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

What are the structural differences between RSV-A and RSV-B Small Hydrophobic proteins?

The SH protein of RSV contains 64 amino acid residues in RSV subgroup A and 65 amino acid residues in RSV subgroup B . Both variants are type II transmembrane proteins that share functional similarities but display sequence variations that may affect their interactions with host proteins . The size difference results from subtype-specific amino acid composition that influences the protein's membrane topology and potentially its functional properties.

When designing experiments involving recombinant SH proteins, researchers should account for these structural differences, particularly when comparing cross-reactivity of antibodies or evaluating functional conservation between subtypes. Sequence analysis demonstrates that while both proteins maintain core functional domains, variations in amino acid sequences may influence their antigenic properties and potentially their interactions with host cellular components.

How does the SH protein function in RSV pathogenesis?

The SH protein plays a significant role in RSV pathogenesis through multiple mechanisms:

• TNF-α signaling inhibition: Research demonstrates that the SH protein of RSV functions similarly to PIV5 SH by inhibiting TNF-α signaling pathways . This inhibition likely contributes to immune evasion during infection.

• Anti-apoptotic activity: Studies using recombinant RSV lacking SH (RSVΔSH) show that SH protein prevents infected cells from undergoing apoptosis . When cells were infected with RSVΔSH, more pronounced cytopathic effects were observed compared to wild-type RSV infection, indicating SH's protective function against cell death .

• Virus attenuation: RSVΔSH is attenuated in animals despite growing as well as wild-type virus in cell culture, confirming SH's importance in pathogenesis .

Methodologically, researchers studying SH function should employ multiple complementary approaches, including genetic deletion studies, cell death assays (TUNEL), and cytokine signaling reporter systems to comprehensively assess SH's contributions to RSV pathogenesis.

What expression systems are most effective for producing recombinant RSV-B SH protein?

For recombinant expression of RSV-B SH protein, researchers should consider the following methodological approaches:

• Bacterial artificial chromosome (BAC) systems: These have proven successful for cloning full-length RSV-B antigenomes, offering enhanced sequence stability compared to traditional plasmid vectors . BAC systems allow for easy manipulation via recombineering protocols and facilitate the generation of stable RSV-B constructs containing SH modifications .

• Hybrid viral vectors: For functional studies, researchers have successfully used PIV5 vectors lacking their own SH but containing RSV SH (from RSV strain B1) in its place (PIV5ΔSH-RSV SH) . This approach allows evaluation of SH function in a viral context while controlling for other variables.

• Mammalian cell expression: For biochemical and structural studies, expression in HEp-2 cells using MVA-T7 polymerase system has shown success for expressing RSV proteins including SH .

When selecting an expression system, consider:

• The intended experimental application (functional studies vs. protein production)

• Required protein modifications (glycosylation patterns vary between systems)

• Scale of production needed

• Need for membrane association to maintain native conformation

What are the key methodological considerations when generating recombinant RSV containing modified SH genes?

When generating recombinant RSV with modified SH genes, researchers should consider:

• Vector selection: BAC-based systems show greater stability than traditional plasmid vectors for cloning full-length RSV genomes . For RSV-B-9671, researchers found that "Full-length clones with the correct consensus sequence were only obtained following prolonged growth of transformed bacteria (Stbl3) at room temperature with increased concentrations of carbenicillin" .

• Transcription initiation optimization: Insertion of three guanine (G) residues following the T7 promoter significantly increases the efficiency of minigenome rescue .

• Reporter gene insertion: For tracking infection, rescue of RSVs expressing fluorescent proteins such as EGFP or mScarlet from an additional transcription unit inserted between the P and M genes has proven successful .

• Cell line selection: HEp-2 cells at 75% confluency have shown optimal results for rescue of recombinant RSVs .

• Helper plasmid balance: Successful rescue requires optimal ratios of helper plasmids encoding N, P, M2-1, and L proteins .

A typical rescue protocol involves transfecting HEp-2 cells (infected with MVA-T7) with pSMART-BAC vector containing a full-length cDNA copy of the RSV antigenome together with subtype-specific helper plasmids, followed by monitoring for syncytium formation and passaging the virus 5-10 days post-transfection .

How can researchers effectively assess the role of SH protein in TNF-α signaling inhibition?

For rigorous evaluation of RSV-B SH protein's role in TNF-α signaling inhibition, researchers should employ the following methodological approaches:

• Reporter gene assays: Transfect cells with a luciferase gene under the control of an NF-κB-responsive promoter along with a plasmid expressing RSV-B SH . This allows quantitative measurement of SH's ability to block TNF-α-induced NF-κB activation.

