 3D variability analysis

  • The pentameric assembly identified through molecular dynamics simulations can be visualized directly

  • Different conditions (pH, membrane composition) can reveal environment-dependent conformational changes

• Solid-state NMR spectroscopy: This approach allows study of membrane proteins in native-like lipid environments:

  • Can detect subtle conformational changes in the transmembrane helices

  • Measures dynamics on various timescales (ns-ms) relevant to channel function

  • Chemical shift perturbation experiments can map binding sites of potential inhibitors

  • Can be combined with specific isotope labeling to focus on key residues identified in the pentameric model

• Single-molecule FRET (smFRET) with membrane proteins: By examining FRET efficiency distributions in single molecules rather than ensemble averages:

  • Can identify multiple conformational states and their interconversion

  • Works under physiological conditions in artificial membranes

  • Can track dynamics in real-time during channel activation

  • May reveal heterogeneity in the behavior of individual pentamers not detectable in ensemble measurements

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique measures the accessibility of backbone amide hydrogens to exchange with deuterium:

  • Identifies flexible regions and protein-protein interfaces

  • Can be performed in various membrane mimetics to assess environment effects

  • Time-resolved experiments track conformational changes during function

  • May reveal how SH structure changes when interacting with TNF-α signaling components

• Cryo-electron tomography (cryo-ET): For studying SH in its native context:

  • Can visualize SH distribution and organization in virus particles

  • Sub-tomogram averaging can reveal in situ structure

  • Correlative light and electron microscopy can link structure to function

  • May resolve discrepancies regarding SH's role in membrane fusion by visualizing it in the context of fusion events

• Microfluidic diffusional sizing: This emerging technique measures hydrodynamic radius changes:

  • Can detect subtle conformational changes in membrane proteins

  • Works with extremely small sample amounts

  • Allows rapid screening of different conditions affecting oligomerization

  • Could help understand how mutations affect the stability of the pentameric assembly

These advanced structural techniques, when combined with the existing computational models of the pentameric SH channel and functional data on TNF-α signaling inhibition , will provide a comprehensive understanding of SH protein dynamics in different contexts and potentially reveal new opportunities for therapeutic intervention.

What role might artificial intelligence and machine learning play in predicting RSV SH protein variants and their functional implications?

Artificial intelligence (AI) and machine learning (ML) approaches are poised to significantly advance our understanding of RSV SH protein variants and their functional implications through several methodological applications:

• Variant effect prediction: Deep learning models can be trained on existing data about RSV SH variants to predict the functional consequences of novel mutations:

  • Neural networks can integrate sequence, structural, and functional data

  • Attention-based models can identify critical residues for pentamer formation and stability

  • Ensemble methods can predict changes in ion channel conductance properties

  • Graph neural networks can model how mutations propagate effects through the protein structure

  This approach could extend the understanding gained from the 45 different molecular dynamics simulations that identified the conserved pentameric structure .

• Molecular dynamics acceleration: AI methods can enhance traditional molecular dynamics approaches:

  • Machine learning potentials can extend simulation timescales by orders of magnitude

  • Active learning frameworks can efficiently explore conformational space

  • Neural network-based analysis can identify cryptic binding sites not obvious in static structures

  • Generative models can propose stable alternative conformations for experimental testing

  These methods could build upon existing molecular dynamics studies of the RSV SH protein to explore conformational dynamics inaccessible to conventional simulations.

• Drug-target interaction prediction: AI can accelerate the discovery of SH protein inhibitors:

  • Deep learning models can screen billions of compounds in silico

  • Graph neural networks can predict binding modes and affinities

  • Generative chemistry can design novel molecules specifically for the pentameric channel

  • Multi-objective optimization can balance channel blocking ability with TNF-α pathway inhibition

• Evolutionary analysis and sequence-function mapping: Advanced ML models can extract patterns from sequence data:

  • Unsupervised learning can identify co-evolving residues critical for function

  • Language models trained on protein sequences can predict functional effects of mutations

  • Attention mechanisms can identify regions under selective pressure

  • Transfer learning approaches can leverage data from related viroporins

• Integration of heterogeneous experimental data: AI methods excel at finding patterns across diverse datasets:

  • Multi-modal deep learning can integrate structural, functional, and phenotypic data

  • Bayesian methods can handle uncertainty in experimental measurements

  • Self-supervised learning can extract features from unlabeled experimental data

  • Explainable AI approaches can generate testable hypotheses about structure-function relationships

• In silico mutagenesis and phenotype prediction: Comprehensive computational mutagenesis can be performed:

  • Deep mutational scanning in silico can predict effects of all possible mutations

  • Clustering algorithms can identify mutation patterns with similar functional outcomes

  • Models can predict both structural stability of the pentamer and TNF-α inhibitory function

  • Sensitivity analysis can identify residues most critical for maintaining dual functionality

Implementing these AI/ML approaches could significantly accelerate research on RSV SH protein by efficiently navigating the vast space of possible variants and their functional implications. This would build upon current understanding of SH's pentameric structure and TNF-α inhibitory function to develop more effective antiviral strategies.