• Comparative analysis with RSVΔSH: Generate and compare wild-type RSV with RSVΔSH to assess differences in TNF-α production and signaling during infection . Consider using ELISA or multiplexed protein assays to measure cytokine production.

• Complementation studies: Use PIV5ΔSH viruses expressing RSV-B SH to determine if RSV SH can functionally replace PIV5 SH in blocking TNF-α signaling .

• Domain mapping experiments: Create chimeric or mutated SH constructs to identify regions critical for TNF-α inhibition, providing insight into the mechanism of action.

• Co-immunoprecipitation assays: Identify host proteins interacting with SH during TNF-α signaling by using tagged versions of SH protein followed by mass spectrometry analysis.

When interpreting results, researchers should consider that "Both RSV A and RSV B subgroups substantially contribute to the global RSV burden" , making it important to compare SH proteins from both subgroups in parallel experiments.

What techniques are most reliable for studying the anti-apoptotic functions of RSV-B SH protein?

For robust investigation of RSV-B SH protein's anti-apoptotic functions, researchers should implement:

• TUNEL assays: Terminal deoxynucleotidyltransferase-mediated dUTP-fluoroscein isothiocyanate nick end labeling (TUNEL) has successfully demonstrated increased apoptosis in RSVΔSH-infected cells compared to wild-type RSV infection .

• Co-infection experiments: Researchers have shown that expressing RSV SH in cells infected with rPIV5ΔSH prevents apoptosis, confirming SH's anti-apoptotic function through complementation . This approach allows assessment of whether RSV-B SH can functionally replace other viral anti-apoptotic proteins.

• Apoptotic marker analysis: Measure caspase activation, cytochrome c release, and mitochondrial membrane potential to identify specific apoptotic pathways affected by SH protein.

• Time-course studies: Compare the kinetics of apoptosis between wild-type and RSVΔSH infection to determine when SH's anti-apoptotic effects are most pronounced.

• Tissue-specific effects: When extending to in vivo models, consider that "RSVΔSH is attenuated in animals, indicating that RSV SH plays an important role in viral pathogenesis" .

How do mutations in the RSV-B SH protein affect its membrane topology and function?

When investigating how mutations affect RSV-B SH protein topology and function:

• Predictive analysis: Begin with computational predictions of transmembrane domains and protein topology based on amino acid sequence, recognizing that RSV-B SH contains 65 amino acid residues compared to 64 in RSV-A .

• Glycosylation mapping: Use site-directed mutagenesis to modify potential glycosylation sites, then analyze protein migration patterns to determine membrane orientation. Previous studies have successfully identified "glycosylated and nonglycosylated RSV SH from each strain" using radioimmunoprecipitation .

• Epitope tagging and accessibility assays: Insert epitope tags at various positions within SH and assess their accessibility to antibodies in permeabilized versus non-permeabilized cells to map membrane topology.

• Structure-function correlation: Generate a panel of SH mutants and test their ability to inhibit TNF-α signaling or prevent apoptosis to identify functional domains. This approach can build on existing research showing that "RSV SH has a function similar to that of PIV5 SH" .

• Cross-subtype chimeras: Create chimeric proteins containing domains from RSV-A and RSV-B SH to identify subtype-specific functional differences, particularly important since "RSV subgroups vary primarily in the mucin-like domains" .

For accurate interpretation, remember that membrane protein topology can be influenced by experimental conditions and expression systems, necessitating validation through multiple complementary approaches.

How can reverse genetics systems be optimized for studying RSV-B SH protein functions?

For optimization of reverse genetics systems to study RSV-B SH protein:

• BAC vector utilization: Employ bacterial artificial chromosome (BAC) vectors rather than traditional plasmids, as "This resulted in enhanced sequence stability of pRSV-B-9671 and enabled construction of a stable full-length cDNA clone" .

• Transcription optimization: "Insertion of three guanine (G) residues following the T7 promoter significantly increased the efficiency of minigenome rescue" . Consider this modification when designing rescue constructs.

• Reporter gene insertion strategies: Successfully rescued RSVs express reporter proteins such as "EGFP, mScarlet (mSct), or NanoLuc luciferase (Nluc)" from transcription units inserted between the P and M genes, allowing for easy tracking of infection.

• Growth conditions for clone stability: For RSV-B constructs, "full-length clones with the correct consensus sequence were only obtained following prolonged growth of transformed bacteria (Stbl3) at room temperature with increased concentrations of carbenicillin" .

• Helper plasmid optimization: While some reverse genetics systems require codon optimization of helper plasmids, research shows "Codon optimization of N, P, M2-1, and L helper plasmids was not necessary for successful virus rescue" .

These optimizations allow researchers to generate recombinant RSV-B viruses with modified SH proteins to investigate structure-function relationships in relevant experimental models, moving beyond older laboratory-adapted strains to "clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain)" .

How should researchers validate antibodies targeting RSV-B SH protein?

For proper validation of antibodies against RSV-B SH protein:

• Specificity testing: Validate antibodies against both glycosylated and non-glycosylated forms of RSV-B SH, as both forms exist during infection . Research has successfully used "antisera specifically recognized glycosylated and nonglycosylated RSV SH from each strain from either rPIV5ΔSH-RSV A2 SH- or rPIV5ΔSH-RSV B1 SH-infected cells by radioimmunoprecipitation" .

• Cross-reactivity assessment: Test antibodies against both RSV-A and RSV-B SH proteins to determine subtype specificity, particularly important since there are structural differences between the 64-amino acid RSV-A SH and 65-amino acid RSV-B SH .

• Multiple detection methods: Confirm antibody performance across different techniques (immunoblotting, immunoprecipitation, immunofluorescence, flow cytometry) to ensure versatility.

• Controls: Always include:

  • Positive controls: Cells transfected with SH expression vectors

  • Negative controls: Uninfected cells and cells infected with RSVΔSH

  • Specificity controls: Pre-immune serum, isotype controls, and peptide competition assays

• Reproducibility verification: Test antibody performance across different batches and in different experimental systems to ensure consistent results.

The small size and hydrophobic nature of SH protein make antibody validation particularly challenging, requiring rigorous controls and multiple validation approaches.

What are effective protocols for purifying recombinant RSV-B SH protein while maintaining its native conformation?

For purification of recombinant RSV-B SH protein with preserved native conformation:

• Expression system selection: Consider using mammalian expression systems to ensure proper post-translational modifications, particularly since RSV SH is known to be glycosylated . HEp-2 cells have proven effective for RSV protein expression .

• Detergent optimization: Due to SH's hydrophobic nature as a transmembrane protein, careful detergent selection is critical:

  • Initial solubilization: Try mild detergents such as CHAPS or DDM

  • Purification buffers: Maintain detergent above critical micelle concentration throughout purification

  • Consider detergent screening to identify optimal conditions that maintain function

• Affinity purification strategy: Use small epitope tags (His, FLAG) rather than large fusion partners that might disrupt the native conformation of this small (65 amino acid) protein .

• Functional validation: After purification, verify that the recombinant protein retains key functions:

  • TNF-α signaling inhibition using cell-based reporter assays

  • Membrane integration and topology using biophysical methods

• Storage optimization: Determine conditions (temperature, buffer composition, additives) that preserve protein stability during storage.

Remember that the small size and hydrophobic nature of SH make it particularly challenging to purify while maintaining native conformation, necessitating careful optimization of each purification step.

How can researchers accurately quantify SH protein expression in infected cells?

For accurate quantification of SH protein expression in infected cells:

• Radioimmunoprecipitation: This technique has been successfully employed to detect "glycosylated and nonglycosylated RSV SH from each strain" , providing a sensitive method for detecting the low-abundance SH protein.

• Reporter-tagged SH constructs: Generate recombinant RSV containing SH fused to small epitope tags for easier detection, similar to the approach used for "recombinant (r) RSV-B-9671 expressing either enhanced green fluorescent protein (EGFP) or the red fluorescent protein dTomato" .

• RT-qPCR for transcript quantification: Measure SH mRNA levels using RT-qPCR with carefully designed primers specific to RSV-B SH sequences. This approach has been used to confirm viral infection: "RT-PCRs using RNA from infected cells were performed to detect the presence of genomic RNA" .

• Western blotting optimization:

  • Use tricine-SDS-PAGE instead of standard glycine-SDS-PAGE for better resolution of small proteins

  • Optimize transfer conditions for hydrophobic proteins (lower methanol concentration, longer transfer times)

  • Consider transferring to PVDF rather than nitrocellulose membranes

• Flow cytometry: For cell-by-cell quantification, optimize intracellular staining protocols specifically for this small transmembrane protein.

When interpreting results, consider that SH expression levels may vary with cell type, viral strain, and time post-infection, necessitating careful experimental design with appropriate time-course studies and controls.

What controls are essential when conducting functional studies of recombinant RSV-B SH protein?

When conducting functional studies of recombinant RSV-B SH protein, implement these essential controls:

• RSVΔSH comparison: Include RSVΔSH as a critical control to establish baseline phenotypes in the absence of SH protein . Research has shown "more notable CPE was observed in RSVΔSH-infected cells" compared to wild-type RSV infection.

• Cross-subtype controls: Include RSV-A SH protein experiments in parallel to identify subtype-specific functions, particularly important since studies show "Both RSV A and RSV B subgroups substantially contribute to the global RSV burden" .

• Functional positive controls: When studying specific functions:

  • For TNF-α inhibition: Include PIV5 SH as a positive control since "the SH protein of RSV has a function similar to that of PIV5 SH"

  • For apoptosis inhibition: Include established anti-apoptotic proteins (e.g., Bcl-2) as positive controls

• Expression level verification: Always validate that experimental and control proteins are expressed at comparable levels, as functional differences may simply reflect expression differences.

• Cell-type comparisons: Test phenotypes in multiple relevant cell types, particularly since "Infection of L929 cells with RSV resulted in noticeable CPE 1 dpi compared to that for mock-infected cells" .

• Time-course analysis: Evaluate functional outcomes at multiple time points post-infection or transfection to capture the dynamic nature of SH protein activities.

These controls help distinguish specific SH protein functions from non-specific effects and allow proper interpretation of experimental results within the broader context of RSV biology.

Why might recombinant RSV-B SH protein show different functional properties compared to native protein?

When recombinant RSV-B SH protein exhibits different functional properties than native protein, consider:

• Post-translational modifications: Native SH protein undergoes glycosylation, as evidenced by detection of "glycosylated and nonglycosylated RSV SH" . Ensure your expression system recapitulates these modifications, particularly if using bacterial expression systems that lack glycosylation machinery.

• Membrane environment differences: As a transmembrane protein, SH function depends on proper membrane integration. Different expression systems may provide different membrane compositions affecting protein topology and function.

• Protein folding issues: The small size (65 amino acids in RSV-B) and hydrophobic nature of SH make proper folding challenging, especially when expressing recombinant protein. Consider:

  • Expression temperature optimization

  • Addition of chaperone co-expression plasmids

  • Use of mild solubilization conditions

• Epitope tag interference: Tags added for purification or detection may interfere with function. If using tagged constructs, validate with multiple tag positions or removable tags.

• Protein concentration effects: Recombinant systems may produce non-physiological concentrations of SH, potentially leading to aggregation or artificial interactions not present during normal infection.

For troubleshooting, systematically test different expression systems, purification methods, and functional assays while maintaining appropriate controls using either wild-type RSV infection or PIV5ΔSH-RSV SH chimeric viruses .

How can researchers address instability issues when cloning RSV genome segments containing the SH gene?

To overcome instability issues when cloning RSV genome segments containing the SH gene:

• Use BAC vectors instead of traditional plasmids: Research demonstrates that "the full-length antigenome of RSV-B-9671 and partial antigenome of RSV-A-0594 were transferred from the pBluescript II KS(+) vector to pSMART-BAC" which "resulted in enhanced sequence stability of pRSV-B-9671 and enabled construction of a stable full-length cDNA clone of RSV-A-0594 to be completed" .

• Optimize bacterial growth conditions: For RSV-B-9671, "full-length clones with the correct consensus sequence were only obtained following prolonged growth of transformed bacteria (Stbl3) at room temperature with increased concentrations of carbenicillin" .

• Use specialized bacterial strains: Employ recombination-deficient strains like Stbl3 to reduce unwanted recombination events that lead to deletions .

• Consider segmentation strategies: If full-length cloning remains problematic, divide the genome into segments that can be reassembled through unique restriction sites or Gibson assembly.

• Remove toxic elements: If specific regions consistently cause instability, consider temporary modifications during cloning that can be restored during virus rescue.

Researchers have noted that "analogous cloning procedures for RSV-A-0594 resulted in clones containing full or partial deletions of the G gene" , highlighting the potential for gene-specific stability issues that may require tailored approaches.

What approaches work best for resolving experimental inconsistencies when studying SH protein functions?

When encountering experimental inconsistencies in RSV-B SH protein functional studies:

• Standardize viral stocks: Ensure consistent passage history and titration methods, as "Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long" which "are not representative of currently circulating genotypes and have a convoluted passage history" .

• Control for RSV subgroup variations: Remember that "Both RSV A and RSV B subgroups cause severe disease" but may have subtle functional differences. Specify and consistently use either RSV-A or RSV-B SH in experiments to avoid conflating subgroup-specific effects.

• Validate reagents across experimental systems: Test antibodies, expression vectors, and functional assays in multiple systems to ensure robust performance, particularly important when studying the small (65 amino acid) SH protein .

• Implement internal controls: Include appropriate positive and negative controls in each experiment, such as comparing RSVΔSH with wild-type RSV .

• Consider cell-type dependencies: SH protein functions may vary between cell types. For example, TUNEL assays demonstrating SH's anti-apoptotic effects were performed in L929 cells , and results may differ in other cell types.

• Document experimental timing precisely: SH's effects on TNF-α signaling and apoptosis may be time-dependent, requiring careful experimental scheduling and documentation.